Performance of Diapausing Parasitoid Wasps, Habrobracon Hebetor, After Cold Storage ⇑ ⇑ Haoliang Chen A,B,C,D, Hongyu Zhang A, , Kun Yan Zhu C, James Throne D

Biological Control 64 (2013) 186–194

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Biological Control

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Performance of diapausing parasitoid wasps, Habrobracon hebetor, after cold storage ⇑ ⇑ Haoliang Chen a,b,c,d, Hongyu Zhang a, , Kun Yan Zhu c, James Throne d,

a State Key Laboratory of Agricultural Microbiology, Institute of Urban and Horticultural Pests, and Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People’s Republic of China b Institute of Plant Protection and Agro-Products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, People’s Republic of China c Department of Entomology, Kansas State University, Manhattan, KS 66506, USA d USDA, Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, KS 66502, USA

highlights graphical abstract

" Diapause female survival is greater than for nondiapause females during cold storage. " Diapause females produce more progeny than nondiapause females after cold storage. " Female percentage of F1 offspring is lower than in the culture after cold storage. " Diapause females can be stored at 5 °C for up to 8 weeks.

article info abstract

Article history: The ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is an important potential bio- Received 1 September 2011 logical control agent for lepidopterous pests of stored products. We investigated the effects of long-term Accepted 26 November 2012 cold storage of diapausing and nondiapausing H. hebetor on their performance after cold storage. Mortal- Available online 2 December 2012 ity during storage increased with increasing storage duration, and the mortality of diapausing females was lower than that of nondiapausing females after 8, 12, and 16 weeks of storage. Longevity, egg laying, Keywords: number of progeny produced, and time to 50% egg laying were all reduced, as compared with the culture Parasitoid females when parasitoids were reared at conditions that do not induce diapause. But, for females reared Braconidae at 20 °C at conditions that induce diapause, all of these quality parameters did not differ from those of Reproductive diapause Quality culture insects when the storage duration was 8 weeks or less. The percentage of female F1 offspring Long-term storage was always lower for cold stored insects than for the culture insects. Presence of a male after cold storage Biological control did not impact any of the quality parameters measured. Thus, rearing parasitoids at 20 °C and 10L:14D and then storing them for up to 8 weeks at 5 °C would produce parasitoids that are similar to culture parasitoids, except that the percentage of females is lower than that in the cultures (36% vs. 52%). Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction in warehouses due to its rapid population growth (Press et al., 1982; Balevski, 1984; Huang, 1986; Keever et al., 1986; Brower The ectoparasitoid Habrobracon hebetor (Say) (=Bracon hebetor) and Press, 1990). It has been used to suppress moth populations (Hymenoptera: Braconidae) is considered to be one of the most in stored products (Press et al., 1982; Balevski, 1984; Huang, important biological control agents of several pyralid moth pests 1986; Brower and Press, 1990; Cline and Press, 1990; Garba and

⇑ Corresponding authors. Fax: +86 27 87396057 (H. Zhang), fax: +1 785 537 5584 (J. Throne). E-mail addresses: [email protected] (H. Chen), [email protected] (H. Zhang), [email protected] (K.Y. Zhu), [email protected] (J. Throne).

