Aspects of the biology and reproductive strategy of two Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii

Accepted Manuscript Aspects of the biology and reproductive strategy of two Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii Xin-Geng Wang, Alexandra H. Nance, John M.L. Jones, Kim A. Hoelmer, Kent M. Daane PII: DOI: Reference:

S1049-9644(18)30077-X https://doi.org/10.1016/j.biocontrol.2018.02.010 YBCON 3719

To appear in:

Biological Control

Received Date: Revised Date: Accepted Date:

2 January 2018 8 February 2018 9 February 2018

Please cite this article as: Wang, X-G., Nance, A.H., Jones, J.M.L., Hoelmer, K.A., Daane, K.M., Aspects of the biology and reproductive strategy of two Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii, Biological Control (2018), doi: https://doi.org/10.1016/j.biocontrol.2018.02.010

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For: Biological Control Article Type: Research Paper

Aspects of the biology and reproductive strategy of two Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii

Xin-Geng Wang a*, Alexandra H. Nance a, John M. L. Jones a, Kim A. Hoelmer b, Kent M. Daane a

a

Department of Environmental Science, Policy and Management, University of California

Berkeley, California, USA

b

USDA Agricultural Research Service, Beneficial Insects Introduction Research Unit, Newark,

Delaware, USA

*Correspondence author: E-mail address: [email protected] (X.G. Wang)

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Abstract Drosophila suzukii Matsumura (Diptera: Drosophilidae) is native to eastern Asia, but has established in Europe, North and South America, where it is a pest of numerous berry and other small fruit crops. As a part of a classical biological control program, two larval D. suzukii parasitoids, Ganaspis brasiliensis Ihering and Leptopilina japonica Novković & Kimura (Hymenoptera: Figitidae), were imported from South Korea to a California quarantine for evaluation. Here, we report on aspects of their reproductive strategy, including egg maturation dynamics, host age preference and suitability, and life-time fecundity. Adult females of both species emerged with a high mature egg-load that peaked 1–2 days post emergence. Both parasitoid species preferred to attack young host larvae (1–2 day old), although host age did not affect the parasitoid offspring’s sex ratio or fitness (survival, developmental time and body size of female wasps). Held at 22 ± 2 C with honey-water and D. suzukii larvae in artificial diet, as well as a constant source of adult males, G. brasiliensis adult females survived 17.7  1.4 days and produced 98.3  11.8 offspring per female, while L. japonica survived 18.7  1.1 days and produced 107.2  9.9 offspring per female. The proportion of female progeny decreased with increasing maternal age for both parasitoid species. Estimated demographic parameters were similar for both G. brasiliensis and L. japonica: net reproduction rate was 39.9 and 47.3, intrinsic rate of increase was 0.130 and 0.138, mean generation time was 28.5 and 28.1 days, and doubling time was 5.4 and 5.0 days, respectively. This information is being used to compare exotic D. suzukii parasitoids and determine their value as potential biological control agents.

Keywords: Adult parasitoid longevity, Ganaspis brasiliensis, host suitability, Leptopilina japonica, parasitoid fecundity, spotted wing drosophila

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Graphical Abstract See submitted files

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Highlights  Two figitid parasitoids, Ganaspis brasiliensis and Leptopilina japonica, were evaluated as potential parasitoids for classical biological control of Drosophila suzukii.  G. brasiliensis and L. japonica adult females eclosed with a high mature egg-load.  Both adult parasitoid species preferentially attacked younger host larvae, but host age did not affect their fitness (survival, developmental time, and body size) or sex ratio.  Demographic parameters, such as lifetime fecundity, were determined for both parasitoid species and showed strong similarity in their performance as parasitoids of D. suzukii.

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1. Introduction Spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), is native to East Asia, but has established widely in Europe, North and South America (Andreazza et al., 2017; Asplen et al., 2015; Cini et al., 2014). Drosophila suzukii is highly polyphagous, attacking important fruit crops such as blackberries, blueberries, raspberries, strawberries and stone fruits (Burrack et al., 2013; Lee et al., 2011; Stewart et al., 2014; Van Timmeren et al., 2017), as well as more than 100 reported wild plants (Haye et al., 2016; Kenis et al., 2016; Lee et al., 2015; Poyet et al., 2015). The fly’s rapid development (one generation in 13–14 days at 22°C) and high reproductive potential (> 600 eggs per female) can lead to explosive population increases and significant economic losses to commercial crops (Hamby et al., 2016; Tochen et al., 2014; Walsh et al., 2011). Current programs primarily rely on insecticides that target adult flies, and this practice can be effective (Beers et al., 2011; Van Timmeren and Isaacs, 2013), but also presents pre-harvest restrictions because of fruit residues, insecticide resistance issues for target and nontarget pests, and negative impacts on beneficial organisms (Biondi et al., 2012; Diepenbrock et al., 2016a). Furthermore, D. suzukii populations can reside in nearby non-crop habitats that represent a pest reservoir for reinvasion of the treated crop (Diepenbrock et al., 2016b; Klick et al., 2014; Wang et al., 2016c). Therefore, area-wide IPM strategies that reduce population densities at the landscape level should be developed for this highly mobile, polyphagous pest. Biological controls may be a key component of area-wide programs by reducing fly populations in natural habitats, thereby reducing the number of flies that migrate into crop habitats and improving the effectiveness of other control tools. Parasitoids play an important role in population regulation of Drosophila species (Carton et al., 1986). The majority are larval parasitoids belonging to Asobara (Braconidae), Ganaspis and

