Development and survivorship of a predatory lady beetle, Coccinella novemnotata, on various aphid diets

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1 BioControl DOI /s Development and survivorship of a predatory lady beetle, Coccinella novemnotata, on various aphid diets Danielle M. Brandt Paul J. Johnson John E. Losey Michael A. Catangui Louis S. Hesler Received: 28 March 2014 / Accepted: 10 November 2014 Ó International Organization for Biological Control (IOBC) 2014 Abstract Ex situ rearing of Coccinella novemnotata Herbst (Coleoptera: Coccinellidae), a Nearctic native that has declined precipitously, may be important for experimentation and conservation. Rearing success may depend on optimizing an aphid prey diet. The objective was to compare development, survivorship and adult size of C. novemnotata reared on diets of various aphid (Hemiptera: Aphididae) species. Diets of Acyrthosiphon pisum (Harris) and A. pisum? Rhopalosiphum padi L. were most suitable for rearing C. novemnotata. Single species diets of other aphids had low suitability for C. novemnotata. Combining A. pisum with one of the other aphid species improved diet suitability compared to diets of other species Handling Editor: Patrick De Clercq. D. M. Brandt P. J. Johnson Department of Plant Science, South Dakota State University, Brookings, SD 57007, USA J. E. Losey (&) Department of Entomology, Cornell University, Ithaca, NY 14853, USA jel27@cornell.edu M. A. Catangui South Clubhouse Road, Sioux Falls, SD 57108, USA L. S. Hesler North Central Agricultural Research Laboratory, Agricultural Research Service, USDA, 2923 Medary Avenue, Brookings, SD 57006, USA alone. Knowledge of C. novemnotata development and survival on various aphid species may not only aid in ex situ rearing, but could also be used to identify habitats where C. novemnotata may be extant or those with suitable prey for reestablishment or augmentation of C. novemnotata. Keywords Coccinella novemnotata Coccinellidae Invasive species Biological control Conservation Introduction Several studies have documented a decline in native species of predaceous lady beetles in North America (Wheeler and Hoebeke 1995; Harmon etal. 2007). One of the most dramatic declines involves Coccinella novemnotata Herbst, the nine-spotted lady beetle, which was distributed throughout much of America north of Mexico and was regarded as a common predator of aphids and other insect pests in crop fields and orchards (Wheeler and Hoebeke 1995; Gordon 1985). This species became rare throughout much of its historic range over the last two decades, while some introduced coccinellids, including Coccinella septempunctata L., became prominent throughout much of North America (Wheeler and Hoebeke 1995; Harmon et al. 2007). Ex situ rearing of C. novemnotata may be advantageous to (a) supply beetles for experiments that test

2 D. M. Brandt et al. hypotheses about the decline, (b) to mass produce beetles for reestablishment in field settings, and (c) to maintain captive populations to offset continued decline in the wild (New 2010; Losey et al. 2012). Successful ex situ rearing of C. novemnotata will depend on knowledge of its development and survival on various aphid species. Coccinella novemnotata is generally aphidophagous (Wheeler and Hoebeke 1995), but little is known about the relative suitability of various aphid prey for its development and survival. The beetle has been reared for short-term studies on relatively few species of aphids (Hodek 1996). The pea aphid, Acyrthosiphon pisum (Harris), has been used as a suitable prey for C. novemnotata (McMullen 1967b). This aphid is native to Europe and was introduced into North America in the 1800s (Thomas 1878). Historically, C. novemnotata was reported as a common predator of A. pisum in North America (Pack 1925; Knowlton et al. 1938). Empirical assessment of prey suitability for aphidophagous coccinellids is typically necessary to optimize rearing procedures (Michaud 2005). As such, additional knowledge about relative suitability of aphid diets may be critical to successful ex situ rearing of