Affect of food provisioning on survival and reproductive success of the olive fruit fly parasitoid, Psyttalia lounsburyi, in the field
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Conservation biological control offers approaches that can be integrated into classical biological control programs to enhance pest suppression. Food subsidies, such as nectar and honeydew, can increase a parasitoid’s fecundity either by extension of the reproductive lifespan, increasing the rate of egg maturation, or both. The olive fruit fly, Bactrocera oleae, is a major pest of olives in California, where a classical biological control program is currently underway using an exotic parasitoid, Psyttalia lounsburyi. We conducted a field study where female–male pairs of P. lounsburyi wasps were caged with B. oleae-infested olives, and provisioned either with or without food. Our study showed that adult feeding is crucial to P. lounsburyi survival and fecundity under field-cage conditions. Food provision increased P. lounsburyi survival and several components of the wasp’s reproductive success; nevertheless, parasitism rates and offspring production were relatively low. This was probably due, at least in part, to location of host larvae in enemy-free space ‘beyond the reach’ of the wasp’s ovipositor. Sex ratio of offspring was male-biased, perhaps due to inbreeding in the laboratory colony from which P. lounsburyi was sourced. Female wasps carried ca. 25–35 eggs at their time of death, suggesting that they were time limited rather than egg limited. Integration of conservation biological control (e.g., food provisioning) and classical biological control (release of an exotic natural enemy) have promise to suppress olive fruit fly populations. Evaluation of the effect of food provisioning on P. lounsburyi reproductive success under open field conditions is warranted.
KeywordsFood subsidy Survival analysis Reproductive success Parasitoid Biological control Bactrocera oleae
Most parasitoids require carbohydrate food sources to satisfy metabolic energy needs. Theoretical and empirical evidence suggests that parasitoid food sources play an important role in regulation of host population dynamics (Heimpel and Jervis 2005; Sabelis et al. 2005). Parasitoids deprived of food often die within a few days, while those provided with carbohydrate subsidies live longer and have greater reproductive success (Sivinski et al. 2006; Furtado et al. 2016; Benelli et al. 2017). This increase in fecundity is a result of either greater longevity, increased rates of egg maturation, or a combination of these factors. Laboratory studies of carbohydrate food subsidies on the longevity and reproductive success of parasitoids provide useful information under standardized conditions (Irvin et al. 2007; Lee and Heimpel 2008a). However, laboratory studies do not account for the effects of natural conditions, such as differential host availability, parasitoid food and host searching behavior, parasitoid mortality factors, and fluctuating climatic factors (Winkler et al. 2006; Heimpel and Casas 2008). Although relatively few field studies have been conducted on the effect of adult parasitoid feeding on longevity and reproductive success, these studies generally demonstrate survival and reproductive benefits of food-provisioning (e.g., Winkler et al. 2006; Lee et al. 2008b; Géneau et al. 2013; Jamont et al. 2014). Consequently, the presence of suitable carbohydrates, usually in the form of nectar or honeydew, for foraging parasitoids is an important factor in the development of habitat management strategies that enhance conservation biological control, and thus, the effectiveness of biological control agents against agricultural pests (Landis et al. 2000; Orre Gordon et al. 2012; Benelli et al. 2017; Gurr et al. 2017).
The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is a major pest of olives worldwide, and this invasive pest was first recorded in California olives in 1998 (Rice et al. 2003; Daane and Johnson 2010). Foreign exploration for natural enemies of B. oleae led to the discovery of several parasitoids with potential for controlling this pest (Copeland et al. 2004; Daane and Johnson 2010). Subsequent biological evaluation of these candidates led to the selection of the specialist Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae) for release in California olives (Sime et al. 2006a, b, c, 2007; Daane et al. 2008; Wang et al. 2009a, 2011a; Yokoyama et al. 2011). Releases of P. lounsburyi in California began in 2006, and thus far, the parasitoid has become established in coastal regions of the state having a relatively mild Mediterranean climate, as compared to the more extreme conditions in the inland regions of olive production (Daane et al. 2015). These coastal locations are characterized by relatively few olive trees grown as ornamentals within a diverse matrix of suburban vegetation that includes many species with floral resources that might serve as food sources for P. lounsburyi. This classical biological control program for the protection of California olives relies on insectary production of P. lounsburyi under quarantine conditions at the USDA-ARS European Biological Control Laboratory (EBCL) in Montferrier-sur-Lez, France (La-Spina et al. 2018). Although this valuable classical biological control program may continue to benefit from production and release of P. lounsburyi wasps, further benefits may be gained by integration of conservation biological control strategies into the existing program. However, little is known about the interactions between the nutritional ecology of P. lounsburyi and its reproductive capacity.