Gaoh, 2008; Zhang, 2009) and in field crops (Uwais et al., 2006; hole covered with fine mesh screen. Parasitoids were reared at Imam et al., 2007). 27.5 ± 0.5 °C, 65 ± 5% RH, and 16L:8D photoperiod. There are two problems which reduce the desirability of H. hebetor as a potential biological control agent. One problem is obtain- 2.2. Obtaining diapause and nondiapause H. hebetor for cold storage ing sufficient numbers for release (Coudron et al., 2007), and another problem is a shortage of host larvae for mass production To obtain H. hebetor eggs for experiments, 10 pairs of adults of H. hebetor when the demand is high. When demand is low, over- from the culture were allowed to oviposit for 24 h on 30 last instar production of host larvae can be wasteful. Cold storage has been P. interpunctella. Females are differentiated based on the presence considered as an option to overcome these problems for other par- of an ovipositor. The larvae with H. hebetor eggs were placed in asitoid species (Foerster and Nakama, 2002; Pitcher et al., 2002; Percival I-36VL chambers set at either 17.5 or 20 °C, 65% RH, and Bradley et al., 2004; Rundle et al., 2004; Tezze and Botto, 2004; Le- either 10L:14D (diapause-inducing conditions) or 16L:8D (condi- vie et al., 2005; López and Botto, 2005; Chen et al., 2008). Cold stor- tions that do not induce diapause) (Chen et al., 2012). Adult emer- age provides flexibility and efficiency in mass production gence was checked every 3.5 days. After emergence, females and (Greenberg et al., 1996; Leopold et al., 1998; Tezze and Botto, males were left together at their treatment conditions for 3.5 days 2004), and minimizes the costs of maintaining a colony during to mate. After the 3.5–7.0-day post-emergence period, 450 females periods when releases are not required (Coudron et al., 2007). Cold and 450 males from each condition were stored with the sexes sep- storage also improves genetic stability because fewer generations arated at 5.0 °C. We only stored mated females because they can are reared. produce both male and female progeny without additional mating, A concern with storing parasitoids is possible loss of vitality which would be desirable in a mass rearing program. There were during storage. Storing diapausing insects is one possible method 30 adults per container. Tests at 17.5 and 20 °C were conducted for maintaining vitality during cold storage. Diapause is one of sequentially. the major strategies used as a means to survive unfavorable environmental conditions, and it is well documented that insect chilling resistance is enhanced during diapause (Foerster and Doetzer, 2.3. Quality parameters measured after cold storage 2006; Denlinger, 2008). However, diapause in H. hebetor has not been well studied (Adashkevich and Saidova, 1985). Our previous After 4, 8, 12, 16, and 20 weeks of storage, three replicates of 30 study showed that adult H. hebetor can enter reproductive dia- females and 30 males from each storage condition were removed pause when reared at low temperature and short photoperiod from cold storage. Mortality during cold storage was recorded. (Chen et al., 2012). However, we don’t know whether reproductive We determined longevity and fecundity of females after cold diapause prolongs the length of time for which H. hebetor can be storage in two ways. First, we determined the ability of females stored at low temperature without loss of vitality. to mate and lay eggs after cold storage by pairing females with a In this study, we evaluate the quality of H. hebetor adults after 0- to 3-day-old male from the cultures in a 15-dram vial containing cold storage. Adults were reared at conditions that appear to in- 9 P. interpunctella last instars. The vials were placed at 27.5 °C, duce reproductive diapause, and then emerged adults were stored 65 ± 5% RH, and 16L:8D photoperiod, and the adults were moved at cold temperatures for varying periods of time. Several quality to a new vial containing 9 new P. interpunctella last instars every parameters were measured to determine the effects of different 3.5 days until the female died. After removal of the parasitoids, lengths of cold storage on reproduction and development. numbers of H. hebetor eggs on larvae were counted. Sex ratio of adults emerging from these eggs was determined. There were 15 pairs of adults set up for this test at each condition. We also deter- 2. Materials and methods mined whether sperm in the female’s spermatheca were still active after cold storage using exactly the same experimental techniques, 2.1. Insect rearing except that the females were placed in vials without males. We determined longevity and fertility of males after cold stor- 2.1.1. Hosts age by pairing one male that had been removed from cold storage The Indianmeal moth, Plodia interpunctella (Hübner) (Lepidop- with one 0- to 3-day-old unmated female from the cultures. Proce- tera: Pyralidae), was reared in 3.8-l glass jars on an artificial diet dures used were the same as for females above. Unmated females consisting of cracked wheat (1000 g), wheat shorts (1000 g), wheat were obtained by placing individual pupae from the culture in a germ (100 g), brewer’s yeast (80 g), glycerine (240 ml), honey small petri dish. (240 ml), and 120 ml of water (McGaughey and Beeman, 1988). Pu- To determine whether longevity and fecundity differed from pae were collected from the stock culture by placing corrugated that of culture insects, 15 pairs of 0- to 24-h-old adults from the cardboard (2-cm high by 5.7-cm diameter) in the glass jar as a culture were treated as above. pupation site (larvae crawl into the corrugations to pupate), and then putting the pupae in another glass jar for adults to emerge 2.4. Statistical analysis and lay eggs. About 50 mg of eggs (ca. 2000 eggs) were then trans- ferred to a 500-ml glass jar filled about one-third with artificial Mortality during cold storage, longevity, fecundity, time of 50% diet. The culture was maintained at 30.0 ± 0.5 °C, 65 ± 5% relative egg laying, total number of progeny produced, and F1 offspring fe- humidity (RH), and 16L:8D photoperiod. Last instar larvae were male percentage were analyzed with a two- or three-way ANOVA. used as hosts for parasitoids in the experiments. When interactions were significant, we then used one- or two-way ANOVA’s to examine effects of one factor within each level of the 2.1.2. Parasitoids other factor. Tukey’s-b test (P = 0.05) was used for mean compari- A field strain of H. hebetor used in the experiments was col- sons (SPSS Inc., 2007). We used Dunnett’s-t test to determine lected in Parlier, CA, in October 2009, and the studies described whether treatment means differed from those for the culture in- in this manuscript were conducted with this strain during the next sects. Only replicates in which there were more than 50 progeny year. Adult parasitoids were introduced into 15-dram plastic vials adults were used to calculate the sex ratio. Biological parameters (3.2-cm diameter by 8.3-cm high) containing 30 last instar P. inter- were compared separately among different photoperiods at each punctella. Vials were covered with lids that had a 0.5-cm diameter temperature. 188 H. Chen et al. / Biological Control 64 (2013) 186–194