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Leptopilina (both Figitidae). Several studies have surveyed for or evaluated extant parasitoids that are newly associated with D. suzukii in North America (Kaçar et al., 2017; Miller et al., 2015; Wang et al., 2016a; Wang et al., 2016b) and Europe (Knoll et al., 2017; Mazzetto et al., 2016; Rossi Stacconi et al., 2015). Overall, these studies showed few effective indigenous parasitoids attacking D. suzukii larvae in its invaded regions. The key problem is that most common larval parasitoids in North America or Europe, such as Asobara tabida Nees, Leptopilina heterotoma Thomson, and Leptopilina boulardi Barb. et al., will attack fly hosts breeding in fermenting substrates (e.g., Drosophila melanogaster Meigen), but are unable to develop from D. suzukii, possibly due to the fly’s immune resistance (Chabert et al., 2012; Kacsoh and Schlenke, 2012; Poyet et al., 2013). Only two common pupal parasitoids, Trichopria drosophilae Perkins (Diapriidae) and Pachycrepoideus vindemiae (Rondani) (Pteromalidae) readily attack D. suzukii in newly invaded regions. Although these pupal parasitoids can be effective under laboratory conditions (e.g., Kaçar et al., 2017; Rossi Stacconi et al., 2017), field parasitism of D. suzukii pupae in sentinel traps was < 5% (e.g., Miller et al., 2015). Predators have also been evaluated for their ability to attack D. suzukii larvae or pupae (e.g., Woltz and Lee, 2017). Still, extant natural enemies are generally considered to be unable to effectively suppress D. suzukii due to low parasitism or predation rates (Haye et al., 2016). The lack of effective indigenous parasitoids in North America led to foreign exploration in East Asia for native parasitoids of D. suzukii. Previously, Mitsui et al. (2007) reported 15 drosophilid parasitoid species collected from fruit-baited traps in Japan, including two larval parasitoids Asobara japonica Belokobylskij and Ganaspis xanthopoda Ashmead that were collected from D. suzukii. Asobara japonica can be effective on D. suzukii (Biondi et al., 2017), but it also attacks many other drosophilid hosts (Mitsui and Kimura, 2010; Nomano et al., 2015).

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Moreover, several different biotypes of G. xanthopoda co-exist in Japan, with one biotype collected from D. suzukii in wild cherries in central Japan (Kasuya et al., 2013). More recently, Daane et al. (2016) explored South Korea in 2013 and 2014, and among several parasitoids collected from D. suzukii, reported that Ganaspis brasiliensis Ihering and Leptopilina japonica Novković & Kimura were the dominant larval parasitoids reared from D. suzukii in wild Rubus fruits. Recent explorations in southern China also found G. brasiliensis and L. japonica were the most common larval parasitoids reared from D. suzukii and the closely related species D. pulchrella Takamori (Giorgini M. et al., unpub. data). A recent genetic analysis by Nomano et al. (2017) revealed that some Ganaspis previously referred to as G. xanthopoda were, in fact, G. brasiliensis (e.g., Carton et al., 1986; Kasuya et al., 2013; Kimura and Novkovic, 2015; Kimura and Suwito, 2015; Mitsui and Kimura, 2010; Schilthuizen et al., 1998). The genetic analysis also suggests the occurrence of several different lineages of G. brasiliensis with varying host specificity – one lineage is a specialist of D. suzukii and has been previously collected in Japan (Kasuya et al., 2013) and South Korea (Buffington and Forshage, 2016), while other lineages may be more of a generalist (Nomano et al., 2017) or even do not attack D. suzukii (Mitsui and Kimura, 2010). Leptopilina japonica is further divided into two subspecies, L. japonica japonica Novković & Kimura and L. japonica formosana Novković & Kimura; the former was collected in Japan, South Korea and China while the later has been collected in Taiwan and South Korea (Daane et al., 2016; Novkovic et al., 2011). Leptopilina j. japonica has been observed to parasitize D. suzukii and several other Drosophila species, predominately from the melanogaster species group in Japan (Kimura and Suwito, 2015; Mitsui and Kimura, 2010; Novković et al., 2011).

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Information is generally lacking on the basic biology of G. brasiliensis and L. japonica. We aimed to systematically evaluate the efficiency and host specificity to determine their potential for classical biological control of D. suzukii in North America. As a part of this classical biological control program, we report here on aspects of their biology, including egg maturation dynamics, host age preference and suitability, and age-specific lifetime fertility of the female parasitoids.