Psyttalia lounsburyi is synovigenic and has a haplodiploid reproductive system. Bactrocera oleae eggs are laid beneath the skin of the olive fruit, and larvae feed on the olive pulp. Psyttalia lounsburyi oviposits through the olive skin into the host larva (3rd instar is preferred) and completes development in the puparium (Daane et al. 2008). Psyttalia lounsburyi oviposition rate peaks at 6–10 days after emergence and rapidly decreases to almost zero at 30 days post-emergence (Daane et al. 2008). More than 90% of the reproductive lifespan of P. lounsburyi occurs in the first 20 days of the wasp’s life (Daane et al. 2008). Adult wasps do not host feed, but their longevity is increased by feeding on carbohydrates (Daane et al. 2008). Williams et al. (2015) reported that even a single sucrose meal significantly increases the lifespan of P. lounsburyi, while unfed wasps die in a few days. The findings of these laboratory studies, coupled with the success of the field release program, suggest that adult P. lounsburyi rely on food sources such as nectar and honeydew in the field and that successful sugar-foraging has contributed to the success of the release program. A better understanding of the effect of sugar-foraging on the reproductive success of P. lounsburyi would help guide future efforts to integrate conservation biological control and classical biological control for suppression of olive fruit fly (Paredes et al. 2013; Benelli et al. 2017; Gurr et al. 2017).
Our study aims at evaluating the effect of food provision for adult P. lounsburyi on the parasitoid’s longevity and fecundity under field-cage conditions. Our goal was to evaluate the per capita reproductive success of P. lounsburyi during the major portion of the reproductive lifespan of the parasitoid, i.e., the first 20 days post-emergence. A honey + bee pollen mixture was used as a proxy for naturally occurring food sources, such as nectar or honeydew. Finally, use of field cages allowed per capita assessment of P. lounsburyi biology, but introduced some artificiality, e.g., exclusion of natural enemies, which perhaps resulted in overestimation of parasitoid longevity. The field cages may have also restricted the foraging and dispersal behavior of the wasps. Nevertheless, our methods allowed us to evaluate the extent to which adult P. lounsburyi food sources increase individual wasp’s realized lifetime fecundity under relatively natural conditions. We predicted that beneficial effects of food provision would increase survivorship and fecundity of P. lounsburyi.
Materials and methods
The P. lounsburyi used in this study originated from B. oleae on wild olive, Olea europaea L. subsp. cuspidata (Wall. ex G. Don), collected in the Burguret Forest (west of Mt. Kenya, ca. 1960 masl), Kenya in 2005. Since then, the parasitoids were maintained as a laboratory colony in the quarantine facility of EBCL. After the initial collection in Africa, parasitoids were reared for a few months on B. oleae-infested olives, but since May 2005 the parasitoids had been reared continuously (~ 95 generations) on the factitious host, Ceratitis capitata (Weidemann) (Diptera: Tephritidae) (Thaon et al. 2009) on artificial diet (Wong and Ramadan 1992) (22 °C ± 1, 60% 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: 1:1 female/male) for 40 h 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. 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.
Bactrocera oleae used in this study were reared from infested olives growing as ornamentals in the environs of Montpellier, France, in July and August 2013. Infested olives were held in ventilated Plexiglass cages (26 cm × 26 cm × 20 cm) on a laboratory bench (23 °C ± 2, 45% r.h.) with ambient light provided by a nearby window. Emerging flies, ca. 40 per cage, were provisioned ad libitum with a dry diet (2.67 g sucrose:1 g yeast hydrolysate) and paper toweling moistened with distilled water. Each cage also contained ca. 30 green, uninfested olives (ca. 1.5 cm length) to stimulate mating. Flies used in the study were 2–3 weeks old, ensuring that they were near their peak oviposition period (Yokoyama 2012).
