Nutritional Manipulation of Adult Female Orius Pumilio (Hemiptera: Anthocoridae) Enhances Initial Predatory Performance

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BIOLOGICAL AND MICROBIAL CONTROL Nutritional Manipulation of Adult Female Orius pumilio (Hemiptera: Anthocoridae) Enhances Initial Predatory Performance

1,2 3 1 JEFFREY P. SHAPIRO, STUART R. REITZ, AND PAUL D. SHIRK

J. Econ. Entomol. 102(2): 500Ð506 (2009) ABSTRACT Commercial shipments of Orius spp. (Hemiptera: Anthocoridae) commonly include water and protein, the latter typically supplied by eggs from a moth such as Ephestia kuehniella (Zeller). To determine whether modiÞed dietary conditions might improve predation, newly eclosed adult female Orius pumilio (Champion) were fed on E. kuehniella eggs plus encapsulated water, encapsulated 5% sucrose solution only, or encapsulated water only for periods of 24, 48, or 72 h. Feeding activity was assessed by measuring the area of a crop in digital images of dissected digestive tracts. Adult females fed continuously on eggs had larger crops than did females fed on encapsulated sucrose solution. When females were prefed encapsulated water or sucrose and then fed 3 h on eggs, their crops became highly engorged and were larger than those in females fed continuously on eggs for the same periods. In behavioral choice tests, adult females prefed on encapsulated water or 5% sucrose solution spent a larger portion of time in contact with eggs, presumably feeding, whereas females prefed on eggs showed no preference between eggs or encapsulated water. After overnight shipping, females prefed on water or sucrose and held for 48 h total consumed 3.6- and 4.3-fold, respectively, more western ßower thrips, Frankliniella occidentalis (Pergande), in 3 h than those prefed on eggs. Survival rates when prefed on sugar or water were comparable with prefeeding on eggs. Thus, inundative releases of Orius can be enhanced by starvation, because females initially feed much more voraciously after shipment with no apparent reduction in Þtness.

KEY WORDS predatory behavior, digestion, nutrition, thrips, Ephestia kuehniella

When applied to current practices of arthropod pest control agent for the management of increasing pest control, inundation and inoculation biological control populations, and control is achieved primarily through depend on efÞcient rearing of native insect predators the feeding rates of released individuals rather than or parasitoids for release (Zehnder et al. 2007). The their progeny (Eilenberg et al. 2001). Therefore, high release of a biological control agent at the initial plant- rates of prey consumption or mortality are critical for ing of a crop is intended to provide extended efÞcient the success of the inundative treatment. biological control throughout the growing season. State-of-the-art insect rearing requires managed pro- Through an inoculative release, a reproductive pop- cesses, assessed through quality control practices that ulation is established that suppresses pest arthropods ensure speciÞed biological standards (Van Lenteren on the crop (Eilenberg et al. 2001). This approach has 2003). The emphasis is on a high-quality product and not been successfully used in biocontrol for vegetables on a product that meets variable end-user requirements. and ornamentals grown in greenhouses (Eilenberg et However, this should not exclude tailoring selected qual- al. 2000) as well as in Þeld crops (Zehnder et al. 2007). ities to meet speciÞc needs in the Þeld. In particular, The impact of an inoculative release relies on an in- conditions might be selected to maximize prey consump- creasing population rather than on the feeding rates of tion for inundative control, or to maximize fecundity for the released parental generation. Consequently, the inoculative control. Such customization is rarely prac- fecundity of the parental generation is more critical ticed, although many conditions can be controlled in an