2. Materials and Methods 2.1. Insects Colony maintenance and all trials were conducted under controlled laboratory conditions (22  2°C, 40–60% RH, and light fixtures set 14L: 10D with additional natural light through glass windows) at a University of California Quarantine Facility (Berkeley, California, USA). A colony of D. suzukii was initiated from field collections of infested cherries during 2013 in Parlier, California (Wang et al., 2016b). Field-collected D. suzukii were introduced into the colony periodically to maintain vigor. Adult flies were held in Bug Dorm cages (BioQuip Products Inc., Rancho Dominguez, CA, USA) and supplied with a 10% honey-water solution as food. Petri dishes (1.5 cm high, 8 cm diameter) containing a standard cornmeal-based artificial diet (based on Dalton et al., 2011) were exposed to adult flies for 24 h; after the resulting progeny had developed into 1–2 day old larvae, they were used for the rearing or experimental trials. Colonies of G. brasiliensis and L. j. japonica (hereafter referred to L. japonica) were initiated from field collections of D. suzukii-infested wild Rubus spp. in South Korea (Daane et al., 2016). Both parasitoid species were identified by Dr. Matthew Buffington (USDA Agricultural

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Research Service, Systematic Entomology Laboratory, Washington D.C., USA). The G. brasiliensis colony consisted of the host specific lineage (Buffington and Forshage, 2016). For the colonies, 40 1–2 day old D. suzukii larvae were transferred from the clean colony to a plastic vial (25 mm diameter, 95 mm high) filled with 2 cm standard cornmeal-based artificial diet. The hosts were exposed to two mated 3–4 day old female parasitoids for 2 days. The parasitoidexposed larvae were then held for the emergence of adult flies (emerged in 3–5 days) and parasitoids (emerged in 20–25 days). Emerged adult parasitoids were transferred to plastic vials supplied with 50% honey water streaked on the vial plug to begin the next generation. Unless otherwise indicated, all trials were conducted under these quarantine conditions and used 1–2 day old host larvae and 3–4 day old female parasitoids that had been kept with males since emergence (i.e., no oviposition experience, and assumed to be mated with a high load of mature eggs). Our preliminary observations showed that host larvae travelled and fed at any depth in the diet and the parasitoid located its host through sensing host movement and/or probing the diet substrate. For this reason, thinner diet vials (1–2 cm) were used for all trials to facilitate the parasitoid’s access to the host in the diet. The vials were also streaked with 50% honey-water as food for the parasitoid.

2.2. Female parasitoid egg maturation dynamics To determine mature egg load in adult female G. brasiliensis and L. japonica, groups of 20– 30 wasps were dissected after their emergence from D. suzukii. Parasitoids were reared using the same methods as described above for the colony. Newly emerged male and female parasitoids were collected every 12 h from 15–20 rearing vials and placed in clean plastic vials provisioned with 50% honey-water, but no host material. Thereafter, female parasitoids were collected at six

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different time periods (12 h, 1, 2, 4, 6 and 8 days post emergence), killed at -20°C, and then dissected to determine the number of mature eggs at the six age categories. The mature eggs were easily identified by the thin, translucent and smooth chorion, and the stalk on the anterior end that is typical of the Eucoilinae (a Figitidae subfamily) (e.g., Kopelman and Chabora, 1984). To consider potential effects of female body size on the mature egg load, the hind tibia length was measured with an ocular micrometer.

2.3. Host age preference and suitability Both no-choice and choice tests were conducted to determine G. brasiliensis and L. japonica host age preference and host age suitability. Because D. suzukii larvae developed into pupae in 5–6 days under our quarantine room conditions, four different age categories (1, 2, 3 or 4 day old larvae) were used in the no-choice test. To obtain the four age categories in separate dishes simultaneously, a new diet dish was exposed to the fly colony for 6 h each day over 4 consecutive days. Ten larvae from each age category were then collected and transferred to plastic vials (separate vials for each age category) for a 6 h exposure period to a single adult female G. brasiliensis or L. japonica. After which, half of the exposed hosts (i.e., 5 hosts) from each vial were dissected within 24 h to determine the presence or absence of parasitoid eggs, while the other half were held for the emergence of adult flies or wasps. The difference between percentage parasitism estimated from dissected hosts and percentage parasitism determined from insects reared to the adult stage provides information on the parasitoid larval mortality, or initial parasitism and realized adult parasitoid emergence (Wang and Messing, 2002). After adult emergence had ceased, all remaining host pupae were reconstituted in water for 1 day and then dissected under a microscope to determine the presence or absence of developed flies or

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parasitoids (to determine if flies or wasps had failed to emerge from the fly puparium). Percentage parasitism of the reared hosts was estimated based on the number of developed flies and wasps from both reared adults and the reconstituted dissections. There were 25 initial replicates for each host age and parasitoid species combination tested. However, because there was lower parasitism of ‘old hosts’ (age categories 3 or 4 days), we added additional replicates for these age categories to obtain samples of 20–30 emerged females for the measurement of developmental time and female body size (i.e., hind tibia length). The developmental time (egg to adult) and sex of emerged wasps were also obtained from this no-choice trial. Using the same methodology, a choice test was conducted that simultaneously presented 5 young (1 day old) and 5 old (4 day old) larvae to a female wasp for 6 h. All exposed hosts were dissected within 48 h following the exposure to determine the parasitism of each age group (20 replicates).