C. novemnotata. Hodek(1996) listed a relatively short development time, a high rate of survival, and large female size as three measures that indicate diet suitability for lady beetles. Subsequently, Ungerová et al. (2010) devised a diet suitability index based on the ratio of female mass to developmental time. The objective of this study was to compare development, survivorship and female size of C. novemnotata among various aphid diets, and to use those measures in determining suitable diets for rearing C. novemnotata. Materials and methods Diet treatments Development and survival of C. novemnotata were measured from egg hatch to adulthood on diets of six aphid species offered alone or in two-species combinations for a total of 11 aphid-diet treatments. The six aphid species are relatively common crop pests in North America (Blackman and Eastop 2000), and respectively included three species that infest leguminous plants (i.e., pea aphid, A. pisum; soybean aphid, Aphis glycines Matsamura; cowpea aphid, Aphis craccivora Koch) and three that infest cereals and other graminaceous plants [bird cherry-oat aphid, Rhopalosiphum padi (L.); greenbug, Schizaphis graminum (Rondani); and yellow sugarcane aphid, Sipha flava (Forbes)]. Rhopalosiphum padi and S. flava are native to North America (Blackman and Eastop 2000; Halbert and Voegtlin 1998), and the other four aphids are not (Foottit et al. 2006). Treatments of two-species combinations of aphids consisted of feeding C. novemnotata a diet of A. pisum on one day and then one of the other five aphid species on alternating days. A twelfth treatment of coccinellid eggs was used as a control (Michaud 2005) and consisted of eggs of C. novemnotata (first two weeks of experiment) and C. septempunctata (last two weeks), based on availability in the respective weeks. Afterwards, eggs were alternated between species every other day. For simplification, reference to this latter treatment uses only the genus name, Coccinella. Experimental insects Various host plants were used to maintain individual colonies of the six aphid species. Acyrthosiphon pisum was reared on pea (Pisum sativum L., var. Austrian winter, Millborn Seeds, Brookings, South Dakota, USA); A. glycines on soybean (Glycine max (L.) Merr., var. 91B91, Pioneer Seed, DuPont Corp., Johnston, Iowa, USA); A. craccivora on black-eye pea [Vigna unguiculata (L.) Walp., var. California Blackeye #5, Burpee Seed Company, Warminster, Pennsylvania, USA]; and R. padi, S. graminum, and S. flava separately on barley (Hordeum vulgare L., var. Robust, Albert Lea Seed House, Inc., Albert Lea, Minnesota, USA). All aphids were obtained from the field and maintained separately as multiclonal stock colonies of each species in growth chambers (Conviron, Winnipeg, Manitoba, Canada) at 19 C, 50 % RH, and 16:8 h (L:D) for grass-feeding aphids, and at 23 C, 50 % RH, and 16:8 h (L:D) for legume aphids. Care was taken to ensure that aphids were free of plant-disease viruses, and rearing plants were monitored to ensure absence of obvious viral disease symptoms and by annual testing of plant tissue using ELISA (Agdia Inc., Elkhart, Indiana, USA). Stock colonies of C. novemnotata and C. septempunctata were reared separately but identically on a largely aphid diet using methods in Hesler et al. (2012). Briefly, first and early second instars were

3 Development and survivorship of Coccinella novemnotata reared on R. padi, and older larvae were fed A. pisum. Adult beetles were fed A. pisum and supplemental food that included several small granules of bee pollen (Fresh Bee Pollen Dietary Supplement; Y.S. Organic Bee Farms, Sheridan, IL, USA), powdered meal (Good Bug Powder Meal; Arbisco Environmentals, Tucson, AZ, USA), and ad libitum eggs of the Mediterranean flour moth, Ephestia kuehniella Zeller. Two to three rumpled, white or brown, paper hand towels were added to each adult rearing container to serve as an oviposition substrate. Lady beetle eggs laid on this substrate were removed, and portions of towels with individual clutches were excised, placed into small cylindrical, vented, plastic holding containers (6-cm ht 9 15-cm diam), and held in a growth chamber until hatching (CMP 3245, Conviron, Winnipeg, Canada) at 23 C, 50 % RH, 16:8 h (L:D) (Hesler et al. 2012), or stored for a few days in the dark in a refrigerator at 9.5 C, 20 % RH, for later use. Experimental procedures For the experiment, newly hatched larvae (\24-h old) of C. novemnotata were randomly selected from egg clutches in the stock colony and carefully transferred by a soft-bristled paint brush to individual 30-ml plastic cups (Crystal Clear Portion Cups; WNA, Chelmsford, MA, USA) with a punctured lid for air exchange and a cm paper-towel cutting. Each cup contained a single larva. Because of the relatively large number of larvae needed across all diet treatments, the experiment was implemented over three days: six larvae were assigned to each diet on the first day, 18 on the second day, and the remaining six individuals per diet treatment on the third day. Neonates were arbitrarily selected from multiple egg clutches and placed individual into experimental cups. Thus, any two given individuals may or may not have arisen from different parental stock. First instars that died within their first two days were discarded, not included in analysis, and replaced with randomly selected neonate larvae from the stock colony. A moderately to heavily aphid-infested plant cutting or coccinellid egg clutch was added to each cup so that larvae could feed ad libitum. Cups were placed into 30-slot trays, and cups were arranged in a restricted completely randomized design with a maximum of two cups per treatment in each tray. Trays were placed into a growth chamber at 23 C, 50 % RH, and 16:8 h (L:D). To avoid disease and monitor progress, each cup was replaced with a clean cup, new paper towel section, plant material, and aphids or coccinellid eggs daily. Lids were replaced after soiling had occurred. Individuals remained in the same cups from pupation to adult eclosion. Food was not added to cups during pupation. Each larva was examined daily for molting or death. Molting was counted on each day that an exuvium was found, and each exuvium was discarded. The pre-pupal period was recorded when a fourth instar was immobile for over one day but responsive to a gentle prod. Dead individuals were recorded and removed. Survival was recorded for individual growth stages through adulthood. When an adult beetle died, the entire cup was removed and placed into a freezer before discarding. At termination of the diet experiment, live adult beetles in their cups were frozen to death. Individuals were sexed by examining the fifth ventrite for curvature of the distal edge in males and lack of curvature in females (Gordon 1985; Stellwag and Losey 2014). All dead female adults were placed into a drying oven (Model 50, Precision Thelco Oven, Winchester, VA, USA) at 40 C for 48 h. Individual female beetles were then weighed at room temperature to the nearest g (Ohaus Analytical Plus Electric Balance #AP250D, Ohaus Corporation, Florham Park, NJ, USA), and their length and width at elytral apices were measured to the nearest 0.1 mm using a stereomicroscope (Stemi SV6, Carl Zeiss Microscoopy, Thornwood, NY, USA) fitted with a reticle. Female size was used as a surrogate measure of fitness, as body size is related to reproductive capacity in Coccinella beetles (Dixon and Guo 1993; Kajita and Evans 2010; Bista and Omkar 2013). All insect rearing and experimentation was conducted at the United States Department of Agriculture North Central Agricultural Research Laboratory, Brookings, South Dakota, USA. Statistical analysis Data were compiled for the 12 diets regarding duration of individual life stages (individual instars, combination of all four instars, prepupa, pupa, and combination of stages from first instar to adult eclosion), proportion surviving to successive life stages (second instar to adult), proportion female (number of females per 30 neonates), and mass, length and width of adult