than its consumption rates. As an alternative, inunda- insectary during rearing, and these conditions can affect tive releases require large numbers of the biological the ultimate characteristics of the biological control agent. Developmental rates, cohort synchrony, repro- The use of trade, Þrm, or corporation names in this publication is duction, predatory behavior, sex ratios, and overall yield for the information and convenience of the reader. Such use does not and quality of the arthropod product can all be affected constitute an ofÞcial endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of (Grenier and De Clercq 2003), and the ideal for each of any product or service to the exclusion of others that may be suitable. these attributes may vary according to the requirements 1 Center for Medical, Agricultural, and Veterinary Entomology, for control of a pest. Additionally, during even a brief USDAÐARS, 1600Ð1700 SW 23rd Dr., Gainesville, FL 32608-1069. shipment period the states of hydration and nutrition, 2 Corresponding author, e-mail: [email protected]. 3 CMAVE, USDAÐARS, Center for Biological Control, Florida response to temperature, and opportunities for canni- A&M University, Tallahassee, FL 32308. balism can affect the Þnal numbers, quality, and perfor- April 2009 SHAPIRO ET AL.: NUTRITION ENHANCES PERFORMANCE OF O. pumilio 501 mance of the product. As a corollary, during both rearing was 13Ð16 d. Adults that had molted from last instar and shipping, control and management of selected con- nymphs within the preceding 24 h were collected for ditions may result in changes in the yields and speciÞc experimentation. qualities of the product. Oviposition. Newly eclosed (Ͻ24 h) adult female O. One critical factor that can be readily managed and pumilio were separated into two treatment groups that has a signiÞcant impact on the quality of the insect with 10 replicate jars for each treatment containing six product is nutrition. With the goals of optimizing num- females and two males per jar. In treatment 1, the bers of insects produced and decreasing the costs of insects were fed with 0.25 g/day 1Ð2-mm-diameter 5% rearing, diet quality and nutrition have been examined sucrose-Hydrocapsules for 72 h and then fed 10 mg of for several predators available internationally from E. kuehniella eggs daily for 6 d. In treatment 2, the commercial suppliers (De Clercq and Degheele 1993, insects were fed 10 mg of E. kuehniella eggs for 6 d. Cohen 2000, Callebaut et al. 2004, Ferkovich and Sha- Water-Hydrocapsules (0.25 g/d) were added daily to piro 2004a, Coudron et al. 2006, SighinolÞ et al. 2008). both treatments during the experimental period. At This laboratory has examined nutritional parameters the conclusion of the 6-d egg-feeding period, the num- affecting the fecundity of adult female Orius ber of survivors in each jar was recorded, and each (Hemiptera: Anthocoridae), as they pertain to the replicate was provided with a fresh green bean for 24 h development of economical artiÞcial diets. Because to facilitate oviposition. The total number of eggs the nutritional value of eggs from the pyralid moths oviposited in each bean was counted at the end of the Ephestia kuehniella (Zeller) and Plodia interpunctella oviposition period. (Hu¨ bner) have proven to provide the greatest value, Alimentary Tract Morphology. Four groups of 14 efforts have focused on isolating speciÞc biochemical adult female O. pumilio (Ͻ24 h after eclosion) were components that confer their nutritional value (Ferk- collected and placed into four 100-ml (6.6-cm height ovich and Shapiro 2004a,b, 2005a,b, 2007). As an ex- by 5.9 cm-diameter) plant tissue culture glass jars tension of that work, the feeding habits of Orius fe- (Sigma, St. Louis, MO) with Ϸ1.5 g of shredded wax males were examined and the ad lib feeding behavior paper as a substrate plus treatments consisting of 1) showed considerable unpredictability for the timing 0.25 g of water-Hydrocapsules, 