Influence of Diet on Long-Term Cold Storage of the Predator Podisus

Biological Control 42 (2007) 186–195 www.elsevier.com/locate/ybcon

Inﬂuence of diet on long-term cold storage of the predator Podisus maculiventris (Say) (Heteroptera: Pentatomidae) q

T.A. Coudron a,*, M.R. Ellersieck b, K.S. Shelby a

a Biological Control of Insects Research Laboratory, USDA-Agriculture Research Service, 1503 S. Providence Road, Columbia, MO 65203-3535, USA b Agricultural Experiment Station Statisticians, University of Missouri, Columbia, MO 65211, USA

Received 26 December 2006; accepted 23 April 2007 Available online 29 April 2007

Abstract

Long-term storage could aid in the cost-effective mass production of beneficial insects. Pre-conditioning, insect developmental stage and environmental conditions should be considered when selecting storage conditions in order to obtain the highest performance after storage. We evaluated the influence of nutrient quality on the response of eggs, nymphs and adults of Podisus maculiventris when exposed to two temperatures most likely to be used for long-term storage. Natural prey-fed insects were compared to artificial diet-fed insects. The results of our study showed that eggs survived storage at 10 °C better than 4 °C, and that eggs from diet-fed insects survived storage at 10 °C significantly better than eggs from prey-fed insects. Nymphal survival of cold storage treatment was slightly higher than for eggs, with a similar pattern of response to temperature, nutrient quality and time of storage. However, of the three developmental stages tested, the best survival was obtained with adults. Similar to eggs and nymphs, adults survival was higher at 10 °C. However, different from eggs and nymphs was that the prey-fed adults survived better than diet-fed adults. Our results show that prey-fed adults were able to withstand cold storage at 10 °C for 4 weeks with very little loss of survival, fecundity or viability of eggs oviposited by those adults. Longer periods of storage resulted in a decline in survival, fecundity and egg viability and that decline was greatest at 4 °C. For the longer intervals of storage at 10 °C, egg viability was highest for eggs from diet-fed adults. Collectively, these results demonstrate an influence of nutrient quality on the response to cold storage at different developmental stages, and that an optimal food source for continuous rearing is not necessarily an optimal food source for cold storage. Published by Elsevier Inc.

Keywords: Podisus maculiventris; Cold storage; Diet; Nutrient quality; Survival; Fecundity

1. Introduction respond to undulating and unpredictable demands. Cold storage may be a useful technique to enable insectaries One of the primary obstacles to the use of insect natural and researchers to store insects for extended periods rather enemies for biological control is the difficulty and expense than to continuously rear a colony during those times when of rearing these insects in sufficient numbers and quality for the colony is not in use. Additionally, cold-storage could fluctuating markets. More efficient storage methods would assist in accumulating sufficient numbers of insects for substantially improve the options available to insectaries to innundative releases, or minimizing costs of retaining a colony between inoculative releases, off-season demands and q Mention of trade names or commercial products in this publication is between research studies. Tolerance to cold may also be solely for the purpose of providing specific information and does not considered a desirable attribute for shipment and for fitness imply recommendation or endorsement by the US Department of when used in early or late season releases. Agriculture. All programs and services of the US Department of The response of insects to cold temperatures has Agriculture are offered on a nondiscriminatory basis without regard to received considerable interest, with the majority of studies race, color, national origin, religion, sex, age, marital status, or handicap. * Corresponding author. Fax: +1 573 875 4261. focusing on diapause. However, the role of nutrient quality E-mail address: [email protected] (T.A. Coudron). in diapause is not well understood (Ruberson et al., 1998).

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2007.04.014 T.A. Coudron et al. / Biological Control 42 (2007) 186–195 187