3. Results and 16 weeks of cold storage than after 8 and 12 weeks of cold storage. The reason that they lived longer after 16 weeks of cold 3.1. Mortality during cold storage storage than after 8 and 12 weeks of cold storage is because the data for 4, 8, and 12 weeks included females reared at both pho- Mortality during cold storage of females reared at 20 °C varied toperiods, while no females reared at 16L:8D survived 16 weeks with photoperiod during rearing and duration of cold storage, of cold storage. Longevity of females reared at 16L:8D was shorter and the interaction was significant (Table 1). Because interaction than those of culture females after all storage durations [mean was significant, we used one-way ANOVA’s to examine the effects (±SE) difference was À16.0 ± 2.5 d after 4 weeks of storage, of storage duration on mortality within each level of rearing pho- À25.4 ± 2.5 d after 8 weeks, and À26.5 ± 3.0 d after 12 weeks], toperiod, and to examine the effects of rearing photoperiod within and longevity of females reared at 10L:14D was shorter than that each level of storage duration. Female mortality increased with of culture females after 12 weeks of storage (À12.4 ± 4.1 d) storage duration at both photoperiods (Table 2). Female mortality (Fig. 1A). was greater throughout most of the storage period when parasit- Longevity after removal from cold storage of females reared at oids had been reared at 16L:8D than when they were reared at 17.5 °C varied with the photoperiod at which they were reared, 10L:14D. Storage duration affected mortality of males reared at but not with the duration of cold storage or whether they were 20 °C, but photoperiod did not affect mortality and the interaction paired with a male after cold storage and no interactions were sig- was not significant (Table 1). Mortality of males reared at 20 °C ex- nificant (Table 4). Longevity after removal from cold storage of fe- ceeded 80% after only 8 weeks of storage, and no males survived males was greater when females were reared at 10L:14D than 16 weeks of cold storage (Table 2). when reared at 16L:8D (Fig. 1B). Longevity of experimental females Mortality during cold storage of parasitoids reared at 17.5 °C was shorter than that of culture females when reared at 16L:8D was similar to that of parasitoids reared at 20 °C(Tables 2 and (À28.1 ± 2.7 d) and at 10L:14D (À20.8 ± 2.6 d). 3). Both photoperiod during rearing at 17.5 °C and storage duration affected mortality of both females and males, and the interaction was significant for both sexes (Table 1). Because interaction was 3.3. Egg laying and progeny production of diapause and nondiapause significant, we used one-way ANOVA’s to examine the effects of females after cold storage storage duration on mortality of each sex within each level of rearing photoperiod, and to examine the effects of rearing photoperiod Egg laying after removal from cold storage of females reared at within each level of storage duration. Female mortality increased 20 °C varied with the photoperiod at which they were reared and with storage duration at both photoperiods (Table 3). Female mor- the duration of cold storage, but not with whether they were tality throughout most of the storage period was greater when par- paired with a male after cold storage (Table 5). Duration Ã photope- asitoids were reared at 16L:8D than at 10L:14D. Male mortality riod interaction was significant (Table 5), so we reanalyzed the increased with storage duration at both photoperiods (Table 3) data using a two-way ANOVA without including presence or ab- and was greater when parasitoids were reared at 16L:8D than at sence of males as a factor. Egg laying of females after removal from 10L:14D after 8 and 12 weeks of storage. No males survived cold storage still varied with the photoperiod at which they were 16 weeks of cold storage. The only parasitoids that survived reared (F = 110.5; df = 1, 171; P < 0.001) and with duration of cold 16 weeks of cold storage were females that were reared at storage (F = 22.1; df = 3, 171; P < 0.001), and interaction was signif- 10L:14D, and, of these, only females reared at 20 °C survived icant (F = 3.9; df = 2, 171; P = 0.022). So, we reanalyzed the data 20 weeks of cold storage (Tables 2 and 3). using one-way ANOVA’s. Females laid more eggs after removal from cold storage when they had been reared at 10L:14D than at 3.2. Longevity of diapause and nondiapause females after cold storage 16L:8D (Fig. 2A; 4 weeks: F = 28.4; df = 1, 58; P < 0.001. 8 weeks: F = 123.8; df = 1, 58; P < 0.001. 12 weeks: F = 16.1; df = 1, 41; Longevity after removal from cold storage of females reared at P < 0.001). When females were reared at 10L:14D, egg laying after 20 °C varied with the photoperiod at which they were reared and 4 and 8 weeks of cold storage was greater than that of females that the duration of cold storage, but not with whether they were had been stored for 12 and 16 weeks (F = 12.5; df = 3, 100; paired with a male after cold storage and no interactions were sig- P < 0.001). When females were reared at 16L:8D, egg laying after nificant (Table 4). Because the presence of males did not affect lon- 4 weeks of cold storage was greater than that of females that had gevity, we reanalyzed the data using a two-way ANOVA without been stored for 8 and 12 weeks (F = 29.1; df = 2, 71; P < 0.001). including presence or absence of males as a factor. Longevity of fe- The numbers of eggs laid by females reared at 16L:8D were re- males after removal from cold storage still varied with the photo- duced by 135.0 ± 25.8 (mean ± SE), 281.2 ± 25.8, and 299.3 ± 30.4 period at which they were reared (F = 82.6; df = 1, 171; P < 0.001) after 4, 8, and 12 weeks of storage, respectively, and the numbers and with duration of cold storage (F = 8.2; df = 3, 171; P < 0.001), of eggs laid by females reared at 10L:14D were reduced by and interaction was not significant (F = 1.4; df = 2, 171; P = 0.262) 150.7 ± 38.8 and 132.9 ± 44.5 after 12 and 16 weeks of storage, (Fig. 1A). Females lived longer after removal from cold storage respectively, as compared with the number of eggs laid by culture when they were reared at 10L:14D, and they lived longer after 4 females.

Table 1 Analysis of variance results for effects of photoperiod at which parasitoids were reared at 17.5 and 20 °C and duration of cold storage on mortality of Habrobracon hebetor during cold storage.

Variable 20 °C 17.5 °C Female Male Female Male F df PFdf PFdf PFdf P Photoperiod 86.5 1, 35 <0.001 0.2 1, 20 0.652 184.7 1, 19 <0.001 19.0 1, 19 <0.001 Duration 281.0 4, 35 <0.001 147.4 4, 20 <0.001 510.8 4, 19 <0.001 115.6 4, 19 <0.001 Photoperiod Ã Duration 15.7 4, 35 <0.001 0.3 4, 20 0.905 34.4 4, 19 <0.001 3.5 4, 19 0.027 H. Chen et al. / Biological Control 64 (2013) 186–194 189

Table 2 Mortality (mean ± SE) of Habrobracon hebetor reared at two photoperiods at 20 °C and then stored at 5 °C for different durations.

Storage duration (weeks) % Female mortality % Male mortality 10L:14D 16L:8D F df P 10L:14D 16L:8D 4 2.5 ± 2.5Ac 5.4 ± 3.9Ad 0.35 1, 6 0.571 24.4 ± 7.3c 22.2 ± 4.0b 8 19.1 ± 5.5Bc 48.0 ± 2.5Ac 26.7 1, 6 0.001 81.1 ± 2.9b 84.4 ± 8.0a 12 39.6 ± 7.5Bb 86.3 ± 1.6Ab 47.1 1, 6 <0.001 95.6 ± 2.2ab 100.0 ± 0a 16 82.0 ± 3.4Ba 100.0 ± 0Aa 19.6 1, 6 0.007 100.0 ± 0a 100.0 ± 0a 20 97.9 ± 1.3Aa 100.0 ± 0Aa 3.6 1, 6 0.100 100.0 ± 0a 100.0 ± 0a F 78.6 346.9 77.4 62.0 df 4, 15 4, 20 4, 15 4, 15 P <0.001 <0.001 <0.001 <0.001

For females, means within columns followed by the same lowercase letter and within rows followed by same uppercase letter are not significantly different (P > 0.05, Tukey’s-b). For males, where effects of photoperiod and interaction between storage duration and photoperiod were not significant, means within columns followed by the same lowercase letter are not significantly different (P > 0.05, Tukey’s-b).