2) 0.25 g of 5% sucrose- and quantity of feeding (J.P.S. and P.D.S., unpub- Hydrocapsules, or 3) 20 mg of E. kuehniella eggs plus lished). To provide a more controlled physiological water-Hydrocapsules. Jars were covered with rip-stop response to feeding, a period of nutritional deprivation fabric under plastic lids and then placed in incubators after adult eclosion was established, allowing rapid at 25.5 Ϯ 1ЊC and 75 Ϯ 5% RH for 24-, 48-, or 72-h synchronized feeding and engorgement. prefeeding periods. After the prefeeding periods, in- This controlled response to nutritional deprivation sects in two of the jars were fed Ϸ10 mg of E. kuehniella suggested a potential beneÞt through enhanced pre- eggs for 3 h, whereas insects in the other two jars were dation during inundative releases. This work describes not fed. Insects were anesthetized with a CO2 anes- our analyses of predatory behavior after restricted thetizing apparatus (Flystuff.com, San Diego, CA), feeding of adult female Orius pumilio (Champion) and the numbers of surviving insects were counted. with only water or sugar solution, thus depriving them The females were then dissected to isolate their di- of protein and lipid and delaying reproductive devel- gestive systems in a drop of phosphate-buffered saline opment. Our results demonstrate that dietary restric- (0.05 M Na2HPO4 and 0.15 M NaCl, pH 7.2) on a glass tion results in immediate predatory activity upon re- microscope slide treated with Sigmacote (Sigma- lease, with a short-term four-fold increase in predation Aldrich). The digestive tracts were digitally photo- and no impact on reproductive or survival rates. graphed through a Nikon SMZ800 stereomicroscope equipped with a darkÞeld base and set at 63ϫ mag- niÞcation. A Nikon Coolpix 4300 camera was mounted Materials and Methods atop a Coolpix MDC lens on the trinocular camera Rearing. Colonies of O. pumilio were reared in mod- mount and set at full optical zoom. Digital photo- iÞed plastic food storage containers (12 cm height by graphs were analyzed for total gut area and total crop 22 cm width by 32 cm length) Þtted with screened lids area in pixels by using ImageJ software version 1.39 to permit air circulation. Each generation was started (National Institutes of Health, Bethesda, MD). Com- with 3,000Ð5,000 eggs oviposited into green beans over bined midgut-hindgut areas were determined by sub- a 2Ð3-d period (Shapiro and Ferkovich 2006). Sub- tracting the total crop area from total gut area of each colonies were started weekly in 15-cm square by 3.5- photograph. cm-high storage containers from eggs laid into green Prey-Seeking Behavior. Small groups (5Ð10) of beans, Phaseolus vulgaris L., placed in colonies for 24 h. adult female O. pumilio (Ͻ24 h after eclosion) were Colonies and subcolonies were regularly maintained set up as described above and held for 24, 48, or 72 h in incubators at 25.5 Ϯ 1ЊC and 75 Ϯ 5% RH and under each of the three feeding regimes. Two 60-mm provided with 0.25 g/day 1Ð2-mm-diameter water- plastic petri dishes were prepared as video arenas by Hydrocapsules (ARS Inc., Gainesville, FL), E. kueh- Þrst coating the inner walls with Fluon to prevent the niella eggs (BeneÞcial Insectary, Redding, CA; re- escape of insects (Chen and Wei 2007). The arenas ceived frozen on dry ice), and fresh green beans on were placed into a 190- by 100-mm crystallizing dish Mondays, Wednesdays, and Fridays. Under these cul- covered by a Plexiglas sheet with a circular lens open- ture conditions, developmental time from egg to adult ing. PVC pipe (10 cm in length by 2 cm in diameter) 502 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 102, no. 2

Table 1. Choice test for individual adult female O. pumilio during a 3-h videographic session, measuring preferences for E. kuehniella eggs over water-Hydrocapsules