The intentional cooling of insects for the purpose of stor- chamber. The prey-fed colony used in this study had been age has focused mostly on beneficial insects with some of maintained for ca. 80 generations on excess quantities of the earlier studies investigating parasitoids (Flanders, coddled fourth instar larvae of Trichoplusia ni (Hu¨bner) 1938; DeBach, 1943). More recently two studies have doc- (Coudron et al., 2000) and the diet-fed colony had been umented that host quality influences the response of bene- maintained for ca. 10 generations on excess quantities of ficial insects subjected to cold temperatures. In one study a zoophytogenous artificial diet (Coudron et al., 2002). involving the predatory pentatomid, Podisus maculiventris These two food sources were used for this study to provide (Say) (Heteroptera: Pentatomidae) it was reported that an optimal nutrition (prey-fed) that has been previously suboptimal food increased the number of individuals that used to maintain a continuous colony of P. maculiventris entered a putative diapause condition (Goryshin et al., for more than 150 generations and suboptimal nutrition 1988). In another study, the presence of cryoprotectants (diet-fed) that has been previously used to maintain a con- in the host were credited with improving the cold hardiness tinuous colony of P. maculiventris for more than 100 gen- in larvae of the fly pupal parasitoid, Nasonia vitripennis erations (Coudron et al., 2002, 2006; Yocum et al., 2006). (Walker) (Hymenoptera: Pteromalidae) (Rivers et al., Eggs, nymphs and adults from the prey-fed and diet-fed 2000). colonies that were used in each cold storage treatment were The spined soldier bug, P. maculiventris, is a polypha- cohorts (Birch, 1948; Wittmeyer et al., 2001) collected as gous predator of many agricultural pests and is native to eggs from control adult females that ranged in age from North America (McPherson, 1980, 1982). P. maculiventris 6 to 20 days old. has often been a preferred predator for research, sale and use because of its high reproductive capacity and its ability 2.2. Temperature treatments to be reared on artificial diets. Parallel with interests in storing insects has been a long-established interest in the Eggs, nymphs and adults were subjected to cold storage use of artificial diets to reduce the production costs and in continuous darkness and 75% RH within incubators set maintain stable colonies of insects. As with most beneficial at either 4 °Cor10°C. These temperatures prevent embry- insects, current methods for mass rearing P. maculiventris onic development in eggs that are <1 day post oviposition involve rearing on a natural host. However, several artifi- (Goryshin and Tuganova, 1989; De Clercq and Degheele, cial diets have been tested for rearing P. maculiventris, cul- 1992). A RH of >75% has been shown to be critical for sur- minating with a zoophytogenous diet that has proven to be vival. Pre-conditioning for the induction of diapause was cost-effect and has been successfully used to maintain a col- not attempted because this had previously been shown ony for more than 200 consecutive generations. The zoo- not to occur for P. maculiventris (Couturier, 1938; Vlasova phytogenous diet has not been fully optimized for et al., 1980; De Clercq and Degheele, 1993). Stored insects continuous rearing and at its original formulation resulted were provided water absorbed on a dental wick but were in an increase in developmental time and a decrease in not provided with prey or diet. Unless otherwise stated, fecundity (Coudron et al., 2002). Other possible effects random samples (Snedecor and Cochran, 1989) of three were indicated by altered gene expression patterns (Yocum replicates of 20 individuals were removed from each treat- et al., 2006; Coudron et al., 2006). ment at designated time points over a 10 week period for In the present study, the potential of nutrient quality on observation and recording. Samples removed for observa- responses of P. maculiventris to storage at two reduced tions were not returned to the study. temperatures were evaluated by measuring survival, fecun- Eggs from prey-fed and diet-fed females were collected dity and egg viability of P. maculiventris reared on larvae of at ca. 1 day post oviposition before being subjected to cold Trichoplusia ni or an artificial diet. In addition to Proc storage treatment. Three groups of 20 eggs were randomly Genmod analyses, the use of odds ratios analyses, i.e., an removed from cold storage treatment at the designated extension of a general linear model analysis, linked to a time points and subsequently held at 26 °C, 75% RH and logit (nonlinear) transformation to provide a good linear observed for hatch over a 7 day period. fit assigning a binomial distribution, enabled the compari- Prey-fed and diet-fed nymphs were subjected to cold son of uneven data sets that resulted from the extensive dif- storage treatment as newly ecdysed third instar nymphs, ferences of each developmental stage in response to the collected within 4–8 h of molting. Survival was recorded treatments. in two groups of 10 nymphs that were randomly removed from cold storage treatment at the designated time points 2. Materials and methods and observed for survival over a 7 day period. Male and female adults 2 days post adult eclosion were 2.1. Insect colonies and diet separated and subjected to cold storage treatment. Survival was recorded in two groups of 10 males and females. Sub- The P. maculiventris colony originated from adults col- sequently, adults removed from cold storage treatment lected in alfalfa in Boone County, Missouri in the summer were held at 26 °C, 16 L:8D photoperiod, and 75% RH of 2000 and subsequently maintained continuously at and survival was recorded 24 h after removal from the cold 26 °C, 16L:8D photoperiod, and 75% RH in a walk-in storage. After 5 days, 20 of the surviving males and females 188 T.A. Coudron et al. / Biological Control 42 (2007) 186–195 from within the same treatment were paired for 18 h and allowed to mate. Mating was repeated every 48 h. Oviposi- tion was recorded for a total of 12 days following the onset of mating. A randomly selected cohort of 100 eggs from each mated pair was collected and observed for hatch.

