Sugar concentration and timing of feeding affect feeding characteristics and survival of a parasitic wasp

Journal of Insect Physiology 79 (2015) 10–18

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Sugar concentration and timing of feeding affect feeding characteristics and survival of a parasitic wasp Livy Williams III a,⇑, Pauline Deschodt b, Olivia Pointurier b, Kris A.G. Wyckhuys c a

USDA-ARS European Biological Control Laboratory, Montferrier sur Lez, France Montpellier SupAgro, Montpellier, France c CGIAR-CIAT, Hanoi, Viet Nam b

a r t i c l e

i n f o

Article history: Received 17 February 2015 Received in revised form 18 May 2015 Accepted 20 May 2015 Available online 27 May 2015 Keywords: Nutritional ecology Carbohydrate subsidy Survival analysis Feeding behavior Stretch receptors Psyttalia lounsburyi Parasitoid Bactrocera oleae

a b s t r a c t The availability of food sources is important for parasitoid survival, especially for those that inhabit ecosystems where nectar and honeydew are spatially or temporally scarce. Therefore, the value of even a single meal can be crucial for survival. Psyttalia lounsburyi is a parasitoid, and biological control agent, of the olive fruit fly, Bactrocera oleae. In order to improve our understanding of the basic nutritional ecology of P. lounsburyi and its role in survival we evaluated the effect of a single sucrose meal on the longevity of female and male wasps. We measured the duration of feeding, volume ingested, sucrose consumption, energy content, and longevity of wasps provided with different concentrations of sucrose (0.5, 1, and 2 M) at different times after emergence (0, 1, 2 or 3 days after emergence). Our results showed that longevity was significantly influenced by sucrose concentration and timing of feeding. For females, feeding on sucrose increased the likelihood of survival to varying degrees, ranging from 32.3% to 95.4%, compared to water-only controls. The longest duration of feeding was observed for the highest sucrose concentrations and oldest wasps. The amount of sugar ingested and energy uptake increased, up to a point, as sugar concentration increased. Our results suggest that P. lounsburyi derived greatest benefit from the intermediate concentration (1 M) of sucrose provided 2 or 3 days after emergence. Our study emphasizes the importance of finding balance between increasing longevity and limiting the duration of feeding, and concomitant uptake of nutrients, that is fundamental for survival of the wasp in nature. Published by Elsevier Ltd.

1. Introduction Many species of parasitic wasps require carbohydrate food sources to satisfy metabolic energy needs. Floral and extrafloral nectar, and honeydew excreted by homopteran insects, provide sugar-foraging wasps with rich sources of carbohydrates which generally increase longevity and subsequent rates of parasitism (Géneau et al., 2013; Irvin et al., 2007; Jamont et al., 2014; Lee et al., 2004; Lee and Heimpel, 2008a; Sivinski et al., 2006; Winkler et al., 2006; Wyckhuys et al., 2008). Theoretical and empirical evidence suggests that parasitoid food sources play an important role in regulation of host population dynamics (Heimpel and Jervis, 2005; Jervis et al., 1996; Sabelis et al., 2005). Consequently, the presence of suitable carbohydrates for foraging parasitoids is an important factor in the development of habitat

⇑ Corresponding author at: USDA-ARS European Biological Control Laboratory, Campus International de Baillarguet, CS 90013 Montferrier-sur-Lez, 34988 St. Gely du Fesc Cedex, France. E-mail address: [email protected] (L. Williams III). http://dx.doi.org/10.1016/j.jinsphys.2015.05.004 0022-1910/Published by Elsevier Ltd.

management strategies aimed at enhancing the effectiveness of biological control agents against agricultural pests (Heimpel and Jervis, 2005; Landis et al., 2000; Orre Gordon et al., 2012). Successful sugar foraging depends on the availability of suitable nectar or honeydew at the time of foraging. However, considerable variability exists in the spatial and temporal availability of food sources, especially in manipulated environments, such as agricultural systems. Despite this, most studies evaluating food resources of parasitoids have permitted feeding ad libitum; little work has been done where feeding frequencies are limited. Most studies that have examined feeding frequency suggest that longevity is optimized when sugar-feeding occurs daily (Azzouz et al., 2004; Fadamiro et al., 2005; Lee and Heimpel, 2008b; Siekmann et al., 2001; but see Dyer and Landis, 1996; Fadamiro and Heimpel, 2001; Krugner et al., 2005). Sugar concentration and composition in nectar and honeydew can also vary greatly within and between individual plants and over time (Baker and Baker, 1983a,b; Byrne et al., 2003; Heil, 2011). The viscosity of a sugar solution generally increases as the concentration increases, (Chirife and Buera, 1997; Kingsolver and Daniel, 1995; Nithiyanantham and Palaniappan,