Mean Prefeeding N Durationb Latencyb Total distance Food source Total visitsb velocityb period (h) (N )a (min) (min) movedb (cm) E (mm/s) E. kuehniella 24 4 (0) 27.8 Ϯ 11.3 63.7 Ϯ 28.7 1.8 Ϯ 26.2 47.0 Ϯ 63.5 1.02 Ϯ 0.27 Eggs 48 11 (1) 6.5 Ϯ 6.8 5.6 Ϯ 17.3 45.1 Ϯ 15.8 23.9 Ϯ 38.3 0.64 Ϯ 0.16 72 5 (3) 11.4 Ϯ 10.1 13.8 Ϯ 25.7 9.1 Ϯ 23.4 42.0 Ϯ 56.8 0.60 Ϯ 0.24 All 20 (4) 15.2 Ϯ 5.5 27.7 Ϯ 14.1 18.7 Ϯ 12.8 37.6 Ϯ 31.1 0.75 Ϯ 0.13 Sucrose (5%) 24 5 (0) 5.4 Ϯ 10.1 52.5 Ϯ 25.7 59.8 Ϯ 23.4 21.7 Ϯ 56.8 0.45 Ϯ 0.24 Hydrocapsules 48 9 (0) 8.8 Ϯ 7.5 46.0 Ϯ 19.1 64.8 Ϯ 17.4 66.1 Ϯ 42.3 0.79 Ϯ 0.18 72 6 (2) 22.2 Ϯ 9.2 98.6 Ϯ 23.4 13.1 Ϯ 21.4 39.1 Ϯ 51.8 0.63 Ϯ 0.22 All 20 (2) 12.1 Ϯ 5.2 65.7 Ϯ 13.2 45.9 Ϯ 12.1 42.3 Ϯ 29.3 0.62 Ϯ 0.13 Water 24 3 (3) 4.0 Ϯ 13.0 84.8 Ϯ 33.1 13.7 Ϯ 30.2 45.6 Ϯ 73.3 0.44 Ϯ 0.32 Hydrocapsules 48 10 (0) 33.3 Ϯ 19.0 78.1 Ϯ 18.2 61.4 Ϯ 16.5 100.5 Ϯ 40.1 0.58 Ϯ 0.17 72 7 (1) 18.3 Ϯ 8.5 126.3 Ϯ 21.7 17.8 Ϯ 19.8 25.2 Ϯ 48.0 0.45 Ϯ 0.21 All 20 (4) 18.5 Ϯ 5.7 96.4 Ϯ 14.5 30.9 Ϯ 13.2 57.1 Ϯ 32.1 0.49 Ϯ 0.14

Females were prefed on E. kuehniella eggs, sucrose-Hydrocapsules, or water-Hydrocapsules. a Number of insects that did not enter the egg-raft zone, excluded from analysis. b Data presented as mean Ϯ SEM (unweighted). connected the lens (3.3Ð8-mm focal length, placed 14 cap screened lids. Each container was stocked with cm from subjects) of a black-and-white closed circuit shredded wax paper as a substrate and one of the three video camera to the chamber. The chamber was lit following treatments: 1) 0.25 g of water-Hydrocap- from above by incandescent lighting and from below sules, 2) 0.25 g of 5% sucrose-Hydrocapsules, or 3) by an infrared LED array (Tracksys Ltd., Nottingham, 0.25 g of E. kuehniella eggs plus 0.25 g of water-Hy- United Kingdom). Humidity was maintained at 66 Ϯ drocapsules. These insects were shipped from Gaines- 4% RH by using a cotton wick saturated with NaCl ville to Tallahassee, FL, by overnight mail in a Styro- solution at a temperature of 26 Ϯ 1ЊC. foam shipping box with a cold pack. On receipt, the E. kuehniella egg-rafts were constructed by punch- insects were incubated at 25ЊC in a humid chamber for cutting 3- ϫ7-mm points from cellulose paper, wetting an additional day and the number of surviving adults the point with 2 ␮l of water, and coating them with a was recorded. Each individual female was then placed monolayer of E. kuehniella eggs. A single E. kuehniella into a 60-mm petri dish feeding chamber with Ϸ15 egg-raft was placed in each arena by wetting the petri adult female thrips (Frankliniella occidentalis Per- dish with a 2-␮l drop of water and placing the raft on gande), ensuring that all E. kuehniella eggs and water- top. Four water-Hydrocapsules were placed in a clus- Hydrocapsules were excluded. After a 3-h exposure of ter Ϸ35 mm from the egg-raft in each arena to provide the adult female O. pumilio to prey, survival was again a choice for the insect once it was placed inside the recorded. All feeding chambers were placed into an arena. At various times between 11 a.m. and 4 p.m., two ultralow freezer to kill all insects, and the numbers of adult females from a treatment group were separately consumed and nonpredated thrips were recorded. placed into two arenas and videographed for3hby The entire experiment was repeated three times. using Mediacruise video software (Canopus Corp., Statistics. STATISTICA version 7.1 (StafSoft, Tulsa, San Jose, CA). Behaviors in each arena were then OK), using the general linear model (GLM) for anal- analyzed using EthoVision software (version 3.1, Nol- ysis of variance (ANOVA), was used for statistical dus Information Technology, Leesburg, VA; Noldus et analyses, except where otherwise noted. Variation is al. 2002) to assess preferences for E. kuehniella eggs represented by standard error bars in all graphs. over water-Hydrocapsules. Each of the following parameters is reported (Table 1) as the mean Ϯ SEM for N females, each observed for a 3-h period: 1) Total Results visits, the total number of times that a female entered the circumscribed zone containing the egg-raft; 