2.3. Statistical analysis

All statistics were performed utilizing Statistical Analy- sis Software (SAS) vers. 9.1 (SAS Institute, 2004). Proc Genmod was used for the analysis of the survivorship data of eggs, nymphs, adults and egg viability (dependent variables) where the independent variables were temperature (4 °C and 10 °C), food source (prey-fed and diet-fed), life- stage, sex and length of time in cold storage (week 1–10). Odds ratios (i.e., likelihood ratios) for Type III analysis Fig. 1. Percentage hatch of eggs from prey-fed (P) and diet-fed (D) were used to estimate magnitude and precision of associa- P. maculiventris after storage of the eggs at 4 °Cor10°C. Survival of eggs from the same colonies of P. maculiventris reared at 26 °C (Control) is tion among treatments and allowed for more comparisons shown for comparison. between treatments where significant differences were found. The odds ratios were calculated by taking the anti- log of the difference between two treatment logit values. either 4 °Cor10°C. At week 2 the highest percentage The reported P values reflect the b coefficient in the rele- hatch of 31% occurred at 10 °C with eggs from diet-fed vant logistic model. A mixed-effects statistical model (Proc insects which are significantly better than the lowest per- Mixed, SAS Institute, 2004) analysis was performed for the centage hatch of 3% obtained with eggs from diet-fed repeated measures of adult fecundity and egg viability insects held at 4 °C. Only eggs from diet-fed insects held (dependent variables) where the independent variables were at 10 °C survived at 3 weeks and those had a <5% hatch temperature (4 °C and 10 °C), food source (prey-fed and rate. No survival was obtained at either treatment temper- diet-fed) and length of time in cold storage (week 1–10). ature beyond 3 weeks. Differences between means were determined using Fisher’s Least Significant Difference (LSD) test for significance at P 3.2. Effect of cold-storage on nymphal survival < 0.05. The results for survival of newly ecdysed third instar 3. Results nymphs following cold storage at 4 °Cor10°C is shown in Fig. 2. After 1 and 2 weeks the 10 °C treatment had a At all temperature treatments for nymphs and adults, higher survival than the 4 °C treatment for both prey-fed dead individuals were readily differentiable from live indi- and diet-fed and >90% survived after week 1 which was viduals. Although individuals were more lethargic during comparable to controls. Only prey-fed and diet-fed nymphs cold treatment, normal motion and response to touch held at 10 °C survived beyond week 2 of treatment and the resumed within 1–2 h after being placed at 26 °C and was used to discern survival. Egg, larval and adult results were compared over those weeks of cold storage where there was survival with each treatment. Adult results were compared over a 10 week period.