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2013); in turn, increased viscosity usually slows a parasitoid’s rate of feeding, i.e., the rate of sugar uptake (Siekmann et al., 2001; Wyckhuys et al., 2008). Also, increased time spent feeding or resting afterwards may put a parasitoid at greater risk of attack by natural enemies (Lightle et al., 2010; Völkl and Kroupa, 1997; Wäckers, 2005), and may decrease the time spent searching for and attacking hosts. Thus, the concentration-viscosity relationship represents complicated tradeoffs that will influence a parasitoid’s rate of nutrient uptake and survival, as well as vulnerability to natural enemies and reproductive success. Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae) is an African parasitoid of the olive fruit fly, Bactrocera oleae Gmelin (Diptera: Tephritidae) (Copeland et al., 2004; Wharton and Gilstrap, 1983) that has been imported into California as part of a classical biological control program (Daane et al., 2008). P. lounsburyi is synovigenic, and adults do not host feed. Little is known about the nutritional ecology of this parasitoid. Although observations of adult P. lounsburyi feeding in the field are lacking, wasps apparently rely on food sources such as nectar and honeydew. However, commercial olive orchards are characterized by a paucity of food resources for P. lounsburyi, and thus opportunities for successful foraging are at a premium. Given the low likelihood of food encounters in an olive orchard, the benefit to P. lounsburyi from even a single meal may be crucial for survival. Our study aims at evaluating the effect of a single sucrose meal on the longevity of P. lounsburyi under laboratory conditions. Since wasps can encounter varying concentrations of sugar in the field, meals were offered at three concentrations representative of those found in nature. Also, we tested the temporal effects of sugar meals by provisioning the wasps on different days after emergence. We predicted that optimal beneficial effects of sugar-feeding would occur at a sucrose concentration that permitted maximum rate of energy uptake, and at a wasp age >1 day post-emergence (after depletion of energy reserves accumulated during the larval period). 2. Materials and methods 2.1. Insect rearing The stock of P. lounsburyi used in this study originated from B. oleae on wild olive, Olea europaea L. subsp. cuspidata (Wall. ex G. Don), in the Burguret Forest, Kenya in 2005. Since then, the parasitoids have been maintained as a laboratory colony in the quarantine facility of the USDA-ARS European Biological Control Laboratory, Montferrier-sur-Lez, France. After the initial collection in Africa, parasitoids were reared for a few months on B. oleae in olives, but in May 2005 the parasitoids were reared continuously (90 generations) using a factitious host, Ceratitis capitata (Diptera: Tephritidae) on artificial diet (Wong and Ramadan, 1992) (23 °C ± 1, 45% r.h., L:D 16:8). This colony of P. lounsburyi is infected with two variants of Wolbachia endosymbionts (Cheyppe-Buchmann et al., 2011). After emergence from rearing chambers, male and female parasitoids were placed together (ca. 30 total) to allow mating in a 1-l plastic food container (No. DM32, SOLO Cups Co., Urbana, IL) containing a cotton wick soaked with distilled water. When only females emerged, older males were added to the cage. The food container was covered with women’s hosiery (L’eggs Knee Highs, Sara Lee Hosiery, Rural Hall, NC) to allow ventilation. The parasitoids were provided with only distilled water until experimentation. 2.2. Experimental procedure Three concentrations of sucrose (Fluka Analytical, Inc., catalog No. 84097, >99.5%) solutions were used as food treatments in the