Table 3 Mortality (mean ± SE) of Habrobracon hebetor reared at two photoperiods at 17.5 °C and then stored at 5 °C for different durations.

Storage duration (weeks) % Female mortality % Male mortality 10L:14D 16L:8D F df P 10L:14D 16L:8D F df P 4 2.2 ± 2.2Ae 10.0 ± 1.9Ac 7.0 1, 4 0.057 24.4 ± 4.0Ac 42.2 ± 7.8Ab 4.1 1, 4 0.112 8 18.9 ± 2.2Bd 53.3 ± 5.8Ab 31.0 1, 4 0.005 70.0 ± 5.1Bb 91.1 ± 2.2Aa 14.4 1, 4 0.019 12 46.4 ± 2.0Bc 96.7 ± 1.9Aa 337.2 1, 4 <0.001 85.6 ± 4.0Ba 97.8 ± 1.1Aa 8.6 1, 4 0.042 16 86.0 ± 1.1Bb 100.0 ± 0Aa 169.0 1, 4 <0.001 100.0 ± 0Aa 100.0 ± 0Aa 1.0 1, 4 0.0 20 100.0 ± 0Aa 100.0 ± 0Aa 1.0 1, 4 0.0 100.0 ± 0 Aa 100.0 ± 0 Aa 1.0 1, 4 0.0 F 594.7 195.4 84.8 46.4 df 4, 10 4, 10 4, 10 4, 10 P <0.001 <0.001 <0.001 <0.001

Means within columns followed by the same lowercase letter and within rows within a sex followed by same uppercase letter are not signiﬁcantly different (P > 0.05, Tukey’s-b).

Table 4 Analysis of variance results for effects of photoperiod at which parasitoids were reared at 17.5 and 20 °C, duration of cold storage, and whether females were paired with a male after cold storage on longevity of Habrobracon hebetor females.

Variable 20 °C 17.5 °C F df PFdf P Photoperiod 82.8 1, 166 <0.001 27.5 1, 154 <0.001 Duration 8.2 3, 166 <0.001 2.0 3, 154 0.116 Male 0.1 1, 166 0.802 0.1 1, 154 0.726 Photoperiod Ã Male 0.3 1, 166 0.602 0.1 1, 154 0.737 Photoperiod Ã Duration 1.0 2, 166 0.385 0.5 1, 154 0.461 Duration Ã Male 1.0 2, 166 0.375 1.0 2, 154 0.384 Photoperiod Ã Male Ã Duration 0.6 1, 166 0.459 0.2 1, 154 0.687

Egg laying after removal from cold storage of females reared at Number of progeny produced after removal from cold storage of 17.5 °C varied with the photoperiod at which they were reared and females reared at 20 °C varied with the photoperiod at which they the duration of cold storage, but not with whether they were were reared and the duration of cold storage, but not with whether paired with a male after cold storage and no interactions were sig- they were paired with a male after cold storage (Table 6). Dura- nificant (Table 5), so we reanalyzed the data using a two-way AN- tion Ã photoperiod interaction was significant (Table 6), so we OVA without including presence or absence of males as a factor. reanalyzed the data using a two-way ANOVA without including Egg laying after removal from cold storage of females still varied presence or absence of males as a factor. Number of progeny pro- with the photoperiod at which they were reared (F = 55.4; duced after removal from cold storage still varied with the photo- df = 1, 159; P < 0.001) and with duration of cold storage (F = 10.4; period at which females were reared (F = 121.5; df = 1, 171; df = 3, 159; P < 0.001), and interaction was not significant (F = 0.1; P < 0.001) and with duration of cold storage (F = 15.9; df = 3, 171; df = 1, 159; P = 0.729). Fewer eggs were laid after removal from P < 0.001), and interaction was significant (F = 3.9; df = 2, 171; cold storage when females had been reared at 16L:8D than when P = 0.021). So, we reanalyzed the data using one-way ANOVA’s. Fe- females had been reared at 10L:14D (Fig. 2B). More eggs were laid males produced more progeny after removal from cold storage after 4 weeks of cold storage than after 16 weeks of cold storage. when they had been reared at 10L:14D than at 16L:8D (Fig. 3A; Egg laying of females was lower than that of culture females for 4 weeks: F = 37.0; df = 1, 58; P < 0.001. 8 weeks: F = 131.3; all treatments [mean (±SE) difference when females were reared df = 1, 58; P < 0.001. 12 weeks: F = 16.2; df = 1, 43; P < 0.001). at 16L:8D was À267.5 ± 13.4 after 4 weeks of storage and When females were reared at 10L:14D, number of progeny pro- À304.3 ± 13.4 after 8 weeks of storage, and when females were duced after 4 and 8 weeks of cold storage was greater than that reared at 10L:14D was À176.5 ± 26.0 after 4 weeks of storage, of females that had been stored for 12 and 16 weeks (F = 10.7; À204.3 ± 26.0 after 8 weeks of storage, À246.2 ± 26.0 after df = 3, 101; P < 0.001). When females were reared at 16L:8D, num- 12 weeks of storage, and À277.9 ± 30.1 after 16 weeks of storage]. ber of progeny produced after 4 weeks of cold storage was greater 190 H. Chen et al. / Biological Control 64 (2013) 186–194