2.4. Life-time fecundity The age-specific reproductive potential of G. brasiliensis and L. japonica was measured on larvae of D. suzukii. Newly emerged (< 12 h old) female and male parasitoids were paired, isolated, and provided with 30 or 45 host larvae every 2 or 3 days, respectively, in a fresh plastic vial. At the end of each 2- or 3-day period, surviving wasps were transferred to a freshly provisioned vial and the previously exposed host larvae were held for the emergence of adult flies and wasps. The number, sex and date of emerged wasps were recorded. Each replicate continued until the female parasitoid died (if the male died before the female wasp, a new male was added in case the female would need multiple mating to maximize its life-time reproduction). Dead females were dissected within 24 h, and the number of mature eggs was

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recorded to assess the residual egg load. From this data set we determined female longevity and survival rate, offspring production, offspring sex ratio, and developmental time from egg to adult. Mean number of offspring produced per day was estimated based on the total number of offspring produced during each host exposure time interval. Life table fertility parameters calculated were net reproductive rate (Ro), intrinsic rate of natural increase (r), mean generation time (T), and doubling time (DT). The r was estimated according to the equation: ∑ e-rx lx mx = 1 where x is female age in days, lx is the age-specific survival rate, and mx is the number of daughters produced per female alive at age x. The R0 is given by R0 = ∑ lxmx. The T in days is given by T = ln R0/r, and the TD in days is DT = ln (2)/r (e.g., Wang et al., 2016b). There were 25 replicates (i.e., 25 females) for each wasp species.

2.5. Data analysis Results are presented as mean ± SE. Because body size of female wasps varied among individuals, mature egg load was analyzed using Generalized Linear Model (GLM) with a normal distribution and an identity link function by considering the effects of parasitoid age, body size, and the interaction of age × body size. Differences in mature egg load between G. brasiliensis and L. japonica were compared using Analysis of Variance (ANOVA). For the Host age preference and suitability trial, data for the 1 and 2 day old age groups were pooled (young age group), as were data for the 3 and 4 day old age groups (old age group). This was done to analyze effects of host age on parasitoid offspring fitness because, first, the number of older hosts parasitized was low and, second, there was no difference in parasitism between the two young groups of hosts or between the two old groups of hosts. Effects of host age on the

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offspring fitness (offspring survival, developmental time, body size and sex ratio) and the parasitoid’s host age preference were analyzed by one-way or two-way ANOVA. Prior to analyses, percentage data were logit transformed as needed to normalize the variance. Adult longevity data were subjected to survival analyses with a log-rank test. All analyses were performed using JMP Pro ver13 (SAS 2013, Cary, NC).

3. Results 3.1. Egg maturation dynamics Ganaspis brasiliensis females emerged with 40.5 ± 3.0 mature eggs and mature egg load reached ~80 mature eggs after 2–3 days, whereas L. japonica females emerged with 62.3 ± 5.2 mature eggs and mature egg load peaked after 1–2 days to ~100 mature eggs, when the females were deprived of hosts but provided with a constant source of adult males (Fig. 1). Overall, G. brasiliensis contained fewer mature eggs than L. japonica, regardless of female age (species: 2 = 124.0, df = 1, P < 0.001; age: 2 = 27.7, df = 5, P < 0.001; species × age: 2 = 2.5, df = 5, P = 0.031). Body size (hind tibia length) of dissected females was not different among the six age classes for G. brasiliensis (F5,138 = 1.7, P = 0.142) or L. japonica (F5,154 = 1.1, P = 0.381). After pooling females from all age categories for each species, the hind tibia of G. brasiliensis (0.560 ± 0.003 mm, n = 144) was longer than that of L. japonica (0.538 ± 0.003 mm, n = 160) (F1,302 = 25.00, P < 0.001). Because body size for each species was similar across the six age categories, mature egg load was further analyzed using ANOVA and Tukey's pairwise comparison. The mature egg load increased with the female age (G. brasiliensis: 2 = 134.1, df = 5, P < 0.001; L. japonica: 2 = 55.8, df = 5, P < 0.001; Fig. 1) and body size (G. brasiliensis: 2 = 46.1, df = 1, P < 0.001; L. japonica: 2 = 46.2, df = 5, P < 0.001). The interaction between these two factors

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affected the mature egg load of G. brasiliensis (2 = 17.8, df = 5, P = 0.003), but not L. japonica (2 = 4.6, df = 5, P = 0.461).