This study was conducted in a commercial organic olive domain in southern France in 2013 (43°40′37.23″N, 3°47′39.07″E). This ca. 30-ha olive domaine has been in operation for more than 50 years and encompasses ca. 10,000 olive trees. The study site consisted of a 1.5-ha block of mixed-cultivar olive trees ca. 25 years old. The site had a northwest aspect, a range in elevation from 100 to 120 masl, and a slope of ca. 8%. Rainfall was supplemented with drip irrigation, and weed control was achieved by hand rouging and grazing by livestock. Within this block, 11 trees of cv. ‘Verdale’ were chosen for this study. ‘Verdale’ was developed in southern France and is now also grown in areas with similar climatic conditions, including California, Australia, and Eastern Europe. It is susceptible to B. oleae, has medium vigor and productivity, good tolerance to drought, and is generally considered to be self-sterile. It is grown for both oil and as a table olive.
In early August, 55 organdy sleeve cages (1-m length) were placed on branches with uninfested olives (confirmed by visual inspection) to protect the olives from natural infestation by B. oleae. The number of cages placed on each tree ranged from 1 to 9, depending on availability of branches bearing fruit. Beginning in mid-September, several randomly chosen sleeve cages were infested with the locally reared B. oleae flies (ca. three females and three males/cage) for 24 h, after which the flies were removed but the sleeve cages remained intact to ensure that no additional oviposition by feral B. oleae occurred. The following day, several more of the sleeve cages were infested with B. oleae flies for 24 h. This procedure was repeated daily until all 55 of the sleeve cages had been infested. While caged, B. oleae flies were provided with the aforementioned dry diet and cages were misted with water daily. This infestation procedure provided a sequential series of host cohorts of known age.
Beginning 7 days after B. oleae oviposition in the sleeve cages, a subset of olives was removed daily and dissected (20–60×) to assess the emergence and size of larvae and thus help choose the appropriate time to begin caging parasitoids. Twelve days after B. oleae oviposition in the sleeve cages, pairs (1 female and 1 male, 48 h post-emergence) of P. lounsburyi wasps were caged on the infested olives for 24 h using cylindrical cages (17 cm long × 9 cm diam) (Williams et al. 2012). Air temperature inside the cages was similar (± 1 °C) to that outside the cages (Williams, unpubl. data). Each of these cages enclosed a portion of a branch with olives (mean = 2.40, SD = 1.11, range = 1–7). Visual inspection of these olives prior to caging confirmed the presence of two to 15 B. oleae oviposition stings/cage. We imposed infestation levels which were similar to natural infestation levels caused by uncaged feral flies. (After the study was complete, we confirmed that on all occasions wasps had more hosts than were parasitized, i.e., P. lounsburyi was not host-limited during the study.) Prior to caging, the foliage, stems, and olives were examined to ensure that: (1) natural enemies and (2) the honeydew-producing olive psyllid, Euphyllura olivina (Costa) (Hemiptera: Liviidae), were not present. In treatment cages (n = 40), a mixture of organic honey and bee pollen granules, purchased at a local health food market (ca. 50 µl, 1:0.5 honey/bee pollen by volume), was streaked onto leaves and stems prior to the introduction of wasps; control cages (n = 12) were not provisioned with food. The composition of honey and bee pollen was not measured, but other studies have reported that honey is mostly (~ 70%) fructose and glucose, and lesser amounts of sucrose, other carbohydrates, and water. Vitamins, minerals, and amino acids occur in much smaller amounts (Bogdanov 2017a). Many of the carbohydrates which occur in honey enhance P. lounsburyi longevity (Williams, unpubl. data). The chemical composition of bee pollen (i.e., pollen collected from flowers by bees for brood provisioning) is mostly protein and carbohydrates, as well as lipids, fiber, minerals, and vitamins (Bogdanov 2017b). All cages were misted with distilled water daily. At this time, a subset of these olives was removed and dissected (20–60×) to measure the depth of the host at the time parasitoids were caged (see below). After the 24-h period, the procedure above was repeated, i.e., the wasps were transferred to a different cage containing olives with a new host cohort of the appropriate age that had not been previously exposed to parasitoids. Transfer of wasps to cages with new host cohorts was repeated daily until both wasps died (note that our supply of experimental host cohorts was exhausted 19 days after wasp emergence. Approximately 80% of the P. lounsburyi females in food-provisioned cages were still alive at this time; they were transferred daily to cages that contained foliage and food, but no olives. This allowed complete assessment of parasitoid survivorship). Upon death, wasps were preserved in 75% ETOH for assessment of egg load (see below). Cages remained on olives after the wasps were transferred to ensure that no additional oviposition by feral B. oleae occurred.