45 A females had been reared at 16L:8D than when females had been Culture reared at 10L:14D (Fig. 3B). Although the ANOVA indicated a differ- 40 20oC,10L:14D ence in number of progeny produced among storage durations, the o 35 20 C,16L:8D Tukey b test showed no differences among durations. Number of progeny produced by females after cold storage was lower than 30 that of culture females for all treatments [mean (±SE) difference 25 when females were reared at 16L:8D was À199.4 ± 7.8 after 20 4 weeks of storage and À214.4 ± 7.8 after 8 weeks, and when females were reared at 10L:14D was À130.8 ± 17.8 after 4 weeks of 15 storage, À148.7 ± 17.8 after 8 weeks, À166.3 ± 17.8 after 12 weeks, 10 and À192.4 ± 20.6 after 16 weeks].

5 B 3.4. Time to 50% egg laying of diapause and nondiapause females after 45 cold storage

Longevity (days) 40 Culture Time to 50% egg laying after removal from cold storage of fe- o 35 17.5 C,10L:14D males reared at 20 °C varied with the photoperiod at which they 17.5oC,16L:8D 30 were reared and the duration of cold storage, but not with whether they were paired with a male after cold storage. Duration Ã photo- 25 period interaction was significant (Table 7), so we reanalyzed the 20 data using a two-way ANOVA without including presence or absence of males as a factor. Time to 50% egg laying after removal 15 from cold storage still varied with the photoperiod at which fe- 10 males were reared (F = 90.6; df = 1, 171; P < 0.001) and with duration of cold storage (F = 6.1; df = 3, 171; P = 0.001), and interaction 5 was significant (F = 5.8; df = 2, 171; P = 0.004). So, we reanalyzed 2 4 6 8 1012141618 the data using one-way ANOVA’s. Females laid 50% of their eggs Storage duration (weeks) more quickly after removal from cold storage when they had been reared at 16L:8D than at 10L:14D (Fig. 4A; 4 weeks: F = 40.9; Fig. 1. Longevity (mean ± SE) of females reared at (A) 20 °C and (B) 17.5 °C and two different photoperiods and then stored at 5 °C for up to 16 weeks. The black circle df = 1, 58; P < 0.001. 8 weeks: F = 103.4; df = 1, 58; P < 0.001. indicates the mean for culture insects. 12 weeks: F = 5.9; df = 1, 41; P = 0.019). When females were reared at 10L:14D, time to 50% egg laying differed with storage duration (Fig. 4A; F = 8.7; df = 3, 100; P < 0.001). Generally, females that than that of females that had been stored for 8 and 12 weeks had been stored longer laid their eggs more quickly. When females (F = 23.6; df = 2, 72; P < 0.001). Number of progeny produced by fe- were reared at 16L:8D, the Tukey’s b test showed no differences males reared at 16L:8D was fewer than those of culture females among storage durations in time to 50% egg laying despite a signif- after 4 [mean (±SE) difference = À123.2 ± 16.8], 8 (À209.5 ± 16.8), icant ANOVA (F = 3.9; df = 2, 71; P = 0.024). Time to 50% egg laying and 12 weeks of storage (À215.3 ± 19.4), and when females were by females reared at 16L:8D was less than that of culture females reared at 10L:14D, number of progeny produced was fewer than after 4 [mean (±SE) difference = À6.8 ± 1.1 d], 8 (À9.3 ± 1.1 d), and that of culture females after 12 (À111.7 ± 27.5) and 16 weeks of 12 weeks of storage (À9.0 ± 1.3 d), and time to 50% egg laying by storage (À87.7 ± 31.5). females reared at 10L:14D was less than that of culture females Number of progeny produced after removal from cold storage of after 12 weeks of storage (À5.1 ± 1.5 d). females reared at 17.5 °C varied with the photoperiod at which Time to 50% egg laying after removal from cold storage of fe- they were reared and the duration of cold storage, but not with males reared at 17.5 °C varied with the photoperiod at which they whether they were paired with a male after cold storage and no were reared and the duration of cold storage, but not with whether interactions were significant (Table 6), so we reanalyzed the data they were paired with a male after cold storage and no interactions using a two-way ANOVA without including presence or absence were significant (Table 7), so we reanalyzed the data using a two- of males as a factor. Number of progeny produced after removal way ANOVA without including presence or absence of males as a from cold storage of females still varied with the photoperiod at factor. Time to 50% egg laying after removal from cold storage of fe- which they were reared (F = 61.0; df = 1, 159; P < 0.001) and with males still varied with the photoperiod at which they were reared duration of cold storage (F = 7.1; df = 3, 159; P < 0.001), and inter- (F = 39.2; df = 1, 159; P < 0.001) and with duration of cold storage action was not significant (F = 0.03; df = 1, 159; P = 0.866). Fewer (F = 2.9; df = 3, 159; P = 0.038), and interaction was not significant progeny were produced after removal from cold storage when (F = 0.4; df = 1, 159; P = 0.552). Females laid 50% of their eggs more

Table 5 Analysis of variance results for effects of photoperiod at which parasitoids were reared at 17.5 and 20 °C, duration of cold storage, and whether females were paired with a male after cold storage on egg laying of Habrobracon hebetor females.