4 D. M. Brandt et al. females. A diet suitability index (DS) was devised to include developmental time, proportion of C. novemnotata that survived to female adults, and dry mass of females, such that DS = (M 9 P)/t (i.e. female dry mass multiplied by proportion of individuals that were female divided by developmental time), based on Hodek (1996) and Ungerová et al. (2010). Measurements of developmental duration and mass, length and width of adult females were individually treated as dependent variables and subjected to separate analyses of variance (ANOVA; PROC GLM) using diet as the independent variable (SAS Institute 2008). Following a significant ANOVA, diet means were separated using Tukey s HSD. Survivorship was compared among diets using logistic regression of the proportion survival within various life stages (PROC GENMOD) (Sokal and Rohlf 1995). Analysis of diet as an explanatory factor was followed by a least square means test with Tukey adjustment to separate survivorship among diet treatments. Results of the means tests are displayed for all stages but only interpreted for stages in which diet was a significant factor. Results Developmental duration Diet affected duration of several life stages of C. novemnotata, including each of the second, third, and fourth instars, the entire larval period, prepupa, and period from neonate larva to adult (Table 1). Duration of the 2nd stadium was longer for larvae fed A. craccivora than those on other diets except a S. graminum diet. Larvae fed A. craccivora also had a longer 3rd stadium than those fed other diets, except for single species diets of S. graminum and A. glycines. The 3rd stadium was longer on single-aphid diets of S. graminum or A. glycines than on a diet of A. pisum. A diet of A. craccivora lengthened the 4th stadium when compared to other diets. Single-species diets of R. padi, S. graminum, and S. flava prolonged the 4th stadium compared to diets of A. pisum, A. pisum? R. padi, A. pisum? S. flava, and A. pisum? A. glycines. Prepupal period was longer for a S. graminum diet than for diets of R. padi and Coccinella eggs. Time from neonate larva to adult eclosion was shorter on a diet of A. pisum compared to diets of R. padi, S. flava, S. graminum, and A. glycines. Diets of A. pisum? R. padi, A. pisum? S. graminum, and A. pisum? A. glycines led to shorter neonate-to-adult development times than diets of S. flava, S. graminum, or A. glycines. Survivorship Diet did not affect C. novemnotata survival during the first three stadia, but differences in survival were significant in later developmental stages (Table 2). More larval fourth instars survived on a diet of A. pisum than on A. craccivora, and survivorship on other diets did not differ from that on these two diets or from one another. Relatively stark contrasts in survivorship of C. novemnotata were seen among diet treatments by prepupal stage, in which survival on A. craccivora was less than that on other diets except Coccinella eggs, R. padi, and A. pisum? A. glycines. Prepupal survivorship on Coccinella eggs was lower than that on A. pisum, S. flava, S. graminum, A. pisum? R. padi, A. pisum? S. flava, and A. pisum? A. craccivora. For pupae, survival on A. craccivora or Coccinella eggs was lower than that on all other diets except R. padi, A. glycines, and A. pisum? A. glycines. More C. novemnotata survived to pupal stage with a diet of A. pisum than on R. padi, A. glycines, A. pisum? A. glycines, A. craccivora, and Coccinella eggs. Survival of C. novemnotata through pupation did not differ among diets of R. padi, S. flava, S. graminum, A. glycines, A. pisum? R. padi, A. pisum? S. flava, A. pisum? S. graminum, A. pisum? A. glycines, and A. pisum? A. craccivora. Survivorship to adulthood on A. pisum (87 %) was greater than that on R. padi, S. graminum, A. glycines, A. craccivora, A. pisum? A. glycines, and Coccinella eggs (all B 40 %). Adult survivorship was also greater on diets of S. flava, A. pisum? R. padi, A. pisum? S. flava, A. pisum? S. graminum, and A. pisum? A. craccivora than on diets of A. craccivora alone or Coccinella eggs. There were no differences among diets in survivorship of females to adulthood (B33 %). Adult measurements Adult mass, length, and width of female C. novemnotata varied by diet (Table 3). Individuals reared on A. pisum had a greater dry mass than those reared on other diets except A. pisum? R. padi, A. pisum? S.