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experiments: 0.5, 1 and 2 M (17.1, 34.2, and 68.5% w/w, respectively). Sucrose is one of the major components of nectar and honeydew (Baker and Baker, 1983a,b; Byrne et al., 2003). Test solutions were prepared in 20 ml glass scintillation vials and were stored at 4 °C. Fresh test solutions were prepared weekly. The study was conducted with female and male wasps. Parasitoids were fed with sucrose only once after emergence, either on the day of emergence (day 0) or 1, 2, or 3 days after emergence (after 4 days without food most wasps would have starved to death). Thus, days 0, 1, 2, and 3 are days of feeding, hereafter referred to as ‘wasp age’. For each wasp age, wasps (ca. 20 females; ca. 17 males) were assigned to each food treatment, i.e., to one of the three sucrose concentrations and to distilled water only controls. For each assay, a known volume, ranging from 2 to 10 ll of sucrose solution (warmed to 23 °C) was pipetted into a 45 ml plastic vial (12 dram, No. 55-12, Thornton Plastics Co., Salt Lake City, UT). A wasp was aspirated into the vial, after which the vial was capped, and wasp behavior was observed at 20 under a dissecting scope. Each wasp was allowed 3 min to explore the vial and encounter the sugar droplet; after this time a wasp that had not encountered the droplet was discarded and a new trial was begun. Feeding was observed as uninterrupted ingestion, i.e., movement of the mouthparts on the droplet and distention of the abdomen. Wasps were allowed to feed uninterrupted until satiated. Termination of feeding was defined as withdrawing the mouthparts from the droplet, followed by walking away from the droplet and/or grooming. Sometimes wasps fed in several bouts; in this case a wasp would withdraw its mouthparts but remain oriented toward the droplet for several seconds before resuming ingestion. For each trial, we recorded the total duration of feeding (measured to the nearest second with a lab timer). After a trial was terminated, the wasp was gently aspirated from the vial, and was transferred alone into a ventilated 1-l plastic food container as described above, where it was provided with distilled water-soaked cotton and held in a climate-controlled room (23 °C ± 1, 45% r.h., L:D 16:8) until death. Water was added to the cotton twice a day (0900–1000 h and 1700–1800 h) when wasp mortality was also recorded. Wasps not exhibiting repetitive (non-reflex) movement were considered dead. The volume of sugar solution ingested was determined for ca. 70 of the 2 day old female wasps by measuring the amount of solution which remained in the vial after cessation of feeding. This was accomplished by using a micropipet (10- or 20-ll) to recover the remaining solution not ingested by the wasp. The length of the solution column was measured under the dissecting scope using a graduated reticle. This number was then divided by the length of the micropipet from the tip to the calibration mark and multiplied by the capacity of the micropipet to give volume (ll) sugar solution collected. The difference between the volumes of sugar solution at the beginning and end of each trial represented the volume ingested by the wasp. A clean, dry micropipet was used for each trial. From these data, the amount of sucrose consumption (mg sucrose = molarity  ll ingested  molecular weight  0.001) and energy content (Joule (J) ingested = 16.8 J  mg sucrose ingested) were calculated for each wasp. Value for energy content (16.8 J/mg sucrose) reported by Dafni (1992). 2.3. Data analysis Survival data were analyzed by the Cox Proportional Hazards Analysis, where sucrose concentration and wasp age were tested as covariates (i.e., explanatory variables) (SAS Institute, 2003). After the initial analysis establishing differences for gender, wasp age and sucrose concentration, survival analyses were conducted for each gender  wasp age combination. Differences in survival curves for each day were analyzed by likelihood ratio tests. After this,

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L. Williams III et al. / Journal of Insect Physiology 79 (2015) 10–18

Fig. 1. Cox Proportional Hazards Analysis of female P. lounsburyi longevity after a single meal on one of three different sucrose concentrations (0.5, 1 or 2 M) or a water-only control at one of 4 days after emergence (A) day 0, (B) day 1, (C) day 2, and (D) day 3. The ‘‘0’’ on the x-axis indicates the day of emergence and the arrow indicates the day of feeding. Different letters next to concentrations in the legend indicate significant differences in survival.

we estimated the effect of sucrose concentration on P. lounsburyi survival for each day (i.e., wasp age) (Cox and Oakes, 2001; Spruance et al., 2004). The risk ratio (sometimes referred to as the hazard ratio) is the quantitative effect of a variable on survival, and it characterizes the risk of death between two treatment groups, for example, between two sugar concentrations or a sugar diet vs a control, to characterize the relative risk of starving after feeding. In the present study it was calculated to characterize the relative effect of the different feeding treatments on survival of P. lounsburyi. A risk ratio of 1 indicates that the sucrose concentration has no effect on survival. A risk ratio <1 indicates a lower likelihood of death, while a risk ratio >1 shows a higher probability of death. The effect of sugar concentration and wasp age on feeding duration, volume ingested, sucrose consumption, energy content, and rates of ingestion, sucrose consumption, and energy uptake were analyzed by analysis of variance (SAS Institute, 2003). Since wasps were water-satiated before the trials, they did not ingest water offered in the control treatment; therefore, data related to feeding behavior were only analyzed for the sugar treatments. Data were log-transformed (X0 = log(X + 1)) prior to analysis, and a planned