3.1. Eﬀect of cold-storage on egg survival

The hatch results for P. maculiventris eggs following varying periods of storage at 4 °Cor10°C is shown in Fig. 1. After 1 and 2 weeks, eggs held at 4 °Cor10°C from both prey-fed and diet-fed insects survived the cold treatments. No significant difference was obtained after 1 week for eggs from prey-fed and diet-fed insects held at either 4 °Cor10°C; with values ranging from 59% to 61% hatch for these treatments. However, this was significantly lower than the 76% and 91% hatch for diet-fed and prey-fed con- Fig. 2. Survival of prey-fed (P) and diet-fed (D) P. maculiventris nymphs trols, respectively. There was a significant drop in the per- after storage at 4 °Cor10°C. Survival of nymphs from the same colonies centage hatch for all treatments after 1 week of storage at of P. maculiventris reared at 26 °C (Control) is shown for comparison. T.A. Coudron et al. / Biological Control 42 (2007) 186–195 189 percentage survival was higher for the diet-fed insects with p > v2 < 0.0001). There was nearly an equal likelihood of 65% and 20% survival after 3 and 4 weeks respectively. survival for prey-fed and diet-fed individuals at both tem- Nymphs did not survive at either temperature beyond 4 peratures during the 2 week storage. The greatest difference weeks. was found after week 2 where adults were 41.5 times more likely to survive than eggs (v2 = 91.38; p > v2 = 0.0017). 3.3. Effect of cold-storage on adult survival Overall, the likelihood of survival was affected by life stage (v2 = 164.03; p > v2 < 0.0001), temperature The pooled survival results of adult males and females (v2 = 18.46; p > v2 < 0.0001), and time (v2 = 38.32; subjected to cold storage at 4 °C and 10 °C are shown in p > v2 < 0.0001), but not by food source (v2 = 0.49; Fig. 3. After 2 weeks, >80% survived all treatments, which p > v2 = 0.4822). The only interactions that were statisti- was the same as for similar controls (data not shown). cally significant were those between stage and temperature However, beyond 2 weeks diet-fed adults survived longer (v2 = 17.7; p > v2 < 0.0001); stage and time (v2 = 10.98; than prey-fed adults when held at 4 °C with 15% surviving p > v2 = 0.0041); and stage, temperature, and food source at 6 weeks. By comparison, the prey-fed adults did not sur- (v2 = 13.3; p > v2 = 0.0013). vive past 4 weeks at 4 °C. In contrast, after 6–10 weeks at 10 °C the survival of prey-fed adults remained at 100% and 3.5. Comparison of adult survival during 10 weeks of cold surpassed the 80% survival of prey-fed controls at 9 weeks. storage By comparison, the diet-fed adults held at 10 °C did not survive past 10 weeks (data not shown). Adults tolerated cold temperatures better than eggs or nymphs and consequently it was possible to calculate the 3.4. Comparison of egg, nymphal and adult survival after 1 odds ratio statistics for adult survival over the first 4 weeks and 2 weeks of cold-storage of cold storage at both 4 °C and 10 °C; and for the first 10 weeks of storage at 10 °C. The odds ratios for the associa- Statistical analysis comparing the survival of all stages tion of adult survival with a full complement of indepen- after 2 weeks of cold storage could not be performed due dent variables (i.e., temperature, food source, time and to high mortality of eggs and nymphs at both 4 °C and sex) over the first 4weeks and a selected complement of 10 °C. The odds ratios for the association of survival with independent variables (i.e., food source, time and sex) over developmental stage, nutrient and temperature are pre- 10 weeks are presented in Tables 2 and 3, respectively. sented in Table 1, using a Proc Genmod analysis. During During the first 4 weeks adults subjected to 10 °C were a 2 week storage at either 4 °Cor10°C the adults were 17.3 times more likely to survive than adults subjected to 2 2 16.5 times more likely to survive than eggs (v2 = 106.5; 4 °C(v = 50.47; p > v < 0.0001) (Table 2). There was p > v2 < 0.0001) and 6.7 times more likely to survive than no difference in the likelihood of survival after one or 2 2 2 nymphs (v2 = 35.66; p > v2 < 0.0001). By comparison, weeks (v = 0.57; p > v = 0.4511) but adults were 3.0 nymphs were 2.4 times more likely to survive than eggs times more likely to survive after 1 week than 3 weeks (v2 = 12.9; p > v2 = 0.0003). The chance of survival at (v2 = 3.0; p > v2 = 0.0203) and 102.5 times more likely to 2 10 °C was 2.6 times more likely than at 4 °C(v2 = 17.88; survive after 1 week than after 4 weeks (v = 48.65; p > v2 < 0.0001) and the chance of survival after week 1 p > v2 < 0.0001). Diet-fed adults were 6.6 times more likely was 4.1 times more likely than after week 2 (v2 = 37.68; to survive during the first 4 weeks than prey-fed adults (v2 = 22.35; p > v2 < 0.0001) and that adult females were 4.4 times more likely to survive than adult males (v2 = 13.95; p > v2 = 0.0002). The greatest differences during the first 4 weeks were found where adults held at 4 °C for 1 week were 8518 times more likely to survive than adults held for 4 weeks at 4 °C(v2 = 62.96; p > v2 < 0.0001) and adults held for 4 weeks at 10 °C were 5767 times more likely to survive than adults held at 4 °C (v2 = 60.37; p > v2 < 0.0001). Overall, the analysis of the effect of temperature, food source, time, and sex over the first 4 weeks of the storage of adults indicated that the likelihood of survival was affected by temperature (v2 = 21.91; p > v2 < 0.0001), food source (v2 = 3.65; p > v2 = 0.0560), and time stored (v2 = 19.88; p > v2 = 0.0002). The interactions that were significant were between temperature and food source Fig. 3. Survival of prey-fed (P) and diet-fed (D) adult P. maculiventris 2 2 after storage at 4 °Cor10°C. Survival of adults from the same colonies of (v = 3.65; p > v = 0.0560), temperature and time 2 2 P. maculiventris after 9 weeks when reared at 26 °C (Control) is shown for (v = 18.60; p > v = 0.0003), and temperature and sex comparison. (v2 = 3.76; p > v2 = 0.0524). 190 T.A. Coudron et al. / Biological Control 42 (2007) 186–195

Table 1 Odds ratios for the survival of eggs, nymphs and adults during the 2 weeks of storage at 4 °C and 10 °Ca

a stg, stage; temp, temperature; fs, food source; nym, nymph; wk, week; D, diet-fed; P, prey-fed. Rows discussed in the text are highlighted with gray-ﬁll to assist the reader.

During a 10 week treatment of cold storage at 10 °C recover for 1 week at 26 °C and then mated with a similarly (Table 3) prey-fed adults were 2.5 times more likely to sur- treated adult of the opposite sex. The total number of eggs vive than diet-fed adults (v2 = 6.42; p > v2 = 0.0113). The laid per female over a 12-day period post-mating, the num- greatest differences during the 10 week period were found ber of days out of 12 that eggs were deposited (oviposition where adults held for 1 week were 23.3 times more likely days), and the number of eggs laid per female per day of to survive than adults held for 10 weeks (v2 = 12.46; oviposition were determined. These dependent variables p > v2 = 0.0004) and prey-fed adults were 34.6 times more were analyzed using a mixed-effects statistical model likely to survive than diet-fed adults after 10 weeks (SAS Institute, 2004). Due to low adult survival at 4 °C, (v2 = 13.71; p > v2 = 0.0002). fecundity over only the first 4 weeks of cold storage was Overall, the analysis of the effect of time, sex, and food analyzed for comparison at 4 °C with 10 °C, but fecundity source over the first 10 weeks of the storage period on the over the first 10 weeks of cold storage was analyzed for survival of adults held at 10 °C indicated that the likeli- adults held at 10 °C. hood of survival was not affected by sex (v2 = 0.01; During the first 4 weeks of cold storage at 4 °Cor10°C, p > v2 = 0.9268) but was affected by food source the total number of eggs laid per female over the 12 day (v2 = 6.49; p > v2 < 0.0109). There was also a significant oviposition period (Fig. 4) was affected by the storage tem- interaction between food source and time (v2 = 15.55; perature (F = 4.24; p = 0.0418), with adults held at 4 °C p > v2 = 0.0164) that affected survival. The interaction laying lower numbers of eggs after 4 weeks than adults between sex and other parameters over the first 10 weeks stored at 10 °C. Diet-fed adults laid significantly lower of storage was not significant. numbers of eggs over this period than prey-fed adults (F = 77.48; p < 0.0001). Although the length of time stored 3.6. Effect of adult cold storage on fecundity did not significantly affect the total number of eggs laid by diet-fed adults, there was a trend for adults held at 4 °Cto To analyze the effects of adult cold storage on fecundity, lay fewer eggs after 4 weeks than after 1 week of cold stor- adults surviving 4 °C and 10 °C treatment were allowed to age (F = 2.00; p = 0.1175). T.A. Coudron et al. / Biological Control 42 (2007) 186–195 191