5 Development and survivorship of Coccinella novemnotata Table 1 Duration (days ± SE) of successive life stages of Coccinella novemnotata fed various diets Diet 1st Stadium 2nd Stadium 3rd Stadium 4th Stadium All larval stadia Prepupa Pupa Neonate larva to adult Acyrthosiphon pisum 6.7 ± 0.3a 3.3 ± 0.1a 3.5 ± 0.2a 6.3 ± 0.2a 19.7 ± 0.4a 2.2 ± 0.1ab 7.0 ± 0.1a 28.3 ± 0.5a Rhopalosiphum padi 6.0 ± 0.3a 3.8 ± 0.3a 4.5 ± 0.4ab 12.7 ± 1.2d 26.2 ± 1.2bc 1.9 ± 0.2a 7.0 ± 0.3a 34.0 ± 1.7bc Sipha flava 5.9 ± 0.3a 3.4 ± 0.2a 4.9 ± 0.4ab 12.1 ± 0.8d 26.2 ± 1.0bc 2.6 ± 0.3ab 7.0 ± 0.3a 35.5 ± 1.0c Schizaphis graminum 6.5 ± 0.3a 4.2 ± 0.4ab 5.4 ± 0.4bc 11.7 ± 1.0cd 27.3 ± 0.9c 3.1 ± 0.4b 7.0 ± 0.2a 36.3 ± 0.9c Aphis glycines 6.7 ± 0.4a 4.1 ± 0.1a 5.5 ± 0.3bc 11.6 ± 0.9bcd 27.7 ± 0.8c 2.4 ± 0.2ab 7.0 ± 0.4a 35.9 ± 0.8c Aphis craccivora 7.3 ± 0.4a 5.6 ± 0.6b 7.0 ± 1.1c 26.3 ± 3.8e 42.3 ± 1.9d 2.5 ± ± ± 3.5 A. pisum? R. padi 6.2 ± 0.3a 3.6 ± 0.2a 4.1 ± 0.3ab 8.1 ± 0.7ab 21.9 ± 1.0a 2.1 ± 0.1ab 7.0 ± 0.2a 30.1 ± 1.0ab A. pisum? A. pisum 6.6 ± 0.4a 3.5 ± 0.2a 4.1 ± 0.2ab 7.3 ± 0.6a 21.3 ± 0.7a 2.2 ± 0.1ab 6.9 ± 0.1a 31.2 ± 1.3abc A. pisum? S. graminum 6.3 ± 0.3a 3.2 ± 0.1a 3.8 ± 0.2ab 8.3 ± 0.7abc 21.6 ± 0.7a 2.2 ± 0.1ab 6.8 ± 0.1a 30.8 ± 0.8ab A. pisum? A. glycines 5.7 ± 0.4a 3.8 ± 0.3a 3.8 ± 0.4ab 7.6 ± 0.5a 20.4 ± 0.8a 2.3 ± 0.1ab 6.6 ± 0.2a 28.4 ± 0.7ab A. pisum? A. craccivora 6.1 ± 0.4a 3.8 ± 0.2a 4.2 ± 0.3a 9.0 ± 0.8abcd 22.9 ± 1.0ab 2.1 ± 0.1ab 7.1 ± 0.1a 31.8 ± 1.2abc Coccinella egg 6.7 ± 0.4a 3.4 ± 0.3a 4.0 ± 0.4ab 8.3 ± 0.6abcd 20.0 ± 0.7a 1.3 ± 0.3a F value F 11, 304 = 1.71 F 11,278 = 4.73* F 11,245 = 5.67* F 11,194 = 15.41* F 11, 194 = 18.57* F 10,175 = 3.25* F 9,142 = 1.01 F 9,142 = 52.19* * P \ Within each column, means followed by same letter do not differ significantly from one another (Tukey s HSD test) Means not followed by a letter had insufficient replications for analysis. Beetles reared at 23 C, ca. 50 % RH, and 16:8 L:D photoperiod

6 D. M. Brandt et al. Table 2 Percentage of Coccinella novemnotata surviving to successive life stages when fed various diets Diet Larval stadium Prepupa Pupa Adult Female adult 2nd 3rd 4th Acyrthosiphon pisum 93a 90a 90a 90a 90a 87a 27a Rhopalosiphum padi 93a 87a 67ab 50abc 43bc 37bc 13a Sipha flava 90a 87a 80ab 70a 70ab 63ab 27a Schizaphis graminum 100a 87a 80ab 73a 67ab 30bc 23a Aphis glycines 90a 83a 73ab 53ab 47bc 30bc 13a Aphis craccivora 80a 63a 43b 10c 7c 7c 7a A. pisum? R. padi 83a 83a 80ab 70a 63ab 57ab 33a A. pisum? A. pisum 93a 90a 83ab 80a 70ab 60ab 30a A. pisum? 87a 77a 77ab 63ab 63ab 57ab 27a S. graminum A. pisum? 67a 63a 57ab 50abc 47bc 40bc 17a A. glycines A. pisum? 93a 77a 73ab 67a 60ab 47abc 23a A. craccivora Coccinella eggs 100a 83a 60ab 20bc 13c 3c 0a v 2 v 2 11 = 15.2 v 2 11 = 17.0 v 2 11 = 27.8* v 2 11 = 80.3* v 2 11 = 85.0* v 2 11 = 88.6* v 2 11 = 27.0* Data for each life stage based on a starting sample size of n = 30 individuals. Test individuals were arbitrarily selected from multiple egg clutches and placed individual into experimental cups. * P\ Within each column, percentages followed by same letter do not differ significantly from one another (Tukey-adjusted least square means procedure). Beetles reared at 23 C, ca. 50 % RH, and 16:8:D photoperiod Table 3 Mean mass, length, and width (±SE) of female Coccinella novemnotata that developed on various diets, and diet suitability of the various diet treatments Diet treatment n Dry mass (mg ± SE) Length (mm ± SE) Width (mm ± SE) DS Acyrthosiphon pisum ± 2.0b 5.8 ± 0.1c 4.3 ± 0.1d Rhopalosiphum padi ± 0.6a 4.6 ± 0.2ab 3.5 ± 0.2ab Sipha flava ± 1.1a 4.6 ± 0.1ab 3.4 ± 0.1ab Schizaphis graminum ± 1.4a 4.5 ± 0.2ab 3.4 ± 0.2ab Aphis glycines ± 0.7a 4.7 ± 0.1ab 3.5 ± 0.1abc Aphis craccivora ± ± ± A. pisum? R. padi ± 0.8ab 5.2 ± 0.1bc 3.9 ± 0.1bcd A. pisum? A. pisum ± 2.4ab 5.3 ± 0.2bc 3.9 ± 0.2bcd A. pisum? S. graminum ± 0.6ab 5.5 ± 0.2c 4.2 ± 0.1cd A. pisum? A. glycines ± 0.3ab 5.6 ± 0.2c 3.9 ± 0.3bcd A. pisum? A. craccivora ± 0.5a 4.7 ± 0.2ab 4.0 ± 0.2bcd F 9, * 8.52* 5.75* * P \ Within each column, means (±SE) followed by same letter do not differ significantly from one another (Tukey s HSD test). Beetles reared at 23 C, ca. 50 % RH, and 16:8 L:D photoperiod DS: diet suitability index derived by multiplying female dry mass by proportion female reaching adulthood and dividing by mean developmental time; not subjected to statistical analysis 1 Not included in statistical comparisons because of inadequate sample size