3.2. Host age preference and suitability In the no-choice test, host age affected parasitism by G. brasiliensis (F3,96 = 4.1, P = 0.009) and L. japonica (F3,96 = 11.7, P < 0.001), with a greater number of younger (1–2 day old) than older (3–4 day old) host larvae parasitized (Fig. 2). There was no difference in the number of hosts parasitized by either parasitoid within the younger and older host age categories (Fig. 2). Data were pooled for the two younger (1 and 2 days) age categories and for the two older (3 and 4 days) age categories to analyze the effects of host age on parasitoid offspring fitness and sex ratio, as described previously. There was no difference in parasitism levels based on data from dissections of exposed hosts or rearing to adults for either age category for G. brasiliensis (younger: F1,98 = 0.6, P = 0.431; older: F1,98 = 3.4, P = 0.068; Fig. 3A) or L. japonica (younger: F1,98 = 0.001, P = 0.967; older: F1,98 = 0.5, P = 0.495; Fig. 3B). This suggests that host age did not affect offspring survival to emergence. Developmental time was affected by offspring sex, with males developing faster than females for both G. brasiliensis (F1,79 = 6.2, P < 0.001; Fig. 4A) and L. japonica (F1,91 = 9.7, P < 0.001; Fig. 4B). However, within each sex there was no effect of host age on developmental time for G. brasiliensis (F1,79 = 1.3, P = 0.209; Fig. 4A) or L. japonica (F1,39 = 0.2, P = 0.876; Fig. 4B). Between species, L. japonica developed faster than G. brasiliensis for both males (F1,93 = 13.8, P < 0.001) and females (F1,73 = 16.7, P < 0.0001). Host age category did not affect the body size (hind tibia length) of developed female G. brasiliensis (young: 0.543 ± 0.009 mm, old: 0.539 ± 0.005 mm; F1,51 = 0.188, P = 0.667) or L. japonica (young: 0.549 ± 0.003 mm, old: 0.540 ± 0.006 mm; F1,39 = 1.800, P = 0.185). Similarly, host age

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category did not affect offspring sex ratio (% females) for G. brasiliensis (young: 37.4 ± 5.9 %, old: 40.0 ± 9.7%; F1,39 = 0.071, P = 0.791) or L. japonica (young: 49.2 ± 7.2%, old: 61.7 ± 14.5%; F1,35 = 0.026, P = 0.872). In the choice tests, both G. brasiliensis (F1,39 = 5.3, P < 0.05) and L. japonica (F1,39 = 5.3, P < 0.05) preferred to attack younger (1 day old) than older (4 day old) host larvae (Fig. 5). Parasitism of the younger larvae (G. brasiliensis: 47.7 ± 5.6 %; L. japonica: 56.9 ± 7.5 %) was about two times higher than that of the older larvae (G. brasiliensis: 22.4 ± 4.8 %; L. japonica: 21.5 ± 6.6 %).

3.3. Life-time fecundity and demographical indexes Ganaspis brasiliensis adult females survived 17.7  1.4 days (range: 326 days) and produced 98.3  11.8 offspring per female, while L. japonica survived 18.7  1.1 days (range: 730 days) and produced 107.2  9.9 offspring per female. Both parasitoid species began oviposition within 2 days after emergence, with offspring production peaking within 5–10 days and then generally decreasing thereafter (Fig. 6). Similarly, percentage female progeny generally decreased with increasing maternal age (the higher ratio of female offspring for G. brasiliensis after 20 days corresponded to lower offspring production; Fig. 6). Adult longevity (2 = 0.154, df = 1, P = 0.691) and offspring production (F1,49 = 0.349, P = 0.557) were similar for G. brasiliensis and L. japonica. Residual mature egg load in the ovaries of deceased females were 15.4  3.4 and 32.9  5.7 for G. brasiliensis and L. japonica, respectively. The estimated demographic parameters of both parasitoids were similar. Net reproduction rate was 39.9 and 47.3; intrinsic rate of increase was 0.130 and 0.138; mean generation time was 28.5 and 28.1 days; and doubling time was 5.4 and 5.0 days for G. brasiliensis and L. japonica, respectively.