Daily air temperatures and dew points during the study were obtained from a weather station 1 km northeast of the study site in the village of St. Gely-du-Fesc.
Measurement of parasitism and P. lounsburyi reproductive characteristics
The caged olives remained on the plants and were collected in early November, after which they were held in the laboratory to assess parasitism by rearing from and dissection of olives. Olives were individually placed in ventilated transparent 45 ml plastic vials (12 dram, No. 55-12, Thornton Plastics Co., Salt Lake City, UT) and held on the laboratory bench (ambient conditions described above) for 60 days. Vials were observed daily, and any emerging wasps were transferred individually to another vial streaked with honey and containing a moist cotton wick. After 3 days wasps held in this manner were killed and preserved in 75% ETOH. After 60 days, all olives were dissected (20–60×) to search for parasitoids that had died prior to emergence. These dissections provided a more accurate assessment of parasitism levels. Gender was determined for all wasp progeny when discernable. Female wasps used in the field study were dissected in 1% methylene blue in 75% ETOH to aid measurement of egg load (mature eggs in each ovariole) at time of death in the field. To provide points of reference, we included in the dissections two groups of 2-day-old host-deprived wasps (subsamples from the wasps available at the time of field caging) that had been provided either food (honey + bee pollen) and water or water only. The relationship between P. lounsburyi ovipositor length and depth of host larvae was assessed using subsets of wasps available for the field study and olives available the day of caging with parasitoids. Using a dissecting scope fitted with an ocular micrometer, ovipositors were measured to the nearest 0.001 mm. Ovipositor measurements did not include the basal portion of the ovipositor that remains inside the wasp during oviposition. Olives were dissected under the scope, and the depth of host larvae was measured, as well as the thickness of the pulp.
Survival data were analyzed by the Cox Proportional Hazards Analysis, where provisioning with or without food (honey + bee pollen) was tested as the explanatory variable (SAS Institute 2013). After the initial analysis which established gender effect for survival, further survival analyses were conducted separately for each gender. Differences in survival curves for each gender were analyzed by likelihood ratio tests. After this, we estimated the effect of food provisioning on P. lounsburyi survival for each gender. The risk ratio (or hazard ratio) is the quantitative effect of a variable on survival, and it characterizes the risk of death between two treatment groups. In the present study, the risk ratio was calculated to characterize the effect of food provisioning on survival of P. lounsburyi. For example, a risk ratio of 1 indicates that food provisioning has no effect on survival. A risk ratio < 1 indicates a lower likelihood of death with food provisioning. Conversely, a risk ratio > 1 shows a higher probability of death with food provisioning.
Reproductive success of P. lounsburyi was measured by counting the number of wasp progeny and the number of olive fruit fly progeny reared from each cylindrical cage in which a given pair of wasps had been enclosed. The proportion of parasitism for each cylindrical cage was measured as number of wasp progeny/total number of wasp and fly progeny. Offspring production for each cylindrical cage was measured by counting the number of wasps emerging from olives in that cage. Because the total number of P. lounsburyi and olive fruit fly progeny were low, the effects of “sleeve cage” and “cylindrical cage” were ignored and the total numbers of wasp and host fly progeny were obtained for each wasp pair. Therefore, wasp pair was the only source of replication used to compare food versus no food treatment effect on reproductive success. ANOVA (SAS Institute 2013) was used to examine overall parasitism and offspring using a completely randomized design (CRD) with one observation per wasp pair and two treatments (i.e., food versus no food). For analyses examining parasitism and offspring production measured for different ages of a wasp pair, the experimental design was a split plot with the main unit treatment (food, no food) having a CRD design and the sub-unit treatment (age of wasp pair) being a repeated measure. Preliminary ANOVAs indicated violation of the assumption of normality because many wasps had zero parasitism rates and low offspring production. There was no transformation that effectively addressed this issue; therefore, the final analysis was based on a generalized linear mixed model (PROC GLIMMIX) with the response being the proportion of parasitism and offspring, and specifying a binomial distribution with a logit link function. Least square means and test of significance were based on this analysis.