Variable 20 °C 17.5 °C F df PFdf P Photoperiod 108.2 1, 166 <0.001 55.4 1, 154 <0.001 Duration 22.0 3, 166 <0.001 10.1 3, 154 <0.001 Male 0.01 1, 166 0.924 0.5 1, 154 0.471 Photoperiod Ã Male 0.4 1, 166 0.539 0.6 1, 154 0.455 Photoperiod Ã Duration 3.4 2, 166 0.037 0.1 1, 154 0.729 Duration Ã Male 1.0 2, 166 0.381 1.8 2, 154 0.173 Photoperiod Ã Male Ã Duration 0.4 1, 166 0.534 1.3 1, 154 0.255 H. Chen et al. / Biological Control 64 (2013) 186–194 191

400 300 A a* A Culture Culture o a* 20oC,10L:14D 250 a* 20 C,10L:14D o 300 20oC,16L:8D a* 20 C,16L:8D 200 b a * b 200 b 150 * b a 100 100 50 b b b b 0 0 400 B 300 B Culture 17.5oC,10L:14D No. progeny produced 250 Culture o o Egg laying (eggs/female) 17.5 C,16L:8D 17.5 C,10L:14D 300 17.5oC,16L:8D 200

200 150

100 100 50

0 0 2 4 6 8 10 12 14 16 18 2 4 6 8 10 12 14 16 18 Storage duration (weeks) Storage duration (weeks)

Fig. 2. Egg laying (mean ± SE) of females reared at (A) 20 °C and (B) 17.5 °C and two Fig. 3. Number of progeny produced (mean ± SE) by females reared at (A) 20 °C and different photoperiods and then stored at 5 °C for up to 16 weeks. The black circle (B) 17.5 °C and two different photoperiods and then stored at 5 °C for up to indicates the mean for culture insects. Different letters above points indicate 16 weeks. The black circle indicates the mean for culture insects. Different letters differences among the storage durations within a photoperiod (Tukey’s-b at above points indicate differences among the storage durations within a photoperiod P > 0.05), and asterisks indicate differences between photoperiods at the same (Tukey’s-b at P > 0.05), and asterisks indicate differences between photoperiods at storage duration (Tukey’s-b at P > 0.05). the same storage duration (Tukey’s-b at P > 0.05). quickly after removal from cold storage when they had been reared at 16L:8D than at 10L:14D (Fig. 4B). Although the ANOVA indicated 16L:8D from the analysis of whether percentage of female progeny a difference in time to 50% egg laying by females among storage differed among treatments. Analysis of the data for females reared durations after removal from cold storage, the Tukey’s b test at 10L:14D showed no differences among treatments (Fig. 5A; showed no differences among durations. Time to 50% egg laying duration F = 1.2; df = 3, 78; P = 0.331. presence of male F = 0.3; after removal from cold storage by females was shorter than that df = 1, 78; P = 0.571. interaction F = 0.2; df = 2, 78; P = 0.827). The of culture females for all treatments [mean (±SE) difference when percentage of females in the treatments (mean ± SE = 0.36 ± 0.014, females were reared at 16L:8D was À11.0 ± 0.8 d after 4 weeks of n = 104) was lower than that in the cultures (0.52 ± 0.035, n = 15) storage and À11.3 ± 0.8 d after 8 weeks of storage, and when fe- (mean difference ± SE = 0.16 ± 0.040, t = 4.1, df = 117, P < 0.001). males were reared at 10L:14D was À6.5 ± 1.5 d after 4 weeks of Females reared at 16L:8D at 17.5 °C did not produce at least 50 storage, À6.0 ± 1.5 d after 8 weeks of storage, À8.3 ± 1.5 d after progeny after any period of cold storage, so we excluded the data 12 weeks of storage, and À9.3 ± 1.8 d after 16 weeks of storage. for 16L:8D from the analysis of whether percentage of female progeny differed among treatments. Analysis of the data for females reared at 10L:14D showed no differences among treatments 3.5. Female percentage of the F generation for diapause and 1 (Fig. 5B; duration F = 1.1; df = 3, 47; P = 0.339. presence of male nondiapause H. hebetor after cold storage F = 0.8; df = 1, 47; P = 0.378. interaction F = 0.6; df = 2, 47; P = 0.552). The percentage of females in the treatments Females reared at 16L:8D at 20 °C only produced at least 50 (mean ± SE = 0.28 ± 0.025, n = 57) was lower than that in the cul- progeny when stored for 4 weeks, so we excluded the data for

Table 6 Analysis of variance results for effects of photoperiod at which parasitoids were reared at 17.5 and 20 °C, duration of cold storage, and whether females were paired with a male after cold storage on the progeny production of Habrobracon hebetor females.

Variable 20 °C 17.5 °C F df PFdf P Photoperiod 116.1 1, 166 <0.001 60.8 1, 154 <0.001 Duration 15.8 3, 166 <0.001 7.1 3, 154 <0.001 Male 0.02 1, 166 0.881 1.0 1, 154 0.329 Photoperiod Ã Male 0.7 1, 166 0.415 0.2 1, 154 0.639 Photoperiod Ã Duration 3.6 2, 166 0.030 0.03 1, 154 0.866 Duration Ã Male 0.7 2, 166 0.487 1.6 2, 154 0.201 Photoperiod Ã Male Ã Duration 0.2 1, 166 0.625 0.8 1, 154 0.378 192 H. Chen et al. / Biological Control 64 (2013) 186–194

Table 7 Analysis of variance results for effects of photoperiod at which parasitoids were reared at 17.5 and 20 °C, duration of cold storage, and whether females were paired with a male after cold storage on the time to 50% egg laying of Habrobracon hebetor females.