mean comparison was conducted using Tukey’s HSD (Zar, 1996). Data for regression of survival versus sucrose consumption were untransformed prior to analysis. For all data sets untransformed values are presented in the results. 3. Results 3.1. Influence of sucrose concentration and wasp age on P. lounsburyi survival Analysis of survival data for gender effects over all treatment combinations showed that female longevity was significantly greater than male longevity (Likelihood ratio test, n = 594, v2 = 21.23, P < 0.0001); therefore, further analyses were conducted on separate genders. For both genders, there was an effect on longevity for sucrose concentration (Likelihood ratio tests: Female; n = 321, v2 = 92.00, P < 0.0001: Male; n = 273, v2 = 62.91, P < 0.0001), and wasp age (Likelihood ratio tests: Female; n = 321, v2 = 90.32, P < 0.0001: Male; n = 273, v2 = 23.35, P < 0.0001). Generally, a single sucrose meal improved the survival of wasps.

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Fig. 2. Cox Proportional Hazards Analysis of male P. lounsburyi longevity after a single meal on one of three different sucrose concentrations (0.5, 1 or 2 M) or a water-only control at one of 4 days after emergence (A) day 0, (B) day 1, (C) day 2, and (D) day 3. The ‘‘0’’ on the x-axis indicates the day of emergence and the arrow indicates the day of feeding. Different letters next to concentrations in the legend indicate significant differences in survival.

Indeed, the median longevity, i.e., the day after emergence at which 50% of the individuals are dead, was 4 or 5 days, depending on age of wasps, for the controls (Figs. 1 and 2). On day 6 and 7 all controls were dead for females and males, respectively. However, sucrose-provisioned wasps lived up to 9 days after feeding (DAF) (Figs. 1 and 2). For female wasps at age 0 there was a significant reduction in the risk of starving to death for each sucrose concentration, except for 0.5 M vs 1 M (P = 0.7682) (Fig. 1A; Table 1). The other days of wasp age show similar trends as observed at wasp age 0. For male wasps the reduction in risk of starvation was most consistent for sucrose concentrations 0 vs 1 M, 0 vs 2 M, and 0.5 M vs 2 M (Fig. 2; Table 2). For both genders, an increase in survival was generally observed when the concentration increased, beginning 2 or 3 DAF (Figs. 1 and 2). However, for female wasps there was no significant difference for 1 M vs 2 M at wasp age 1 (P = 0.2234) or 2 (P = 0.0611) (Fig. 1A and B; Table 1). For females all sucrose concentrations were significantly different at wasp age 3 (Fig. 1). The effect of sucrose concentration was compared for each concentration pair (e.g., water control vs 2 M) for each wasp age. Characterizing the risk ratio was only relevant for concentration

pairs with a significant difference. For each of those pairs, the risk ratio was lower than 1, indicating that an increase of the sugar concentration enhances the survival of P. lounsburyi (Tables 1 and 2). For females, maximum lifespan was extended from 8 DAF (8 days total), when the single meal occurred at the day of emergence, compared to 9 DAF (12 days total) when wasps were fed 3 days after emergence. For females, treatment pairs with the smallest concentration differences usually exhibited lower risk ratios, i.e., water controls vs 0.5 M, 0.5 M vs 1 M, and water controls vs 1 M (Table 1), although this effect was not as apparent for males (Table 2). For example, the lowest risk ratio for female wasps, 0.046, was found for water control vs 1 M at wasp age 2. That is, compared to controls, feeding a 2 day old female a single meal of 1 M sucrose reduces its risk of starving to death by 95.4% (=1– 0.046  100). Conversely, risk ratios for both genders were generally higher for the largest concentration differences, i.e., water controls vs 2 M, and 0.5 M vs 2 M (Tables 1 and 2). The highest risk ratio, 0.677, was observed for females at age 0 for 0.5 M vs 2 M sucrose. In this case, a female provisioned with a 2 M sucrose meal immediately after emergence reduces its risk of starving to death by 32.3% compared to a wasp fed 0.5 M sucrose.