Data for lifetime fecundity, number of days during which wasps parasitized hosts, reproductive lifespan, and P. lounsburyi ovipositor length versus host depth were square-root transformed (X′ = sqrt(X + 0.5)) prior to t test (one-tailed, unpaired data with unequal variance) (Zar 1996). Data for parasitoid egg load were similarly transformed prior to one-way ANOVA (SAS Institute 2003), and a planned mean comparison was conducted using Tukey’s HSD (Zar 1996). The sex ratio of emerging P. lounsburyi, expressed as proportion of males among the offspring, was calculated with the binomial test (Byers and Wood 1980).
Influence of food provisioning on P. lounsburyi survival
Analysis of survival data for gender effects showed that female longevity was significantly greater than male longevity (Likelihood ratio test: Hazard ratio = 0.082, n = 104, χ2 = 50.7, P < 0.0001); therefore, further analyses were conducted separately on each gender. Wasps provisioned with food lived longer than wasps provided water only (Female: Hazard ratio = 0.024, n = 53, χ2 = 28.4, P < 0.0001; Male: Hazard ratio = 0.167, n = 51, χ2 = 14.9, P < 0.0001) (Fig. 1a, b). Median longevity of female wasps provided with food was 38 days versus 5 days for females provided water only. Median longevity of male wasps provided with food was 8 days versus 5 days for males provided water only.
Influence of food provisioning on P. lounsburyi reproduction
Rearing and dissection resulted in a total of 56 P. lounsburyi offspring from the field study. During the first 3 days of the study, parasitism rates ranged from ca. 15 to 42% and declined thereafter until peaking again at 30% on day 19 (Fig. 2A); production of offspring showed similar trends (Fig. 2B). There was no difference (P > 0.05) in proportion parasitism or offspring production between food provision treatments in the first 4 days of the study (when comparable data were available for both treatments). Also, there was no difference (P > 0.05) in proportion parasitism or offspring production between wasp age in the food provision treatment. The proportion of wasps parasitizing hosts did not differ between the two treatments (P > 0.05); of the 40 female wasps provided with food, 22 parasitized host larvae, while four of 12 wasps without access to food parasitized hosts (Table 1). The lifetime fecundity of P. lounsburyi females with access to food was 1.18 offspring versus 0.67 offspring for the control (T > 0.05) (Table 1). Wasps provisioned with food parasitized hosts for > 2× as long as unfed wasps (mean = 1.00 days versus 0.42 days) (t26 = 2.02, T = 0.0542) (Table 1). The average reproductive lifespan was longer (t40 = 3.30, T = 0.0020) for P. lounsburyi with access to food (5.3 days, range 0–17 days) as compared to the control (1.1 days, range 0–2 days).
Effect of food provisioning on per capita Psyttalia lounsburyi reproduction
Control (water only) mean ± SE (range)*
Treatment (honey + bee pollen + water) mean ± SE (range)**
Number of wasps that parasitized hosts
Lifetime fecundity (offspring per female wasp)
0.67 ± 0.36 (0–4) a
1.18 ± 0.22 (0–5) a
Number of days that wasps parasitized hosts
0.42 ± 0.19 (0–2) b
1.00 ± 0.18 (0–4) a
Reproductive lifespan (average number of days that wasps parasitized hosts)
1.08 ± 0.47 (0–2) b
5.25 ± 0.93 (0–17) a
Egg loads of wasps differed across treatments (F3,57=8.21, P = 0.00013) (Fig. 3). Wasps provisioned with food and used in the field trial had fewer eggs than either group of 2–day-old host-deprived wasps, but not the water only wasps used in the field (Fig. 3). There was no difference in egg load between the right and left ovaries for any of the four treatment groups (P > 0.05) (Fig. 3). The most progeny produced by a single wasp was five, which occurred on one occasion in the food-provisioned treatment. The most progeny produced by a wasp in a single day was three, which occurred on two occasions in the food-provisioned treatment.
Measurements of subsamples of infested olives and P. lounsburyi ovipositors used in the field study revealed that average depth of host larvae (1.93 mm) at the time of exposure to parasitoids was greater than average ovipositor length (0.88 mm) (t39 = 12.59, T < 0.0001) (Fig. 4). The average thickness of the pulp of these olives was 4.37 mm (n = 45, SD = 0.44, range = 3.65–5.3 mm). A male-biased sex ratio was observed for P. lounsburyi progeny. The first progeny produced by caged females had a sex ratio of 2.4:1 (n = 17, P > 0.05), while the sex ratio for all the progeny in the study was 2:1 (n = 39, P < 0.05).