2) Oviposition. For an assessment of the feeding be- duration, the total time that a female spent within the havior, it was necessary to establish the impact of zone containing the egg-raft; 3) latency, the time be- starvation and minimal nutrition on the reproductive fore the female initially entered the zone; 4) total behavior of adult female O. pumilio. Survival and ovi- distance moved, the total distance that a female moved position of starved or fed adult females were tested within the arena; and 5) mean velocity, the mean of all separately with no signiÞcant differences observed for velocities for each female, each velocity measured the conditions used. When females were fed only 5% over a 0.2-s interval. Females that searched but did not sucrose-Hydrocapsules for 72 h and then fed E. kue- enter the zone containing the egg-raft were excluded hniella eggs for 6 d, 6.1 Ϯ 2.8 eggs per female were laid from these analyses. in a 24-h oviposition period, compared with 6.8 Ϯ 1.9 Predation. Adult females (Ͻ24 h after eclosion) eggs per female when fed E. kuehniella eggs for 6 d were collected, and 14 individuals were placed in each with no starvation period (no signiÞcant difference; of three 50-ml polypropylene containers with screw- t ϭϪ0.0162, df ϭ 18, P Ͼ 0.98). April 2009 SHAPIRO ET AL.: NUTRITION ENHANCES PERFORMANCE OF O. pumilio 503

Fig. 1. Digestive tracts dissected from adult female O. pumilio fed E. kuehniella eggs for 72 h (A), fed sucrose- Hydrocapsules for 72 h (B), or fed sucrose-Hydrocapsules for Fig. 2. Relative areas of digitized images of digestive 72 h followed by E. kuehniella eggs for 3 h (C). C, crop; E, tracts (A, total gut area; B, crop area; C, midgut-hindgut area) esophagus; HG, hindgut; MG, midgut; Ov, ovary. Scale bar ϭ dissected from adult female O. pumilio fed on sucrose-Hy- 0.50 mm. drocapsules or on E. kuehniella eggs for 24, 48, or 72 h (mean Ϯ SEM; bars indicated by the same letters are not signiÞcantly different at ␣ ϭ 0.05). Alimentary Tract Morphology. Microscopic exami- nation of adult females after feeding for 72 h demonstrated that the crop, midgut, and hindgut contained a df ϭ 2, P Ͼ 0.80), but digitized areas of those fed uniform distribution of material regardless of whether sucrose-Hydrocapsules were signiÞcantly lower (F ϭ they fed on E. kuehniella eggs (Fig. 1A) or sucrose- 57.6, df ϭ 1, P Ͻ 0.001) than those fed E. kuehniella Hydrocapsules (Fig. 1B). Females that were prefed for eggs (Fig. 2A). When crop areas were measured sep- 72 h on 5% sucrose-Hydrocapsules and presented with E. arately (Fig. 2B), no signiÞcant change occurred over kuehniella eggs for 3 h had highly distended crops ap- time (F ϭ 0.07, df ϭ 2, P Ͼ 0.90), but differences parently from engorging on the eggs (Fig. 1C). Females between treatments with E. kuehniella eggs and su- prefed with water-Hydrocapsules alone and then fed on crose-Hydrocapsules were signiÞcant (F ϭ 44.9, df ϭ E. kuehniella eggs for 3 h also had distended crops similar 1, P Ͻ 0.001). MidgutÐhindgut areas, derived from to those prefed on sucrose-Hydrocapsules. subtraction of crop area from total area, also showed When females were fed continuously on sucrose- no difference with time (F ϭ 0.069, df ϭ 2, P Ͼ 0.90), Hydrocapsules or E. kuehniella eggs for increasing but clear differences between treatments were ob- times, there were no changes with time in the total served (F ϭ 23.7, df ϭ 1, P Ͻ 0.001) (Fig. 2C). Factorial areas of digestive tracts (measured in pixels; F ϭ 0.212, ANOVA followed by NewmanÐKeuls post hoc tests 504 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 102, no. 2

Fig. 4. Consumption of thrips by, and survival of, adult female O. pumilio prefed with E. kuehniella eggs, water- Hydrocapsules, or sucrose-Hydrocapsules for 48 h followed by feeding on thrips for 3 h (mean Ϯ SEM; bars indicated by the letters of the same case are not signiÞcantly different at ␣ ϭ 0.05; means were identical for survival of those fed eggs).