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Table 1 Cox Proportional Hazards Analysis for sucrose concentration and wasp age. Each sucrose concentration is an explanatory variable on the longevity of female P. lounsburyi. Risk ratios were calculated for every possible pair of feeding treatments for each wasp age; values for each treatment pair indicate the risk of starving to death after feeding on the higher concentration diet vs the lower concentration diet. Risk ratios <1 indicate a lower risk, and ratios >1 indicate a higher risk of starvation. Sucrose concentration (M) 0 vs 0.5

0 vs 1

0 vs 2

0.5 vs 1

0.5 vs 2

1 vs 2

Day 0 Risk ratio 95% CI P

0.217 0.072–0.651 0.0064

0.417 0.252–0.689 0.0006

0.435 0.313–0.603 <0.0001

1.172 0.408–3.362 0.7682

0.677 0.460–0.995 0.0474

0.417 0.235–0.740 0.0028

Day 1 Risk ratio 95% CI P

0.192 0.059–0.629 0.0064

0.116 0.042–0.321 <0.0001

0.519 0.358–0.753 0.0005

0.058 0.011–0.313 0.0009

0.583 0.364–0.931 0.0240

1.541 0.768–3.089 0.2234

Day 2 Risk ratio 95% CI P

0.053 0.012–0.223 <0.0001

0.046 0.013–0.169 <0.0001

0.357 0.218–0.585 <0.0001

0.054 0.013–0.232 <0.0001

0.358 0.197–0.647 0.0007

0.521 0.263–1.031 0.0611

Day 3 Risk ratio 95% CI P

0.012 0.001–0.184 0.0016

0.060 0.012–0.299 0.0006

0.309 0.156–0.614 0.0008

0.145 0.023–0.904 0.0386

0.333 0.157–0.706 0.0041

0.312 0.123–0.789 0.0139

Table 2 Cox Proportional Hazards Analysis for sucrose concentration and wasp age. Each sucrose concentration is an explanatory variable on the longevity of male P. lounsburyi. Risk ratios were calculated for every possible pair of feeding treatments for each wasp age; values for each treatment pair indicate the risk of starving to death after feeding on the higher concentration diet vs the lower concentration diet. Risk ratios <1 indicate a lower risk, and ratios >1 indicate a higher risk of starvation. Sucrose concentration (M) 0 vs 0.5

0 vs 1

0 vs 2

0.5 vs 1

0.5 vs 2

1 vs 2

Day 0 Risk ratio 95% CI P

0.506 0.166–1.538 0.2296

0.415 0.233–0.738 0.0028

0.637 0.481–0.843 0.0016

0.296 0.097–0.906 0.0328

0.650 0.452–0.933 0.0196

1.007 0.594–1.708 0.9779

Day 1 Risk ratio 95% CI P

0.537 0.159–1.809 0.3159

0.438 0.219–0.874 0.0192

0.325 0.208–0.507 <0.0001

0.349 0.098–1.240 0.1035

0.246 0.141–0.427 <0.0001

0.258 0.127–0.526 0.0002

Day 2 Risk ratio 95% CI P

0.057 0.009–0.372 0.0027

0.241 0.092–0.646 0.0045

0.184 0.064–0.526 0.0016

0.613 0.142–2.651 0.5128

0.501 0.283–0.887 0.0177

0.465 0.197–1.098 0.0807

Day 3 Risk ratio 95% CI P

0.327 0.032–3.296 0.3432

0.100 0.021–0.475 0.0038

0.430 0.203–0.910 0.0273

0.019 0.001–0.323 0.0061

0.364 0.142–0.932 0.0350

0.593 0.191–1.843 0.3667

3.2. Influence of sucrose concentration and wasp age on feeding behavior, energetics and survival of P. lounsburyi females Two-factor analysis of variance revealed significant differences for feeding duration for both main effects (sucrose concentration: F2,218 = 10.59, P < 0.0001; wasp age: F3,218 = 79.12, P < 0.0001). Feeding duration for the 2 M sucrose concentration was longer than for the lower concentrations (Fig. 3). Feeding duration did not differ between the two lower concentrations. Feeding duration was longest for wasp ages 2 and 3, while wasp age 1 was intermediate, and wasps at the day of emergence (day 0) exhibited the shortest feeding times. Depending on sucrose concentration, feeding duration was 2–4 longer for the 3 day wasp age compared to day 0. For wasps age 2, analysis of variance of sucrose concentration showed significant differences for sucrose consumption (F2,58 = 39.52, P < 0.0001), and energy content (F2,58 = 47.88, P < 0.0001), but not for volume of solution ingested (P > 0.05).