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4. Discussion This study provides a first report on key biological traits of G. brasiliensis and L. japonica, two Asian parasitoids that are candidates for the classical biological control of D. suzukii in North America. We show that both figitids have similar biological parameters, including egg maturation dynamics, host age preference and suitability, and life-time fecundity. We found that adult females of both figitids emerged with high mature egg load, which significantly increased by day 1 (L. japonica) or day 2 (G. brasiliensis) and remained relatively similar thereafter, although G. brasiliensis did have a small increase in egg load on day 8 (Fig. 1). A parasitoid’s total offspring production, rather than egg production, depends on mature egg load, adult female lifespan, hosts encountered, and oviposition behaviors (Ellers et al., 2000). Ovarian dynamics is thus central to understanding parasitoid foraging behavior and efficiency as the ovaries’ physiological status may determine, for example, pre-oviposition duration and oviposition rate (Jervis et al., 2001). Egg maturation varies among parasitoid species; most are synovigenic (emerge with few or a partial life-time complement of mature eggs, with more eggs maturing throughout their adult life), and some proovigenic (emerge with their full lifetime complement of mature eggs) although strict proovigeny is rare (reviewed in Jervis et al., 2001). Among only a few listed proovigenic species (Jervis et al. 2001), two are figitid larval parasitoids, L. boulardi and Trybliographa rapae Westwood (Hymenoptera: Figitidae) (Kopelman and Chabora, 1992). In our study, both G. brasiliensis and L. japonica may be considered as weak proovigenic; both species share features with other proovigenic parasitoids, including a short preoviposition period, short adult life span, and decreasing fecundity with increasing adult female age (Jervis et al., 2001). Proovigenic egg maturation maximizes egg load

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early in the adult parasitoid’s life, which would be beneficial if they were time- rather than egglimited. In fact, D. suzukii often occurs in great abundance for short periods, thus favoring proovigenic parasitoid species. Both G. brasiliensis and L. japonica attacked all larval age categories, but preferred younger hosts (Figs. 2 and 3). However, host age did not significantly affect offspring fitness (survival, development time, body size) or sex ratio. Kopelman and Chabora (1984) report that with increased host age at oviposition, L. boulardi development is accelerated, as ecdysis to the third instar strictly coincides with host pupariation. Similarly, G. xanthopoda larvae molt into the third-instar in synchrony with host pupariation (Melk and Govind, 1999). Although we found that both G. brasiliensis and L. japonica generally developed a little faster on the older than the younger hosts (Fig. 4), the difference was not statistically significant. Because both defense capability and physiological suitability of the host can vary with its age, the parasitoid’s selection of suitable hosts can be critical to its development. The optimal host acceptance of a parasitoid is often based on a combination of host defenses, host qualities, and host endocrine changes (Vinson and Iwantsch, 1980). Some koinobionts can manipulate host physiology and feeding behavior to their own benefit, thus fitness is not always a simple positive function to host size or age and may depend on physiological and nutritional compatibility between the two organisms (Harvey and Strand, 2002). In this case, both G. brasiliensis and L. japonica are solitary koinobiont endoparasitoids, but we saw no impact of host age. A lack of a positive relationship between adult oviposition preference and offspring fitness may result from older host larvae having better defensive systems or burrowing further into the plant substrate, therefore being physically more difficult to reach (e.g., Wang et al., 2009). In the current study, host larvae were provided in 1–2 thick artificial diet substrate and the larvae were observed feeding at various

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depths in the diet. It is possible that old D. suzukii larvae might feed deeper and move more actively than younger larvae in the diet, which could favor the selection of young hosts by both parasitoids. Further studies are needed to determine how host larval feeding behavior and withinfruit distribution in relation to fruit size and host age might affect the host location by these larval Drosophila parasitoids. Life table parameters can be used to estimate a parasitoid’s development and fitness on a host species and have been applied to compare the relative efficiency of several pupal parasitoid species or populations on D. suzukii (e.g., Rossi Stacconi et al., 2015; Wang et al., 2016b). We developed basic life table parameters for G. brasiliensis and L. japonica on D. suzukii and found each to be relatively similar. This was somewhat surprising as the G. brasiliensis population we evaluated is a specialist on D. suzukii (Daane et al., 2016; Nomano et al., 2017) while L. japonica seems to have a wider host range (Kimura and Suwito, 2015; Mitsui and Kimura, 2010; Novkovic et al., 2011). The information gained through this quarantine work can be applied to mass rearing and field release programs for G. brasiliensis and L. japonica if approved for field release in North America. However, further detailed evaluations of these candidate parasitoids are needed to compare their relative efficiency in host fruits, responses to different host densities, host specificity, and climatic adaptability as well as potential interactions among these different candidate parasitoid species.

Acknowledgements Funding for the study was supported by the USDA APHIS (Farm Bill award 14-8130-0463), the U.S. Department of Agriculture National Institute of Food and Agriculture, Specialty Crops Research Initiative Agreement No. 2015-51181-24252, the California Cherry Board, and the

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University of California Agricultural and Natural Research Grant. We thank Kristine Chang, Yeonji Joen, John Hutchins and Dominique Shield (University of California, Berkeley) for assistance with insect rearing and research in the quarantine and Antonio Biondi (University of Catania, Italy) for valuable advice and/or information. We thank Betsey Miller, Jeffrey Miller and Vaughn Walton (Oregon State University), Emilio Guerrieri and Massimo Giorgini (Institute for Sustainable Plant Protection, CNR, Portici, Italy), Matthew Buffington (USDA Agricultural Research Service, Systematic Entomology Laboratory, Washington D.C., USA), and Yoohan Song (Gyeongsang University, Jinju, South Korea) for helping with the importation of parasitoid material from South Korea.