Table 2 Odds ratios for the survival of adults during 4 weeks of storage at 4 °C and 10 °Ca

a temp, temperature; wk, week; D, diet-fed; P, prey-fed. Rows discussed in the text are highlighted with gray-ﬁll to assist the reader.

The total number of days when females oviposited eggs p = 0.0507), and time of storage (F = 11.45; p < 0.0001), (Fig. 5) was aﬀected by parental storage temperature, with a signiﬁcant interaction between temperature and time (F = 17.71; p < 0.0001), food source (F = 3.90; of storage (F = 3.63; p = 0.0152). After 4 weeks of storage

Table 3 Odds ratios for the survival of adults during 10 weeks of storage at 10 °Ca

a fs, food source; wk, week; D, diet-fed; P, prey-fed. Rows discussed in the text are highlighted with gray-ﬁll to assist the reader. 192 T.A. Coudron et al. / Biological Control 42 (2007) 186–195

Fig. 6. Number of eggs laid per day by 10 prey-fed and diet-fed females after storage at 4 °Cor10°C. Fig. 4. Total number of eggs laid over a 12-day oviposition period by 10 prey-fed and diet-fed females after they had been subjected to storage at 4 °Cor10°C. prey-fed females oviposited after 4 weeks of cold storage at 10 °C. However, this drop did not occur in prey-fed at 4 °C, both prey-fed and diet-fed females laid eggs on females stored at this same temperature for 6 weeks or fewer days over the 12-day oviposition period than at the longer, and therefore is likely not a true treatment effect. beginning of the storage period. Additionally, prey-fed As was the case with total eggs laid per female, eggs laid females laid eggs on fewer days than diet-fed females at per day per female were also affected by food source 4 °C. At 10 °C, however, oviposition days were similar in (F = 144.9; p < 0.0001), but not by time of storage prey-fed and diet-fed females, and did not decrease consis- (F = 0.88; p = 0.5153). tently with increasing time stored. The average number of eggs laid per female per day that eggs were oviposited (Fig. 6) was affected by food source 3.7. Effect of adult cold storage on egg viability (F = 135.75; p < 0.0001), with diet-fed females laying significantly fewer eggs each day they oviposited than prey- Due to low adult survival at 4 °C, the viability of eggs fed females. Neither temperature (F = 0.68; p = 0.4114) from adults held over the first 4 weeks of cold storage or time of storage (F = 0.52; p = 0.6664) affected the num- was analyzed for comparison at 4 °C with 10 °C, and the ber of eggs laid per female per day. odds ratios are presented in Table 4. Separately, the viabil- When fecundity of females measured for 10 weeks ity of eggs from adults held over the first 10 weeks of cold after storage at 10 °C(Fig. 4) was analyzed, the total storage was analyzed for adults held at 10 °C and the odds number of eggs laid per female was strongly affected by ratios are presented in Table 5. During the 4 week period, food source (F = 104.54; p<0.0001) but not by time of eggs from adults held at 10 °C were 1.2 times more likely to 2 storage (F = 1.14; p = 0.3422). In contrast, the number hatch than eggs from adults held at 4 °C(v = 12.58; 2 of days out of the oviposition period that females laid p > v = 0.0004). Eggs from prey-fed adults were 1.3 times eggs after storage at 10 °C(Fig. 5) was affected by the more likely to hatch than eggs from diet-fed adults 2 2 time of cold storage (F = 2.76; p = 0.0156); but not by (v = 20.86; p > v < 0.0001). For eggs from prey-fed food source F = 0.86; p = 0.3564). There was a significant adults held at 4 °C, hatch was 3.2 times more likely 2 2 food source by time interaction (F = 4.11; p = 0.0009). (v = 29.10; p > v < 0.0001) after 2 weeks of adult storage This was due to a strong drop in the number of days than after 4 weeks of adult storage. In comparison, for eggs from prey-fed adults held at 10 °C, the likelihood of hatch was nearly the same (v2 = 0.41; p > v2 = 0.5218) after 1 week of adult storage than after 4 weeks of adult storage. Similarly, for eggs from diet-fed adults held at 4 °C hatch was 4.5 times more likely (v2 = 82.75; p > v2 < 0.0001) after 1 week of adult storage than after 4 weeks of adult storage, while for eggs from diet-fed adults held at 10 °C the likelihood of hatch was nearly the same (v2 = 1.22; p > v2 = 0.2696) after 1 week of adult storage than after 4 weeks of adult storage. Eggs from prey-fed adults were 5.3 times (v2 = 141.3; p > v2 < 0.0001) more likely to hatch than eggs from diet- fed adults after 1 week of adult storage at 4 °C but that eggs from diet-fed adults were 2.7 times (v2 = 18.31; 2 Fig. 5. Number of days out of the 12-day oviposition period that prey-fed p > v < 0.0001) more likely to hatch than eggs from and diet-fed females laid eggs after storage at 4 °Cor10°C. prey-fed adults after 4 weeks of adult storage at 4 °C. In T.A. Coudron et al. / Biological Control 42 (2007) 186–195 193