Sucrose consumption and energy content both increased as sucrose concentration increased, with values >3 higher at 2 M than at 0.5 M, and intermediate values at 1 M (Fig. 4). Regression analysis indicated a positive linear relationship between survival (y) and sucrose consumption (x) for 1 M and 2 M concentrations (Fig. 5), but not for 0.5 M sucrose (P > 0.05). Volume of solution ingested showed a non-significant decreasing trend as sucrose concentration increased (F2,58 = 1.02, P = 0.3671) (mean ll ± SE: 0.5 M = 0.757 ± 0.045, 1 M = 0.706 ± 0.050, 2 M = 0.661 ± 0.064). Analysis of sucrose concentration revealed significant differences for rate of sucrose consumption, rate of energy content uptake, and rate of volume ingestion. Rate of sucrose consumption at 1 M was significantly greater than at 0.5 M; the differences between 1 M and 2 M, and between 2 M and 0.5 M were not significant (F2,58 = 5.04, P < 0.0096) (Fig. 6). Rate of energy content uptake was greatest at 1 M, intermediate at 2 M, and was lowest at 0.5 M, with all differences being significant (F2,58 = 13.95, P < 0.0001) (Fig. 6). The rate of volume of solution ingested was >2 slower at

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understory that might contribute nectar or support honeydew-producing insects. Olive trees support insects that produce honeydew (i.e., black scale, Saissetia oleae, and olive psyllid, Euphyllura olivina); however, these insects are usually only present for a few months and often at low densities. Olive pollen is widespread throughout orchards, but is only available for a few weeks of the year, and little is known about its role as a food source for P. lounsburyi. Thus, encounters with food resources are infrequent and unpredictable events for P. lounsburyi wasps. Given the low likelihood of food encounters in an olive orchard, the benefits of even a single meal may be crucial for survival. Our results demonstrated that the timing of the first meal (i.e., age of the wasp at the time of the meal), the sugar concentration, and wasp gender all affect longevity of P. lounsburyi. The concentration of sucrose solution was an explanatory variable of wasp survival; increasing the sucrose concentration

Fig. 3. Duration of feeding (mean minutes + s.e.m.) by P. lounsburyi female wasps for a single meal of sucrose. Three different sucrose concentrations (0.5, 1, and 2 M) were provided at four times after emergence (days 0, 1, 2, and 3). For each sucrose concentration, different letters above bars indicate statistical differences (Tukey’s HSD).

Fig. 4. Sucrose consumption (mean mg + s.e.m., gray bars) and energy content (mean Joule + s.e.m., white bars) for P. lounsburyi female wasps for a single meal of sucrose. Three different sucrose concentrations (0.5, 1, and 2 M) were provided 2 days after emergence. For each dependent variable, different letters above bars indicate statistical differences (Tukey’s HSD).

2 M than at 0.5 M or 1 M, with no difference between the two latter concentrations (F2,58 = 46.66, P < 0.0001); mean ll/min ± SE: 0.5 M = 0.129 ± 0.008, 1 M = 0.117 ± 0.006, 2 M = 0.049 ± 0.004).

4. Discussion Parasitoids of the olive fruit fly that inhabit the commercial olive agroecosystem are confronted by a spatial and temporal disjunction between food sources and host patches that makes survival challenging. Host patches consist of olive fruit infested with B. oleae larvae; they are spatially widespread in orchards but are temporally constrained to ca. 4–5 months of the year. However, food resources, such as nectar, honeydew, and pollen, are in more limited supply. Olive flowers produce little, if any, nectar, and cultivation practices eliminate weeds and other plants in the

Fig. 5. Relationship between female P. lounsburyi survivorship and sucrose consumption (mg) for a single meal of sucrose solution. Three different sucrose concentrations (0.5, 1, and 2 M) were provided 2 days after emergence. Regressions: (A) 1 M sucrose F1,20 = 7.23, P = 0.0145; (B) 2 M sucrose F1,18 = 10.12, P = 0.0055; 0.5 M sucrose P > 0.05. Survival time on y-axis is total survival (2 days after emergence + DAF).