Climatic conditions at the field site
During the period when wasps were caged, the average high and low temperatures were 23° and 10 °C, respectively. The average high and low dew points were 20° and − 2 °C, respectively. Total accumulation of precipitation was 3.10 cm, the majority of which (3.05 cm) occurred on 4 October.
Provisioning caged P. lounsburyi with food had a positive impact on the parasitoid’s survival and reproductive success under field conditions. We found that food-provisioned female P. lounsburyi lived almost 8× longer than unfed females. Food provisioning also increased longevity of P. lounsburyi under laboratory conditions (Daane et al. 2008), as well as P. humilis (Wang et al. 2011b; Yokoyama et al. 2011) and P. concolor (Furtado et al. 2016). Wasps used in the present study were drawn from the same EBCL colony as those released in California olives and that have established and spread there; thus, deleterious affects on P. lounsburyi fitness due to long-term rearing at EBCL, if they exist, did not appear to be a factor in our study. Parasitism rates during the first 4 days of the wasp’s life were similar between wasps with and without food. This may be due to a cage effect resulting in increased success of unfed wasps relative to fed wasps. The relatively small (~ 1-l capacity) cages used for rearing in the laboratory and for exposure to hosts in the field restricted dispersal of and host searching by the wasps, thus greatly limiting their need to engage in flight behavior. Flight is very energy-intensive (Casas et al. 2003) and parasitoids that engage in flight deplete their energy reserves at much faster rates than those that do not fly (Steppuhn and Wackers 2004). Reduced flight by P. lounsburyi in our study may have slowed depletion of energy reserves in unfed wasps and permitted these wasps more time for host foraging than if they had been in open field conditions. Few unfed wasps remained alive after the 4th day of the trial, thus contributing to the difference in parasitism rates between the two treatments. This factor also contributed to the treatment differences in reproductive lifespan and number of wasps parasitizing hosts. The difference in egg load between unfed 2-day-old host-deprived wasps at the beginning of the trial and fed wasps at time of death indicated that egg maturation did not offset oviposition.
Adult P. lounsburyi emerge with limited energy reserves which allow them to survive for only a few days in the absence of food (Daane et al. 2008; Williams et al. 2015). During this time, the parasitoids must balance foraging for food with reproductive behavior, such as mate finding and mating, and host searching and oviposition. Our results indicate that P. lounsburyi is capable of successfully attacking hosts within a few days of emergence, regardless of the condition of its energy reserves. All four unfed wasps that successfully attacked hosts did so on the first day of exposure to hosts, i.e., the third day after their emergence. Similarly, Daane et al. (2008) reported that P. lounsburyi began oviposition the second day after emergence. From a pest management perspective, this is important because it indicates that even newly emerged P. lounsburyi that do not feed are capable of reproduction, even if for a short period, before death. In the present study, wasps provisioned with honey + bee pollen had greatly increased their longevity relative to unfed wasps, which permitted longer reproductive lifespan. In a laboratory study with similar temperatures as our study (25 ± 1 °C), P. lounsburyi oviposition rate peaked 6–10 days after emergence after which it rapidly decreased to almost zero at 30 days post-emergence (see Daane et al. 2008 for detailed laboratory conditions). Conversely, in our study, parasitism rates and offspring production were bimodal, with peaks on days 3–5 and again on day 19 post-emergence. Lifetime fecundity of P. lounsburyi was ca. 10× greater in the laboratory (Daane et al. 2008) than in our field-cage study. Differences between our study and Daane et al. (2008) may reflect the influence of the different conditions in the laboratory (i.e., constant number of olives presented in a petri dish, standardized climatic conditions) vs. the field (i.e., intact, growing olives and presence of foliage, variable climatic conditions) on P. lounsburyi reproduction. Survival of female P. lounsburyi in the food-provisioned treatment at the time that our supply of experimental host cohorts expired (19 days after wasp emergence) was ca. 80%; had additional host cohorts been available it is possible that differences in reproductive lifespan, and other reproductive variables that were measured, would have been greater.