7 Development and survivorship of Coccinella novemnotata flava, A. pisum? S. graminum, and A. pisum? A. glycines. Length of adult C. novemnotata was greater for those that were reared on diets of A. pisum, A. pisum? S. graminum, and A. pisum? A. glycines compared to diets of R. padi, S. flava, S. graminum, A. glycines, and A. pisum? A. craccivora. Width of adults reared on A. pisum was greater than those reared on single-aphid diets. Adults reared on A. pisum? S. graminum were wider than those reared on R. padi, S. flava, and S. graminum. Few females reared on A. craccivora survived to adulthood, and therefore their measurements were not compared to those of beetles reared on other diets. The suitability of diets (DS) for C. novemnotata ranged considerably (Table 3). Suitability of A. pisum alone (DS = 0.137) was markedly superior to other single-species diets (DS B 0.043). Combination diets of A. pisum and one of the other aphid species generally had intermediate suitability (DS = ), with improvements ranging from 2.1- (S. flava) to 11.7-fold (A. craccivora) over single-aphid diets. The diet of A. pisum? R. padi had the second best suitability (DS = 0.118) of all diets. Discussion A relatively short development time, a high rate of survival, and large female size are three measures that indicate diet suitability for lady beetles (Hodek 1996). Using an index based on those three measures, diets of A. pisum and A. pisum? R. padi were most suitable for rearing C. novemnotata in this study. Single species diets of aphids other than A. pisum had low suitability for C. novemnotata. Diets combining A. pisum with one of the other aphid species improved suitability for C. novemnotata development compared to diets of the other aphid species alone. The magnitude of improvement with A. pisum was inversely proportional to suitability of the single species diet, i.e. single species diets with the lowest suitability showed the greatest improvement when A. pisum was added. Offering A. pisum in mixed diets on alternate days may have compensated for nutritional deficiency of the other aphid or mitigated a toxic effect of consuming the other aphid (Mehrparvar et al. 2013), but regardless it resulted mainly in reducing developmental time of C. novemnotata in our study. In contrast, other studies have shown that combining high quality aphids with a poor quality aphid does not significantly improve suitability, largely due to a failure to improve survival rate to adult stages (Nielsen et al. 2002; Mehrparvar et al. 2013). The relatively high suitability of diets containing A. pisum for rearing C. novemnotata in this study is generally consistent with previous use of A. pisum as prey for C. novemnotata in a laboratory study (McMullen 1967b) and with field observations of C. novemnotata exploiting A. pisum in alfalfa fields before establishment of C. septempunctata in the US (Thomas 1878; Pack 1925; McMullen 1967a; Wheeler 1971). However, published studies have not reported C. novemnotata in alfalfa in the last two decades, even though field sampling yielded A. pisum and other coccinellids species (Wheeler and Hoebeke 1995; Harmon et al. 2007; Day and Tatman 2006; Hesler et al. 2005; Hesler and Petersen 2008). Beetles used in this study were collected as adults from rangeland and weedy roadside vegetation, but it is not known what aphid species they had been associated with. Upon collection, they were reared on A. pisum and R. padi in the laboratory (Hesler et al. 2012). However, the use of these two aphids could not have pre-conditioned C. novemnotata for prime