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Fig. 6. Rate of sucrose consumption (mean mg/min + s.e.m., gray bars) and rate energy uptake (mean Joule/min + s.e.m., white bars) for P. lounsburyi female wasps for a single meal of sucrose. Three different sucrose concentrations (0.5, 1, and 2 M) were provided 2 days after emergence. For each dependent variable, different letters above bars indicate statistical differences (Tukey’s HSD).

generally led to an increase in wasp longevity. However, the risk ratio increased, which indicates that the beneficial effect of higher sucrose concentration decreased. Thus, it appears that above a certain concentration threshold, the effect of sucrose on longevity became weaker. In particular, treatment combination pairs that included 2 M sucrose tended to have relatively high risk ratios. The overall loss of effect as concentration increased may be due to the fact that wasps are less able to properly metabolize highly concentrated solutions, resulting in unsuitable osmotic concentration of the hemolymph (Chippendale, 1978). Siekmann et al. (2001) studied the effect of sugar concentration and wasp age on the longevity of Cotesia rubecula. They used three concentrations of honey solution: low (0.7 M or 25% sucrose equivalent), medium (1.4 M or 47% sucrose equivalent) and high (2.5 M or 86% sucrose equivalent) and two ages of wasp (days 0 and 1 post-emergence). They found an opposite tendency in the evolution of the risk ratio compared to the one we observed. In their case, the risk ratio decreased as the sucrose concentration increased, indicating that, for the range of their test concentrations, the positive effect of sucrose is enhanced by an increase in concentration. Differences in results between the studies may represent species effects, or could be due to differences in food sources, i.e., honey (a complex mixture of carbohydrates, proteins, and other compounds) versus the disaccharide sucrose. Comparison of survival probabilities between our study and that of Siekmann et al., 2001 show that for comparable times of feeding, female P. lounsburyi lived about 2x longer than C. rubecula. This may be because P. lounsburyi ingested proportionally more sugar than C. rubecula. Sucrose concentration also affected the duration of feeding, and, correspondingly, the rates of volume ingestion, sucrose consumption, and energy content uptake for female P. lounsburyi. The duration was longest on the highest concentration, 2 M. This response was also observed for C. rubecula, where 1 day old wasps fed ca. 25 min on a 2.5 M honey solution (Siekmann et al., 2001). Viscosity of honey and sugar solutions increases exponentially with concentration (Kingsolver and Daniel, 1995; Nardone et al., 2013), and this increased viscosity can slow the rate of ingestion (Heyneman, 1983; Josens et al., 2006; Siekmann et al., 2001) and thus affect longevity if feeding is interrupted before the wasp becomes satiated. Our observation that the effect of sucrose on longevity is reduced as the concentration increases indicates that highly concentrated sugar solutions are underexploited. This could