Table 4 Odds ratios for the viability of eggs laid after 1–4 weeks of adult storage at 4 °Cor10°Ca

a temp, temperature; fs, food source; wk, week; D, diet-fed; P, prey-fed. Rows discussed in the text are highlighted with gray-ﬁll to assist the reader.

Table 5 Odds ratios for the viability of eggs laid after 1–10 weeks of adult storage at 10 °Ca

a fs, food source; wk, week; D, diet-fed; P, prey-fed. Rows discussed in the text are highlighted with gray-fill to assist the reader. contrast, eggs from prey-fed adults were only 1.3 times (v2 = 10.68; p > v2 < 0.0011), length of storage time more likely to hatch (v2 = 4.65; p > v2 = 0.0311) than eggs (v2 = 657.91; p > v2 < 0.0001), and the interaction of food from diet-fed adults after 1 week of adult storage at 10 °C source and length of time stored (v2 = 228.97; and were only 1.7 times (v2 = 15.11; p > v2 = 0.0001) more p > v2 < 0.0001). likely to hatch than eggs from diet-fed adults after 10 weeks of adult storage at 10 °C. Overall, the likelihood of an egg hatching was affected 4. Discussion by adult storage temperature (v2 = 12.49; p > v2 = 0.0004), food source (v2 = 20.76; p > v2 < 0.0001), and There was a clear effect of food source quality on egg, length of storage time (v2 = 44.39; p > v2 < 0.0001). Inter- nymphal and adult survival, fecundity and egg viability fol- action between food source and week (v2 = 294.59; lowing cold storage. Cold storage treatment of eggs of p > v2 < 0.0001) and temperature, food source, and week P. maculiventris held at 4 °C and 10 °C rapidly lowered (v2 = 80.68; p > v2 < 0.0001) were also significant. the percentage hatch even though in this study higher per- During the 10 week period of storage at 10 °C(Table 5), centages of eggs from prey-fed and diet-fed insects survived eggs from diet-fed adults were 1.1 times more likely to storage at both temperatures for longer periods of time hatch than eggs from prey-fed adults (v2 = 10.64; (i.e., up to 14 days) than had been previously reported p > v2 < 0.0011), although, after 6 weeks of adult storage (De Clercq and Degheele, 1993). The results of our study eggs from diet-fed adults were 2.9 times (v2 = 64.70; show that eggs from diet-fed insects survived cold storage p > v2 < 0.0001) more likely to hatch than eggs from at 10 °C significantly better than eggs from prey-fed insects prey-fed adults. Further, eggs from diet-fed adults were exposed to the same treatment. This would indicate that 3.60 times (v2 = 90.17; p > v2 < 0.0001) more likely to alteration of diet may be a useful method to improve sur- hatch after the adult had been stored for 1 week than after vival for the storage of eggs. This is of particular impor- 10 weeks. Overall, the likelihood of an egg hatching over 10 tance because of the interest in storing eggs for shipment weeks of storage at 10 °C was affected by food source and release exercises. 194 T.A. Coudron et al. / Biological Control 42 (2007) 186–195