development and survival on test diets of A. pisum and A. pisum? R. padi because the development trials were initiated with unfed first instars. Thus, observations suggest that C. novemnotata used in this study were adapted for faster development and higher survival on some aphid species that were tested than others. Knowledge of the development and survival of C. novemnotata on various aphid species may not only aid in successful ex situ rearing, but could also be used in selecting candidate prey and associated habitats for augmentation or re-introduction of C. novemnotata in the field (New 2010). Given the results of this study, habitats conducive to A. pisum (e.g. alfalfa) or to A. pisum? R. padi combinations (e.g., legume-cereal grain intercroppings) could arguably be candidates for C. novemnotata release. However, factors other than type of prey may need consideration, as C. novemnotata has rarely been found in habitats such as alfalfa and legume-grain intercrops in roughly 20 years, despite continued infestation of these crops with suitable aphid prey (Hauge et al. 1998; Day and Tatman 2006; Hesler et al. 2005). Coccinella novemnotata has recently been found in non-agricultural as well as agricultural habitats (McMullen 1967a;

8 D. M. Brandt et al. Wheeler 1971; Hesler et al. 2009; Losey et al. 2007). Besides crop plants, A. pisum infests a wide variety of legumes, and the cereal aphids infest a wide variety of grasses (Blackman and Eastop 2000; Michels 1986; Anstead et al. 2003). Many of the favorable legumes and grasses are located in non-agricultural habitats that may be appropriate sites for strategic releases of C. novemnotata in the future. Moreover, molecular analysis of C. novemnotata gut contents in future studies would be enormously valuable in determining prey and thereby in identifying candidate habitats. The probability of successful reestablishment of C. novemnotata could potentially be increased by combining site selection based on prey with management practices that minimize mortality. Within agricultural systems and adjacent non-agricultural areas, the selection, timing and method of application of insecticides are known to impact the survival of beneficial insects. The susceptibility of C. novemnotata to conventional insecticides has been documented (Travis et al. 1978) and other species of coccinellids (C. novemnotata was not available for testing) have been shown to be negatively impacted by the Bt toxins incorporated into maize (Stephens et al. 2012). Other factors such as invasive species and global climate change would be much more challenging to manage, but reestablishment site selection and management could provide the basis for sustainable populations of C. novemnotata. The populations could provide stability for C. novemnotata in the short term and potentially a source for wider redistribution in the longer term. Acknowledgments Francoise Marie Vermeylen and Cuirong Ren provided statistical advice, and Eric Beckendorf helped perform statistical tests. Eric Beckendorf, Mallory Burtz and Corrinna Schwartz assisted with experiments. Jeff Heinle, Guadalupe Rojas, and Eric Beckendorf reviewed drafts of this paper. Research was supported by funding from the National Science Foundation through the Lost Ladybug Project, award number DRL , and through base funding to the USDA North Central Agricultural Research Laboratory, CRIS Project Number D. References Anstead JA, Burd JD, Shufran KA (2003) Over-summering and biotypic diversity of Schizaphis graminum (Homoptera: Aphididae) populations on noncultivated grass hosts. Environ Entomol 32: Bista M, Omkar (2013) Effects of body size and prey quality on the reproductive attributes of two aphidophagous Coccinellidae (Coleoptera) species. Can Entomol 45: Blackman RL, Eastop VF (2000) Aphids on the world s crop: an identification and information guide. Wiley, New York, USA Day WH, Tatman KM (2006) Changes in abundance of native and adventive Coccinellidae (Coleoptera) in alfalfa fields, in northern New Jersey ( ) and Delaware ( ), U.S.A. 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