References Andreazza, F., Bernardi, D., dos Santos, R.S.S., Garcia, F.R.M., Oliveira, E.E., Botton, M., Nava, D.E., 2017. Drosophila suzukii in Southern neotropical region: Current status and future perspectives. Neotrop. Entomol. 46, 591-605. doi: 10.1007/s13744-017-0554-7. Asplen, M.K., Anfora, G., Biondi, A., Choi, D.S., Chu, D., Daane, K.M., Gibert, P., Gutierrez, A.P., Hoelmer, K.A., Hutchison, W.D., Isaacs, R., Jiang, Z.L., Karpati, Z., Kimura, M.T., Pascual, M., Philips, C.R., Plantamp, C., Ponti, L., Vetek, G., Vogt, H., Walton, V.M., Yu, Y., Zappala, L., Desneux, N., 2015. Invasion biology of spotted wing drosophila (Drosophila suzukii): a global perspective and future priorities. J. Pest Sci. 88, 469-494. doi: 10.1007/s10340-015-0681-z. Beers, E.H., Van Steenwyk, R.A., Shearer, P.W., Coates, W.W., Grant, J.A., 2011. Developing Drosophila suzukii management programs for sweet cherry in the western United States. Pest Manag. Sci. 67, 1386-1395. doi: 10.1002/ps.2279.

19

Biondi, A., Mommaerts, V., Smagghe, G., Vinuela, E., Zappala, L., Desneux, N., 2012. The nontarget impact of spinosyns on beneficial arthropods. Pest Manag. Sci. 68, 1523-1536. doi: 10.1002/ps.3396. Biondi, A., Wang, X.G., Miller, J.C., Miller, B., Shearer, P.W., Zappala, L., Siscaro, G., Walton, V.W., Hoelmer, K.A., Daane, K.M., 2017. Innate olfactory responses of Asobara japonica toward fruits infested by the invasive spotted wing drosophila. J. Insect Behav. 30, 495-506. doi: 10.1007/s10905-017-9636-y. Buffington, M.L., Forshage, M., 2016. Redescription of Ganaspis brasiliensis (Ihering, 1905), new combination (Hymenoptera: Figitidae), a natural enemy of the invasive Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae). Proc. Entomol. Soc. Wash. 118, 1-13. doi: 10.4289/0013-8797.118.1.1. Burrack, H.J., Fernandez, G.E., Spivey, T., Kraus, D.A., 2013. Variation in selection and utilization of host crops in the field and laboratory by Drosophila suzukii Matsumara (Diptera: Drosophilidae), an invasive frugivore. Pest Manag. Sci. 69, 1173-1180. doi: 10.1002/ps.3489. Carton, Y., Boulétreau, B., van Alphen, J.J.M., van Lenteren, J.C., 1986. The Drosophila parasitic wasps. In: M., A., Carson, H.L., Thompson, J.N., Eds.), The Genetics and Biology of Drosophila. Academic Press, London, pp. 347–394. Chabert, S., Allemand, R., Poyet, M., Eslin, P., Gibert, P., 2012. Ability of European parasitoids (Hymenoptera) to control a new invasive Asiatic pest, Drosophila suzukii. Biol. Control 63, 40-47. doi: 10.1016/j.biocontrol.2012.05.005.

20

Cini, A., Anfora, G., Escudero-Colomar, L.A., Grassi, A., Santosuosso, U., Seljak, G., Papini, A., 2014. Tracking the invasion of the alien fruit pest Drosophila suzukii in Europe. J. Pest Sci. 87, 559-566. doi: 10.1007/s10340-014-0617-z. Daane, K.M., Wang, X.G., Biondi, A., Miller, B., Miller, J.C., Riedl, H., Shearer, P.W., Guerrieri, E., Giorgini, M., Buffington, M., van Achterberg, K., Song, Y., Kang, T., Yi, H., Jung, C., Lee, D.W., Chung, B.K., Hoelmer, K.A., Walton, V.M., 2016. First exploration of parasitoids of Drosophila suzukii in South Korea as potential classical biological agents. J. Pest Sci. 89, 823-835. doi: 10.1007/s10340-016-0740-0. Dalton, D.T., Walton, V.M., Shearer, P.W., Walsh, D.B., Caprile, J., Isaacs, R., 2011. Laboratory survival of Drosophila suzukii under simulated winter conditions of the Pacific Northwest and seasonal field trapping in five primary regions of small and stone fruit production in the United States. Pest Manag. Sci. 67, 1368-1374. doi: 10.1002/ps.2280. Diepenbrock, L.M., Rosensteel, D.O., Hardin, J.A., Sial, A.A., Burrack, H.J., 2016a. Seasonlong programs for control of Drosophila suzukii in southeastern US blueberries. Crop Prot. 81, 76-84. doi: 10.1016/j.cropro.2015.12.012. Diepenbrock, L.M., Swoboda-Bhattarai, K.A., Burrack, H.J., 2016b. Ovipositional preference, fidelity, and fitness of Drosophila suzukii in a co-occurring crop and non-crop host system. J. Pest Sci. 89, 761-769. doi: 10.1007/s10340-016-0764-5. Ellers, J., Sevenster, J.G., Driessen, G., 2000. Egg load evolution in parasitoids. Am. Nat. 156, 650-665. doi: 10.1086/316990. Hamby, K.A., Bellamy, D.E., Chiu, J.C., Lee, J.C., Walton, V.M., Wiman, N.G., York, R.M., Biondi, A., 2016. Biotic and abiotic factors impacting development, behavior, phenology,