Experimental studies of the effect of food provisioning on parasitoid reproduction are essential to improve conservation biological control. Laboratory evaluations provide important information under controlled conditions, but the results can be difficult to extend to the field. Greenhouse and field-cage studies are more realistic, but the results may still not provide a true reflection of parasitoid biology in open-field conditions. In a study using relatively large (3 m × 4 m × 2 m) field cages, Winkler et al. (2006) found that food provisioned Diadegma semiclausum (Hellén) (Hymenoptera: Ichneumonidae) wasps had greater longevity and reproductive success (e.g., parasitism rates and lifetime fecundity) than did unfed wasps. Moreover, their results suggested that laboratory studies might actually underestimate the importance of food provisioning for D. semiclausum reproductive fitness. This difference may be partly due to a cage effect, because larger cages better reflect field conditions by allowing parasitoids to move freely between hosts and food sources (although long-distance dispersal is limited). In turn, this would allow the wasps to sample a wide array of hosts and food sources, detect subtle differences between them, and choose those that best suit them. This activity is restricted in smaller cages and may have impacted our assessment of P. lounsburyi reproductive fitness.
The relatively low parasitism rates observed in the present study may have had several sources. Previously mentioned are the restricted choice of hosts imposed by the field cages, and the incomplete supply of host cohorts that did not permit evaluation of parasitism throughout the entire reproductive lifespan. Additionally, it is possible that the low rates of parasitism observed were due to the depth of host larvae in relation to the ovipositor length of P. lounsburyi. Bactrocera oleae eggs are laid under the skin of olive fruits, and as larvae grow, they generally burrow deeper into the olive. Our dissections indicated that some host larvae were already ‘out of reach’ of the parasitoid’s ovipositor when the parasitoid was caged with the host. Had we caged the wasps on fruit with younger host larvae the observed reproductive success of P. lounsburyi in our study might have been higher. The interaction between thick pulp in domesticated olives and a relatively short ovipositor has been noted previously for P. lounsburyi (Daane et al. 2008; Wang et al. 2009b). Wild olives are relatively small compared to cultivated olives (Bartolini and Petruccelli 2002; Tzanakakis 2003), and Neuenschwander (1982) reported that more P. lounsburyi individuals emerged from wild olives than cultivated olives. Parasitism of B. oleae larvae in wild olives can be > 80% (Mkize et al. 2008), probably due in part to the relatively thin pulp of wild olives that offers less of a host refuge than the thicker pulp in cultivated olives. Apparently, the relatively thick pulp of cultivated olives functions as enemy-free space for B. oleae larvae and represents a case of crop domestication that has tipped the balance in favor of the pest. This phenomenon may not be uncommon in situations where wild plants have been domesticated to produce larger fruit, thus creating enemy-free space for fruit feeders (Chen et al. 2015). Fecundity reported in studies with P. ponerophaga (Sime et al. 2007), P. concolor (Sime et al. 2006b), and two Diachasmimorpha species (Sime et al. 2006c) was also higher than in our study. The establishment of P. lounsburyi in California olives may be due to the wasp’s freedom of dispersal during host foraging, allowing the wasps to find and test numerous hosts before parasitizing those that are suitable for attack and still within reach of the ovipositor. The caged wasps in our study were not able to freely disperse and forage, perhaps impacting successful host attack.
The sex allocation of parasitoids can be an important determinant of the success of classical biological control programs, which rely on the successful establishment and spread of the parasitoid. In our field study, we observed a male-biased sex ratio for P. lounsburyi offspring, which is consistent with results from laboratory studies (Thaon et al. 2009; La-Spina et al. 2018). Similarly, during the ca. 12 years that P. lounsburyi has been reared at the European Biological Control Laboratory, the proportion of male offspring has varied from ca. 60 to 70%. However, Daane et al. (2008) reported 60% female P. lounsburyi offspring in a laboratory study. The reasons for the differences in P. lounsburyi sex ratio between the studies are not clear, but may be due in part to laboratory rearing for many generations on a factitious host with minimal infusion of feral wasps. Male-biased sex ratios under these conditions are consistent with complementary sex determination (CSD) which appears to be promoted in laboratory or mass-rearing colonies where inbreeding is prevalent. CSD has been documented in at least 40 species of Hymenoptera and appears to be the ancestral sex determination mechanism for the group (Ode and Harvey 2008). The parasitoids used by Daane et al. (2008) had only been under laboratory rearing for ca. 2 years and thus had been subjected to less selection for male bias than the wasps in our study.