Variable 20 °C 17.5 °C F df PFdf P Photoperiod 96.3 1, 166 <0.001 38.7 1, 154 <0.001 Duration 6.9 3, 166 <0.001 2.9 3, 154 0.036 Male 0.3 1, 166 0.602 1.0 1, 154 0.331 Photoperiod Ã Male 0.6 1, 166 0.441 0.4 1, 154 0.554 Photoperiod Ã Duration 4.2 2, 166 0.017 0.4 1, 154 0.554 Duration Ã Male 0.9 2, 166 0.399 0.9 2, 154 0.407 Photoperiod Ã Male Ã Duration 0.8 1, 166 0.361 0.8 1, 154 0.375

25 they were reared, and interaction was not signiﬁcant (Table 8). The A Culture percentage of females produced by culture females that were * 20oC,10L:14D paired with stored males (Fig. 6B; mean ± SE = 0.28 ± 0.025) was 20 * a o ab 20 C,16L:8D less than that produced by pairs of culture parasitoids bc (0.52 ± 0.035) (mean difference ± SE = 0.24 ± 0.053, t = 4.6, df = 71, 15 c* P < 0.001). a 10 a a 4. Discussion

5 Our results show that H. hebetor reared at 20 °C and 10L:14D and stored for up to 8 weeks did not differ in most measurements 0 of quality from culture parasitoids. Mortality during cold storage and longevity, egg laying, number of progeny produced, and time 25 B to 50% egg laying after removal from cold storage were similar to Culture that of culture parasitoids. The percentage of female progeny pro- 20 o 17.5 C,10L:14D duced was lower than that of culture parasitoids (36% vs. 52%). The o Time to 50% egg laying (days) 17.5 C,16L:8D presence of a male did not impact any of the quality parameters 15 measured. Rearing the parasitoids at 16L:8D or at 17.5 °C generally resulted in a reduction in quality, as did cold storage for more than 8 weeks. Males had low survivorship when stored for more than 10 4 weeks, but, given that their presence did not impact quality parameters of females, storage of males is not important. 5 The longevity of females after cold storage declined with the duration of the cold storage period. A possible explanation for 0 the decline in longevity, and for some of the other reductions ob- 2 4 6 8 10 12 14 16 18 served in quality parameters, after cold storage might be that Storage duration (weeks) increasingly larger amounts of resources were depleted by mainte- nance during longer periods of cold storage at 5 °C, resulting in re- Fig. 4. Time to 50% egg laying (mean ± SE) by females reared at (A) 20 °C and (B) duced performance and survival (Salt, 1961). Also, accumulation of 17.5 °C and two different photoperiods and then stored at 5 °C for up to 16 weeks. toxic metabolites during long cold storage could cause death or re- The black circle indicates the mean for culture insects. Different letters above points indicate differences among the storage durations within a photoperiod (Tukey’s-b duce fitness (Storey and Storey, 1988). Diapausing insects produce at P > 0.05), and asterisks indicate differences between photoperiods at the same fewer and/or less toxic metabolites during storage because metab- storage duration (Tukey’s-b at P > 0.05). olism is lowered (Koštál, 2006). The number of eggs laid during the first time check was always lower for stored insects than for culture insects. One reason for this tures (0.52 ± 0.035, n = 15) (mean difference ± SE = 0.25 ± 0.053, may be that the parasitoids are recovering from chilling injury t = 4.7, df = 72, P < 0.001). after exposure to low temperatures (Danks, 1987; Yocum et al., When culture females were paired with stored males that had 1994; Leopold et al., 1998). Another possible reason is that eggs been reared at 20 °C, percentage of female progeny varied with in the ovary might have been killed during storage, and the eggs the duration of cold storage, but not with the photoperiod at which laid at the first time check might have developed from oocytes pro- they were reared and interaction was not significant (Table 8). A duced after the female wasps were placed at culture conditions. one-way ANOVA that included only duration showed that percent- Egg laying in our study was similar to that obtained in the study age of female progeny was greater when males had been stored for of Jackson and Butler (1984) which reported 326 eggs laid per fe- 4 weeks than when they had been stored for 8 weeks (F = 8.2; male on Pectinophora gossypiella Saunders (Lepidoptera: Gelechii- df = 1, 55; P = 0.006). The percentage of females produced by cul- dae). In our study, culture females laid an average of 311 eggs ture females that were paired with stored males was less than that per female, which was higher than obtained in some other studies produced by pairs of culture parasitoids [Fig. 6A; mean (±SE) differ- – 253 eggs per female on Anagasta kuehniella Zeller (Lepidoptera: ence was À0.20 ± 0.069 after 4 weeks of storage and À0.38 ± 0.070 Pyralidae) (Clark and Smith, 1967), 78.3 eggs per female on Galleria after 8 weeks of storage]. mellonella L. (Lepidoptera: Pyralidae) (Masood and Chi, 2006), and When culture females were paired with stored males that had 66.3 eggs per female on Ephestia kuehniella Zeller (Lepidoptera: been reared at 17.5 °C, percentage of female progeny did not vary Pyralidae) (Masood and Chi, 2006). The differences in fecundity with the duration of cold storage or with the photoperiod at which might be due to the different species of hosts used, to the different H. Chen et al. / Biological Control 64 (2013) 186–194 193

Culture A 20oC, 10L:14D-Female without male B Culture o 60 20oC, 10L:14D-Female with male 60 17.5 C, 10L:14D- Female without male o 20oC, 16L:8D-Female without male 17.5 C, 10L:14D- Female with male 20oC, 16L:8D-Female with male 50 50

40 40 generation 1 30 30

20 20

10 10 % Females in F

0 0 Culture 4 8 12 16 Culture 4 8 12 Storage duration (weeks) Storage duration (weeks)

Fig. 5. Female percentage of F1 offspring (mean ± SE) produced by females reared at (A) 20 °C and (B) 17.5 °C and two different photoperiods and then stored at 5 °C for up to 16 weeks. The single black bar indicates the mean for culture insects.