with time regarding total gut area (Fig. 3A; F ϭ 3.5, df ϭ 2, P Ͻ 0.05) but not in crop area (Fig. 3B; F ϭ 2.1, df ϭ 2, P Ͼ 0.10) or midgutÐhindgut area (Fig. 3C; F ϭ 2.6, df ϭ 2, P Ͼ 0.05). As indicated by factorial ANOVA followed by NewmanÐKeuls, a change with time in total gut area and crop area but not midgutÐhindgut area was due to an increase in areas between 24 and 48 or 72 h only in those adult females prefed on sucrose-Hydrocapsules (Fig. 3). Prey-Seeking Behavior. In 3-h choice tests between E. kuehniella egg-rafts and water-Hydrocapsules, adult females that were prefed for three different periods on eggs, sucrose-Hydrocapsules, or water-Hydrocapsules showed highly variable behavior. Depending on the food source, only one of Þve dependent parameters measured, the duration of contact of adult females with E. kuehniella egg-rafts, was signiÞcantly affected (Table 1, duration). When averaged over the three prefeeding periods, duration increased for females that were prefed on water-Hydrocapsules (P Ͻ 0.001; Newman-Keuls) or sucrose-Hydrocapsules (P Ͻ 0.02) compared with those Fig. 3. Relative areas of gut proÞles (A, total gut area; B, prefed on E. kuehniella eggs (GLM ANOVA; F ϭ 2.04, crop area; C, midgut-hindgut area) dissected from adult fe- df ϭ 10, P Ͻ 0.05). Change in prefeeding period did not male O. pumilio prefed on E. kuehniella eggs, sucrose-Hy- signiÞcantly affect duration. drocapsules, or water for 24, 48, or 72 h and then fed on E. kuehniella eggs for 3 h (mean Ϯ SEM; bars indicated by the Predation. Predation of female O. pumilio on west- same letters are not signiÞcantly different at ␣ ϭ 0.05, except ern ßower thrips signiÞcantly increased with nutri- C, where there was no signiÞcance among treatments). tional deprivation for adult females fed 48 h on sucrose-Hydrocapsules or water-Hydrocapsules (Fig. 4). After the 48-h shipping and holding period, adult showed consistently lower total gut, crop, and midgutÐ females prefed on sucrose-Hydrocapsules or water- hindgut areas in adult females fed sucrose-Hydrocap- Hydrocapsules consumed 2.78 Ϯ 0.02 or 2.30 Ϯ 0.46 sules as opposed to adult females fed E. kuehniella eggs thrips per female, respectively, whereas those prefed at each feeding time, except for crop area at 48 h (Fig. 2). on E. kuehniella eggs consumed 0.64 Ϯ 0.13 thrips per If adult females were treated by prefeeding on E. female. Differences were signiÞcant (F ϭ 32; df ϭ 2; kuehniella eggs, encapsulated sucrose, or encapsulated P Ͻ 0.0001), and sucrose-Hydrocapsule and water- water for 24, 48, or 72 h and then fed eggs for 3 h, Hydrocapsule treatments differed from the egg treat- signiÞcant differences resulted from the effect of ment (P Ͻ 0.0005; Newman-Keuls) but not from each treatment on total gut area (Fig. 3A; F ϭ 25.4, df ϭ 2, other (P Ͼ 0.2). Thus, the consumption rate for fe- P Ͻ 0.001) and crop area (Fig. 3B; F ϭ 36.4, df ϭ 2, P Ͻ males starved on sucrose-Hydrocapsules or water-Hy- 0.001), but not on midgutÐhindgut area (Fig. 3C; F ϭ drocapsules was 4.3- and 3.6-fold greater, respectively, 0.35, df ϭ 2, P Ͼ 0.70). SigniÞcant change occurred than the rate for females prefed on E. kuehniella eggs. April 2009 SHAPIRO ET AL.: NUTRITION ENHANCES PERFORMANCE OF O. pumilio 505

Survival. Overall, there were no statistically signif- prefeeding adult females on sucrose-Hydrocapsules re- icant effects of the type or timing of the feeding resulted in small gut areas at 24 h, which increased at 48 and gimes on the percentage of survival of insects. For the 72 h to areas equal to those of females prefed on water- oviposition assay, 93.3 Ϯ 10% of insects prefed sucrose- Hydrocapsules, again with crop engorgement providing Hydrocapsules for 72 h survived, whereas 86.7 Ϯ 7.4% the primary differentiating parameter. of insects fed continuously with E. kuehniella eggs That starved females had an increased feeding rate survived. For those insects used in the alimentary tract was supported by videographic analyses of adult female morphology assessment, the percentage of