The response of nymphs to cold storage treatment was to cold storage. The results of our studies, when compared slightly better than for eggs, with a higher weekly percent- to previous reports support that concern. For prey-fed age survival and survival out to 4 weeks which was 1 week adults from a colony that originated in Florida, the sur- longer than for eggs. Again, there was a higher survival at vival of males and females following treatment at 9 °C 10 °C than 4 °C as the treatment time increased and, simi- for 8 weeks was 90% and 73%, respectively (De Clercq lar to that for eggs, at 10 °C the diet-fed nymphs survived and Degheele, 1993). By comparison, the insects used in better than the prey-fed nymphs. Yet, it is unlikely with this study were from Missouri and prey-fed adults had a such low survival that there would be much value gained survival for males and females of 100% and diet-fed by subjecting eggs or nymphs to cold storage as a means females had a survival of 50% following treatment at of increasing colony size or as a substitute for maintaining 10 °C for 8 weeks. This is of importance because it suggests a continuous colony. However, this information is also of the possibility that diet-fed females from a cooler climate importance for time spent during shipping and release may out-perform prey-fed females from a temperate cli- operations. mate in cold storage treatments. Additionally, studies done Of the three developmental stages tested, the best sur- thus far have looked at the immediate response of preda- vival was obtained with adults. It is noteworthy that Podis- tory pentatomids to cold treatment. Equally important us spp. are thought to survive cold seasons as adults in a would be a study of the long-term effects over several suc- quiescent state and not a true diapause. Therefore, this nat- cessive generations exposed to some period of cold storage. ural feature may explain the better performance of adults Such studies could determine if repeated sessions of cold to cold storage treatment. For eggs, nymphs and adults storage in successive generations were to result in the selec- there was a higher likelihood of survival at 10 °C. How- tion of more cold-tolerant insects. ever, different from eggs and nymphs the prey-fed adults Our results showed that prey-fed adults were likely to were more likely to survive better over time than diet-fed, withstand cold storage at 10 °C for up to 8 weeks with very with 100% and 30% survival of prey-fed and diet-fed adults little loss of fecundity. Longer periods of storage resulted in at week 10, respectively. Also similar to the results for nym- a likelihood of a decrease in fecundity. The discovery that phal survival at 4 °C, the diet-fed adults were more likely to neither temperature or time of storage affected the number survive longer than prey-fed adults. However, in contrast of eggs laid per female per day of oviposition suggests that to what was observed for eggs and nymphs, at 10 °C the reduction in total eggs per female as a consequence of prey-fed adults were more likely to survive than diet-fed storage at 4 °C was due to a decrease in the number of days adults. The higher mortality for males observed in this that females oviposited, and not due to a reduction in the study is in contrast to what has been previously reported number of eggs produced each day of oviposition. There- for P. maculiventris but similar to what was reported for fore, in this species, a certain egg load may need to be Podisus sagitta (De Clercq and Degheele, 1993). Given that reached before females will oviposit. Consequently, storage nutrient quality affected survival in this study, the differ- at 10 °C not only was less detrimental to adult survival, but ences reported for the likelihood of male versus female sur- also had less effect on adult fecundity, perhaps, by main- vival reported here with those reported previously may taining egg loads in the female such that they are able to reflect differences in the natural prey used in the two stud- oviposit with greater frequency than adults stored at ies. The fact that there are differences reported for cold tol- 4 °C. Regardless of temperature, however, females reared erance between males and females suggests that diet on diet had lower fecundity than adults reared on prey even formulations could be manipulated to affect this difference. though diet seems to have given adults stored at 4 °Can The ability to compare egg, nymphal, and adult survival advantage in terms of survival. is of particular value when considering short-term cold The viability of eggs produced by adults subjected to storage for shipment and release exercises. Our results cold storage was affected by temperature, food source showed that survival of nymphs and adults was the same and length of storage. For intervals of storage less than 4 when stored for 1 week at either 4 °Cor10°C and when weeks, the likelihood of viable eggs produced by females reared on either prey or diet. Equally valuable is the com- held at 10 °C was slightly higher than those produced by parison of adult survival when considering extended stor- females held at 4 °C, was slightly higher for eggs from age to accumulate large numbers or to retain a colony prey-fed adults than from diet-fed adults, and decreased between uses. Our odds ratios analyses showed that sur- with the increase in adult storage time. However, for inter- vival was affected by temperature and food source, with a vals of storage at 10 °C that exceeded 4 weeks, the egg via- higher survival at 10 °C than at 4 °C, with a higher survival bility