Table 8 matheca could retain their vitality for many years. The percentage Analysis of variance results for effects of photoperiod at which parasitoids were of female progeny produced by pairs when the male had been in reared at 17.5 and 20 °C and duration of cold storage on percentage of female progeny cold storage and then was mated with an unmated culture female when culture females were paired with stored males. was lower than that of the culture, indicating that the reproductive Variable 20 °C 17.5 °C ability of the male was reduced after cold storage. F df PFdf P Storage proteins are synthesized shortly before the onset of dia- Photoperiod 1.6 1, 50 0.214 3.0 1, 45 0.092 pause, and they are released into the hemolymph and then remain Duration 7.6 1, 50 0.008 3.5 1, 45 0.067 there throughout diapause (Denlinger, 2002). These proteins disap- Photoperiod Ã Duration 0.7 1, 50 0.411 0.04 1, 45 0.837 pear quickly after diapause is terminated. Storage proteins have been found in diapausing adult Colorado potato beetle, Leptinotarsa decemlineata (Koopmanschap et al., 1995) and the red ﬁre bug Pyr- densities of parasitoids and larval hosts used (Taylor, 1988), or to rochoris apterus (Sula et al., 1995). The fat body is a site of synthesis differences in the parasitoid populations used in the studies. of storage proteins, and the fat bodies were full in H. hebetor fe- In our study, the percentage of female progeny produced by the males reared in diapause conditions. Most diapause-associated female after storage was 20–40%, and was always lower than for proteins are synthesized before the insect enters diapause, and the culture (52%). Ode et al. (1997) and Heimpel et al. (1999) doc- these proteins include not only the proteins utilized during dia- umented female-biased sex allocation in H. hebetor. Yu et al. (2003) pause but also proteins or amino acids used in diapause termina- found the progeny sex ratio remained at approximately 0.5 regard- tion (Denlinger, 2002). Genes encoding diapause-associated less of host density, and this sex ratio was similar to our culture. In proteins would change expression patterns before, during, and a study by Rotary and Gerling (1973) using A. kuehniella as host, the after diapause (Clark and Worland, 2008). Heat-shock proteins percentage of females was found to be 0.39. The difference in sex act as chaperones to stabilize and refold denatured proteins when ratio among different studies might be due to the use of different organisms undergo heat or desiccation stress, and it has been hosts, temperature conditions, densities of H. hebetor, and/or fe- hypothesized that up-regulation of heat-shock proteins is essential males of different ages. In our study, the presence of a male after for cold survival during insect diapause (Rinehart et al., 2007). cold storage did not affect the sex ratio of progeny. This indicates Our results show that diapausing female H. hebetor can be that the sperm could survive in the female for as long as 16 weeks stored at 5 °C for up to 8 weeks without adversely affecting the of cold storage. Lillie (1919) showed that sperm stored in the sper- performance of the parasitoid. The ability to store H. hebetor for

A B 60 Culture 60 Culture 20oC, 10L:14D 17.5oC, 10L:14D o o 50 20 C, 16L:8D 50 17.5 C, 16L:8D

40 40 generation 1 30 30

20 20

10 10 % Females in F

0 0 Culture 4 8 Culture 4 8 Storage duration (weeks) Storage duration (weeks)

Fig. 6. Female percentage of F1 offspring (mean ± SE) produced by culture females mated with males reared at (A) 20 °C and (B) 17.5 °C and two different photoperiods and then stored at 5 °C for up to 16 weeks. The single black bar indicates the mean for culture insects. 194 H. Chen et al. / Biological Control 64 (2013) 186–194 up to two months would contribute to the improved use of H. heb- Heimpel, G.E., Antolin, M.F., Strand, M.R., 1999. Diversity of sex-determining alleles etor as a biological control agent in pest management programs. in Bracon hebetor. Heredity 82, 282–291. Huang, X.F., 1986. Use of Habrobracon hebetor Say in granary pest control. Chinese However, field performance of stored H. hebetor is not known. Re- Journal of Biological Control 2, 78–80. search needs to be conducted on the mechanism of cold resistance Imam, M., Uwais, A., Namat, U., Akbar, A., Ahmat, T., 2007. Influence of Habrobracon in H. hebetor to try to reduce mortality during long-term cold stor- hebetor on Helicoverpa armigera in southern Xinjiang. Natural Enemies of Insects 29, 12–15. age and to try to improve performance after long-term cold Jackson, C.G., Butler, G.D., 1984. Development time of three species of Bracon storage. (Hymenoptera: Braconidae) on the pink bollworm (Lepidoptera: Gelechiidae) in relation to temperature. Annals of the Entomological Society of America 77, 539–542. Acknowledgments Keever, D.W., Mullen, M.A., Press, J.W., Arbogast, R.T., 1986. Augmentation of natural enemies for suppressing two major insect pests in stored farmers stock The authors thank Dr. Judy Johnson (USDA-ARS, Parlier, CA) for peanuts. Environmental Entomology 15, 767–777. Koopmanschap, A.B., Lammers, J.H.M., de Kort, C.A.D., 1995. The structure of the providing the Habrobracon hebetor and for reviewing an earlier ver- gene encoding diapause protein 1 of the Colorado potato beetle (Leptinotarsa sion of this manuscript, and Joel Perez-Mendoza and Ann Redmon decemlineata). Journal of Insect Physiology 41, 509–518. for their technical assistance. This research was supported by the Koštál, V., 2006. Eco-physiological phases of insect diapause. Journal of Insect China National Science and Technology Project of the 11th Five- Physiology 52, 113–127. Leopold, R.A., Rojas, R.R., Atkinson, P.W., 1998. Post pupariation cold storage of Year Plan (2006BAI09B04-06 and 2006BAD02A18-03), the Ear- three species of flies: increasing chilling tolerance by acclimation and recurrent marked Fund for Modern Agro-industry Technology Research Sys- recovery periods. Cryobiology 36, 213–224. tem (CARS-27) to H. Zhang, and a scholarship from the China Levie, A., Vernom, P., Hance, T., 2005. 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