survival behavior. Adult females prefed on sucrose-Hydrocap- ranged from a low of 84% for those fed sucrose-Hy- sules or on water-Hydrocapsules spent a greater amount drocapsules or water-Hydrocapsules for 72 h to a high of time proximate to eggs, which we interpreted as feed- of 100% for insects fed E. kuehniella eggs for 24 h (data ing, than adult females prefed on E. kuehniella eggs. not shown). This also was observed for those insects SigniÞcant changes did not occur with increasing subjected to shipping whether analyzed as one large prefeeding periods up to 3 d, only with prefeeding di- trial or as three individual trials. The survival after a etary treatments of water-Hydrocapsules or sucrose-Hy- 48-h period of shipment and holding (Fig. 4) was not drocapsules, depriving the adult females of proteins and signiÞcantly different for females fed on E. kuehniella lipids. High variability in other behavioral parameters eggs (85.7 Ϯ 0%; N ϭ 36) than for those fed either on indicated a need for larger sample sizes. sucrose-Hydrocapsules (92.8 Ϯ 7.1%; N ϭ 39) or wa- In previous studies, the prey-searching patterns of ter-Hydrocapsules (88.1 Ϯ 8.2%; N ϭ 37) (means Ϯ Orius majusculus (Reuter) were affected by short SD; F ϭ 0.55; df ϭ 2, 4; P Ͼ 0.60). periods of starvation (Henaut et al. 2002). After a 6-h feeding period following eclosion, the adults were starved for varying periods from2hupto12handthen Discussion examined using videography. The shorter periods of Adult female O. pumilio tolerated short-term starva- starvation had greater impact on the search patterns tion without apparent ill effect and subsequently were and led to decreased walking speeds, an increased voracious when presented with prey. Starved adult fe- number of stops and a reduced diffusion rate from the male O. pumilio consumed 3 to 4 times the number of origin. However, all of these effects had disappeared thrips as well-fed adult females. Starvation by feeding on in those starved for 12 h. The source of the nutrients water-Hydrocapsules or sucrose-Hydrocapsules over a is critical as well. O. insidiosus exhibits an ovipositional 72-h period did not signiÞcantly affect survival nor did it preference for beans, which leads to greater survival reduce the number of eggs produced upon refeeding. rates for the nymphs and suggests that omnivory is a These results strongly suggest that suppliers should be critical component during the earlier stages of devel- able to maximize the initial impact of an inundative opment for these predators (Coll 1996). In addition, release of Orius spp. by manipulating their food supply water acquisition from plant feeding was shown to be before and during shipment. necessary for successful feeding by the predatory To assess feeding rates of adult females, the degree of mirid Dicyphus hesperus Knight (Gillespie and gut engorgement after a 3-h exposure to E. kuehniella McGregor 2000) and also may be necessary for O. eggs was semiquantiÞed by calculating the relative areas pumilio. As observed in this study, most of the behav- of digestive tracts in digital images of guts dissected from ioral activities were highly variable for those females adult females. We presumed that the amount of food in subjected to starvation on water or sucrose (Table 1). the gut would reßect active feeding. Obviously, this The increase in predation when starved of protein is method resulted in an inherent underestimate of the likely to be linked with reproduction. We have observed differences because area increases as a squared function, in both O. pumilio (J.P.S. and S. M. Ferkovich, unpub- whereas volume increases as a cubic function and the gut lished) and Podisus maculiventris (Say) (Shapiro et al. is a three-dimensional structure. Although volumetric 2000) that ovarian development occurs only with inges- measurements would yield more accurate and represen- tion of protein in the adult stage, corresponding to similar tative values, area measurements did not require expen- requirements for blood feeders such as anautogenous