be explained by a limited capacity of ingestion of viscous meals, as evoked by a longer duration of feeding. However, we found that volume ingested did not differ between concentrations, which suggests that wasps were able to feed to satiation on all sucrose concentrations. These results also suggest that cessation of P. lounsburyi feeding is triggered by stretch receptors in the digestive tract, as is the case for other insects that imbibe liquid meals (Bowdan and Dethier, 1986). Our results indicate that the gut capacity of P. lounsburyi females is ca. 0.75 ll, similar to that of C. rubecula, a similar-sized wasp (Siekmann et al., 2001). A comparison between our study and that of Siekmann et al. (2001) showed that the trends for rates of volume ingested and sugar consumption were similar despite differences in food sources and their concentrations between the two studies. Age at the time of first meal also affected the longevity of P. lounsburyi females and males. Moreover, the risk ratio tended to decrease as the number of days between emergence and the single meal increased. This trend was most evident in females, and may be because wasps fed on day 0 or 1 post-emergence still held nutrient reserves from larval development, and therefore did not need to feed as much to become satiated. Conversely, wasps fed 2 or 3 days post-emergence had depleted these reserves almost completely, and thus were hungrier, fed more, and lived longer. This is consistent with our results that feeding duration increased with age of wasps, and is supported by changes in the risk ratio. Indeed, the risk ratio tends to be lower when the day of treatment (first meal) occurred later. This reveals a reduction in risk of starving to death when the sucrose treatment is delayed. Similarly, C. rubecula fed at day 1 lived longer than the wasps fed at emergence (day 0) (Siekmann et al., 2001). Both studies suggest that feeding on the day of emergence (=first 24 h of life) may not be necessary for survival. When female P. lounsburyi was provided with a honey-water mixture (50% v/v = ca. 1.4 M sucrose equivalents) and water for the first 2 days of adult life, average longevity of the wasps was 11 days, i.e., 9 days beyond cessation of feeding (Daane et al., 2008). These results are similar to those of the present study for wasps fed 2 days post-emergence on 1 M sucrose; median survival of 8 days after the meal. Under natural conditions extrinsic factors, such as climatic conditions (Weisser et al., 1997) and mortality from natural enemies (Heimpel et al., 1997; Völkl et al., 1999), interact with nutrient uptake and metabolism to mediate survival and fitness of parasitic wasps. Duration of feeding is important because it represents a period when wasps are vulnerable to natural enemies. Therefore, feeding for a short duration should be favored because it would limit a wasp’s exposure to natural enemies (Siekmann et al., 2001). Moreover, feeding on a highly concentrated solution may increase the time required for digestion, and this period of extended inactivity might increase the risk from natural enemies (Lightle et al., 2010). For P. lounsburyi, duration of feeding can be shortened by ingesting sugar solutions of intermediate concentration. However, we have found that the amount of energy content ingested by P. lounsburyi was reduced when duration of feeding was reduced. Theoretically, a sucrose concentration exists that permits an optimal rate of uptake of energy content per unit time for P. lounsburyi. This optimal concentration represents a tradeoff between feeding duration and energy uptake. Our results for 2 day old females indicate that this concentration of sucrose lies between 0.5 and 2 M. Likewise, the optimal concentration for C. rubecula was between 0.7 M (25% sucrose equivalent), and 2.5 M (86% sucrose equivalent) honey solution (Siekmann et al., 2001). Similarly, sucrose intake rate for Camponotus mus ants peaked at 0.9 M (30.8% w/w) (Josens et al., 1998). Optimal frequency of meals would depend on temperature and other abiotic conditions as well as wasp behavior, especially metabolically costly locomotory behavior, such as flight or

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searching for host patches. Regardless of the optimal sugar concentration for P. lounsburyi, sucrose feeding extended their longevity compared to water-only controls. Similar positive effects of sucrose-feeding have also been found on other braconids, such as Cotesia glomerata (Lee and Heimpel, 2008b) which parasitizes Pieris spp. larvae (Lepidoptera: Pieridae), or Apanteles aristoteliae, a parasitoid of the orange tortrix Argyrotaenia franciscana (Lepidoptera: Tortricidae) (Lightle et al., 2010). Provisioning P. lounsburyi with a food source has the potential to enhance the affect of this classical biological control agent on olive fruit fly. Indeed, it allows extending their lifespan, and potentially their affect on B. oleae populations. Examining the effect of the day of feeding would provide a better understanding of the optimal feeding frequency of P. lounsburyi for practical application under field conditions and for mass-rearing. Siekmann et al. (2001) concluded that C. rubecula needs a daily meal to maximize longevity. Aphidus ervi (Hymenoptera: Braconidae), an aphid parasitoid, would benefit most from twice daily meals (Azzouz et al., 2004). Other studies have also reported that sugar-feeding on a daily basis optimizes parasitoid survival (Fadamiro et al., 2005; Lee and Heimpel, 2008b). Our study demonstrated that postponing the day of feeding to 2 or 3 days after emergence is more beneficial to the P. lounsburyi than feeding them earlier. Identification of optimal feeding intervals after the first meal might be a worthwhile topic for future studies. We demonstrated that providing a single sucrose subsidy to P. lounsburyi extends its longevity, and that moderate sucrose concentrations are particularly adapted because they optimize rates of nutrient uptake and thus limit the duration of feeding.

Acknowledgements We thank A. Blanchet and M. La Spina for assistance in rearing parasitoids. We are grateful to J.C. Lee and J.M. Patt for helpful comments on the manuscript. This work was supported by USDA-ARS appropriated funding. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by the USDA for its use. The USDA is an equal opportunity provider and employer. The U.S. Government has the right to retain a non-exclusive, royalty-free license in and to any copyright of this article.

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