was more likely to be higher for eggs from diet-fed for diet-fed than prey-fed during the first 4 weeks of cold adults. This demonstrated that nutrition has a direct effect storage at either 4 °Cand10°C, but with a higher survival on the response to both temperature and time associated for prey-fed than diet-fed after 4 weeks of storage, and a with cold storage. decrease in survival over 10 weeks at 4 °C in contrast to In summary, it is noteworthy that eggs from diet-fed similar survival over time at 10 °C. insects tolerated 4 weeks of cold storage better than eggs Other researchers had expressed some concern that the from prey-fed insects, diet-fed nymphs survived 4 weeks geographical origin of the colony may affect the response of cold storage better than prey-fed nymphs and hatch of T.A. Coudron et al. / Biological Control 42 (2007) 186–195 195 eggs oviposited by diet-fed adults subjected to more than 4 DeBach, P., 1943. The effect of low storage temperature on weeks of cold storage was higher than the hatch of eggs reproduction in certain parasitic Hymenoptera. Pan-Pac. Entomol. oviposited by prey-fed adults subjected to similar cold 19, 112–119. De Clercq, P., Degheele, D., 1992. Development and survival of Podisus treatment. This would suggest that the high protein and maculiventris (Say) and Podisus sagitta (Fab.) (Heteroptera: Pent- low lipid content or the presence of other substances atomidae) at various constant temperatures. Can. Entomol. 124, (e.g., vitamins, antibiotics, etc.) in the diet can be advanta- 125–133. geous for tolerating cold storage. Collectively, these results De Clercq, P., Degheele, D., 1993. Cold storage of the predatory bugs demonstrate different nutritional needs for cold storage Podisus maculiventris (Say) and Podisus sagitta (Fabricius) (Heterop- tera: Pentatomidae). Parasitica 49 (1–2), 27–41. than for continuous rearing and that a diet more optimal Flanders, S.E., 1938. The effect of cold storage on the reproduction of for continuous rearing is not necessarily the better diet parasitic Hymenoptera. J. Econ. Entomol. 31, 633–634. for cold storage. Proteomic and genomic analyses of insects Goryshin, N.I., Tuganova, I.A., 1989. Optimization of short-term storage on different diets following cold treatments would be par- of eggs of the predatory bug Podisus maculiventris (Hemiptera, ticularly informative studies for insight into which nutri- Pentatomidae). Zoologicheskii Zhurnal 68, 111–119. Goryshin, N.I., Saulich, A.K., Volkovich, T.A., Borisenko, I.A., ents enhance tolerance to cold storage and what Simonenko, N.P., 1988. The influence of the food factor on the physiological processes are involved. development and photoperiodic reaction of the predatory bug Podisus maculiventris (Hemiptera, Pentatomidae). Zoologicheskii Acknowledgments Zhurnal 67, 1324–1332. McPherson, J.E., 1980. A list of prey species of Podisus maculiv- We thank the following ARS employees: Maureen entris (Hemiptera: Pentatomidae). Great Lakes Entomol. 13, 17–24. Wright for her extensive contributions to the statistical McPherson, J.E., 1982. The Pentatomidea (Hemiptera) of Northeastern analyses and manuscript, James Smith and John Willen- North America with Emphasis on the Fauna of Illinois. Southern berg for their creative contributions to rearing and techni- Illinois University Press, Carbondale, IL, 240 pp. cal assistance in this project and Dr. David Stanley for his Rivers, D.B., Lee, R.E., Denlinger, D.L., 2000. Cold hardiness of the fly critique of the manuscript. pupal parasitoid Nasonia vitripennis is enhanced by its host Sarcophaga crassipalpis. J. Insect Physiol. 46, 99–106. Ruberson, J.R., Kring, T.J., Elkassabany, N., 1998. Overwintering and References the diapause syndrome of predatory Heteropter. in: Coll, M., Ruberson, J.R. (Eds.), Predatory Heteroptera: Their ecology and use Birch, L.C., 1948. The intrinsic rate of natural increase of an insect in Biological Control. Thomas Say Publ. Entomol. Soc. Am., Lanham, population. J. Anim. Ecol. 17, 15–26. MD. pp. 49–69. Coudron, T.A., Knop-Wright, M.M., Puttler, B., Brandt, S.L., Rice, SAS Institute, 2004. SAS/STATÓ 9.1 User’s Guide. SAS Institute Inc., W.C., 2000. The effect of the ectoparasite Necremnus breviramulus Cary, NC. USA. (Hymenoptera: Eulophidae) and its’ venom on natural and facticious Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods, eight ed. Iowa hosts. Ann. Entomol. Soc. Am. 93, 890–897. State University Press, Ames, Iowa.. Coudron, T.A., Wittmeyer, J.L., Kim, Y., 2002. Life history and cost Vlasova, V.A., Ziskind, L.A., Izhevskii, S.S., 1980. The possibility of the analysis for continuous rearing of Podisus maculiventris (Say) (Hetre- acclimatisation of Podisus. Zashchita Rastenii 4, 46–47. optera: Pentatomidae) on a zoophytophagous artificial diet. J. Econ. Wittmeyer, J.L., Coudron, T.A., Adams, T.S., 2001. Ovarian develop- Entomol. 95, 1159–1168. ment, fertility and fecundity in Podisus maculiventris Say (Heteroptera: Coudron, Yocum, G.D., Brandt, S.L., 2006. Nutrigenomics: A case study Pentatomidae): An analysis of the impact of nymphal, adult, male and in the measurement of insect response to nutritional quality. Entomol. female nutritional source on reproduction. Invertebr. Reprod. Dev. 39, Exper. Appl. 121, 1–14. 9–20. Couturier, A., 1938. Contribution a` l’e´tude biologique de Podisus Yocum, G.D., Coudron, T.A., Brandt, S.L., 2006. Differential gene maculiventris Say, pre´dateur ame´ricain du Doryphore. Ann. Epiphyt. expression in Perillus bioculatus nymphs fed a suboptimal artificial Phytogeń. 4, 95–165. diet. J. Insect. Physiol. 52, 586–592.