sive imaging and analysis systems and yet provided a mosquitoes (Uchida et al. 2001, Hernandez-Martinez et reliable value for assessment. al. 2007) or some omnivores such as house ßies (Browne As controls to establish fed and nonfed gut morphol- 2001). It is likely that predators such as O. pumilio will ogy, the guts of adult females fed strictly on sucrose- demonstrate reduced prey-seeking behavior and pred- Hydrocapsules or on E. kuehniella eggs were examined. atory efÞciency while developing eggs, in favor of in- Adult females fed on sucrose-Hydrocapsules had small creased seeking of oviposition sites. Our behavioral study gut areas, whereas those fed on E. kuehniella eggs had reßected this, because the time spent with eggs de- signiÞcantly larger gut areas, although the crop did not creased after 48 and 72 h of feeding on eggs. Ovarian show any distention. When prefed on water-Hydrocap- development is nearly complete at 72 h (J.P.S. and S. M. sules regardless of the length of time, feeding on E. Ferkovich, unpublished). kuehniella eggs resulted in food accumulating in the crop Predation rates from insects that were shipped and with no signiÞcant changes in the midgutÐhindgut area. held for 48 h substantiated the morphological and Although the total area of the digestive system was in- behavioral results: Females prefed and shipped with creased overall, the data indicated that the crop area was sucrose-Hydrocapsules or water-Hydrocapsules ate the critical differentiating measurement. Similarly, Ͼ3 times the number of thrips than those prefed and 506 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 102, no. 2 shipped with E. kuehniella eggs. Together, these Þnd- egg-laying of Orius insidiosus maintained on artiÞcial diet ings demonstrated that at least the short-term feeding as adults” [Biol. Control 31 (2004) 57Ð64]. Biol. Control performance of O. pumilio may be enhanced by re- 32: 180. stricting their diet to only carbohydrate solution or Ferkovich, S. M., and J. P. Shapiro. 2005b. Erratum to “In- water immediately after adult eclosion. This treatment creased egg-laying in Orius insidiosus (Hemiptera: An- thocoridae) fed artiÞcial diet supplemented with an em- has no negative impact on either survival or oviposi- bryonic cell line” [Biol. Control 31 (2004) 11Ð15]. Biol. tion rates. The efÞcacy of these procedures remains to Control 32: 181. be validated in greenhouse or Þeld-cage assays. Ferkovich, S. M., and J. P. Shapiro. 2007. Improved fecundity in the predator Orius insidiosus (Hemiptera: Antho- coridae) with a partially puriÞed nutritional factor from Acknowledgments an insect cell line. Fla. Entomol. 90: 321Ð326. We thank Jean M. G. Thomas for excellent technical as- Gillespie, D. R., and R. R. McGregor. 2000. The functions of sistance and manuscript review, Lyndall Brezina for assis- plant feeding in the omnivorous predator Dicyphus hes- tance in digital analyses and rearing, and Terry Arbogast for perus: water places limits on predation. Ecol. Entomol. 25: consultation on behavioral studies and manuscript review. 380Ð386. Thanks also to David Carlson and Stephen Ferkovich for Grenier, S., and P. De Clercq. 2003. Comparison of artiÞ- manuscript reviews. cially vs. naturally reared natural enemies and their potential for use in biological control, pp. 115Ð131. In J. C. Van Lenteren [ed.], Quality control and production of References Cited biological control agents: theory and testing procedures. CAB International, Cambridge, MA. Browne, L. B. 2001. Quantitative aspects of the regulation of Henaut, Y., C. Alauzet, and M. Lambin. 2002. Effects of ovarian development in selected anautogenous Diptera: starvation on the search path characteristics of Orius integration of endocrinology and nutrition. Entomol. majusculus (Reuter) (Het., Anthocoridae). J. Appl. En- Exp. Appl. 100: 137Ð149. tomol. 126: 501Ð503. Callebaut, B., E. Van Baal, B. Vandekerkhove, K. Bolckmans, Hernandez-Martinez, S., J. G. Mayoral, Y. Li, and F. G. and P. 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