It is also possible that a male-biased sex ratio may be normal early in the egg-laying sequence of P. lounsburyi and that eggs laid later in the sequence (that we were not able to assess due to exhausted supply of host cohorts) would be female. This sex-biased oviposition sequence was observed for P. concolor (Avilla and Albajes 1984). Further, differential mortality, i.e., one gender, usually females, suffering greater mortality than the other during immature development, sometimes alters observed sex ratios (Wellings et al. 1986), but did not occur in P. concolor (Avilla and Albajes 1984). The conditions under which sex allocation decisions are made also affect the sex ratio of offspring. The sex ratio of P. concolor varied with host size (Avilla and Albajes 1984) and duration of host exposure (Loni 2003), but not with irradiation of hosts (Hepdurgun et al. 2009). Also, sugar foraging generally leads to a higher proportion of female offspring (Singh et al. 2000; Berndt and Wratten 2005; Fuchsberg et al. 2007; Hu et al. 2012), although there are exceptions (Divya et al. 2011; Mutitu et al. 2013). Clearly, the factors that control sex ratio in P. lounsburyi deserve more study.
To maximize reproductive success, parasitoids must achieve a balance between egg limitation and time limitation (Rosenheim et al. 2008). Egg and time limitations are governed by losses due to oviposition, resorption, and risk of mortality (e.g., from starvation and predation), and by gains due to egg maturation. Food provisioned parasitoids generally have greater egg loads than unfed wasps (England and Evans 1997; Tylianakis et al. 2004; Lee and Heimpel 2008b), suggesting the ability to offset losses from oviposition by gains from nutrition-enabled egg maturation. In our study, fed and unfed host-provisioned wasps from the field died with substantial egg loads, suggesting that the fecundity of P. lounsburyi is time limited. The water only 2–day-old P. lounsburyi wasps provide a measure of egg load for wasps when the field experiment began. The difference in egg loads between the water only 2-day-old wasps and the wasps used in the field study represent: (1) losses due to oviposition and resorption, and (2) gains attributed to maturation of new eggs. For the water only controls from the field, gain was probably negligible since these wasps were unfed. Loss due to oviposition was also apparently minimal as indicated by the parasitism measurements. This leaves loss from resorption (to regain nutrients for maintenance) as the primary source of reduction in egg load for water only wasps used in the field. For fed wasps from the field, egg loss from oviposition was probably the major source of the difference because these wasps were provided with food there was little need for resorption of eggs. The amount of gain in egg load due to maturation of new eggs in these food-provisioned field wasps is not known. The significant reduction in egg load between water only 2-day-old wasps and fed wasps from the field cannot be explained solely by our observed parasitism rates. However, while dissecting olives, we observed an increased incidence of host larvae dead from undetermined cause(s) in the fed field wasps compared to the water only field wasps. Thus, it is possible that these hosts represent undetermined cases of parasitism. If this is the case, it appears that loss of eggs from oviposition was not being offset by maturation of new eggs in these wasps, despite having access to food.
Further studies are needed to expand our understanding of P. lounsburyi biology and its interface with olive agroecosystems to better utilize this biological control agent for suppression of B. oleae. Our results from field-cage studies reveal several aspects of P. lounsburyi reproduction that differ between field and laboratory studies. These include the sex ratio of offspring, offspring production and parasitism rates over the wasp’s reproductive lifespan, in addition to the time limitation in realized fecundity that we observed. Characterization of these components of the parasitoid’s reproductive biology under open field conditions with free-ranging hosts and parasitoids would clarify the importance of food provisioning for P. lounsburyi reproduction and help guide future work.
We are grateful to the Vialla family for allowing us to conduct this study at the Domaine de l’Oulivie. We thank D. Boykin (USDA-ARS) for statistical advice, and A. Blanchet and M. La-Spina (both USDA-ARS) for providing P. lounsburyi. Helpful comments on the manuscript were given by E. Riddick (USDA-ARS) and anonymous reviewers. 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.
LW conceived the study, designed the experiments, and analyzed the data. All authors contributed to the laboratory and field research and writing and review of the manuscript.
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