21

and reproductive biology of Drosophila suzukii. J. Pest Sci. 89, 605-619. doi: 10.1007/s10340-016-0756-5. Harvey, J.A., Strand, M.R., 2002. The developmental strategies of endoparasitoid wasps vary with host feeding ecology. Ecology 83, 2439-2451. doi: 10.2307/3071805. Haye, T., Girod, P., Cuthbertson, A.G.S., Wang, X.G., Daane, K.M., Hoelmer, K.A., Baroffio, C., Zhang, J.P., Desneux, N., 2016. Current SWD IPM tactics and their practical implementation in fruit crops across different regions around the world. J. Pest Sci. 89, 643651. doi: 10.1007/s10340-016-0737-8. Jervis, M.A., Heimpel, G.E., Ferns, P.N., Harvey, J.A., Kidd, N.A.C., 2001. Life-history strategies in parasitoid wasps: a comparative analysis of 'ovigeny'. J. Anim. Ecol. 70, 442458. doi: 10.1046/j.1365-2656.2001.00507.x. Kaçar, G., Wang, X.G., Biondi, A., Daane, K.M., 2017. Linear functional response by two pupal Drosophila parasitoids foraging within single or multiple patch environments. PLoS One 12(8): e0183525. doi: 10.1371/journal.pone.0183525. Kacsoh, B.Z., Schlenke, T.A., 2012. High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster. PLoS One 7, e34721. doi: 10.1371/journal.pone.0034721. Kasuya, N., Mitsui, H., Ideo, S., Watada, M., Kimura, M.T., 2013. Ecological, morphological and molecular studies on Ganaspis individuals (Hymenoptera: Figitidae) attacking Drosophila suzukii (Diptera: Drosophilidae). Appl. Entomol. Zool. 48, 87-92. doi: 10.1007/s13355-012-0156-0.

22

Kenis, M., Tonina, L., Eschen, R., van der Sluis, B., Sancassani, M., Mori, N., Haye, T., Helsen, H., 2016. Non-crop plants used as hosts by Drosophila suzukii in Europe. J. Pest Sci. 89, 735-748. doi: 10.1007/s10340-016-0755-6. Kimura, M.T., Novkovic, B., 2015. Local adaptation and ecological fitting in host use of the Drosophila parasitoid Leptopilina japonica. Ecol. Res. 30, 499-505. doi: 10.1007/s11284015-1244-8. Kimura, M.T., Suwito, A., 2015. Altitudinal patterns of abundances and parasitism in frugivorous drosophilids in west Java, Indonesia. J. Nat. Hist. 49, 1627-1639. doi: 10.1080/00222933.2015.1005709. Klick, J., Lee, J.C., Hagler, J.R., Bruck, D.J., Yang, W.Q., 2014. Evaluating Drosophila suzukii immunomarking for mark-capture research. Entomol. Exp. Appl. 152, 31-41. doi: 10.1111/eea.12197. Knoll, V., Ellenbroek, T., Romeis, J., Collatz, J., 2017. Seasonal and regional presence of hymenopteran parasitoids of Drosophila in Switzerland and their ability to parasitize the invasive Drosophila suzukii. Sci. Rep. 7, e40697. doi: 10.1038/srep40697. Kopelman, A.H., Chabora, P.C., 1984. Immature stages of Leptopilina boulardi (Hymenoptera: Eucoilidae), a protelean parasite of Drosophila spp. (Diptera: Drosophilidae). Ann. Entomol. Soc. Am. 77, 264-269. doi: 10.1093/aesa/77.3.264. Kopelman, A.H., Chabora, P.C., 1992. Resource variability and life-history parameters of Leptopilina boulardi (Hymenoptera, Eucolidae). Ann. Entomol. Soc. Am. 85, 195-199. Lee, J.C., Bruck, D.J., Curry, H., Edwards, D., Haviland, D.R., Van Steenwyk, R.A., Yorgey, B.M., 2011. The susceptibility of small fruits and cherries to the spotted-wing drosophila, Drosophila suzukii. Pest Manag. Sci. 67, 1358-1367. doi: 10.1002/ps.2225.

23

Lee, J.C., Dreves, A.J., Cave, A.M., Kawai, S., Isaacs, R., Miller, J.C., Van Timmeren, S., Bruck, D.J., 2015. Infestation of wild and ornamental noncrop fruits by Drosophila suzukii (Diptera: Drosophilidae). Ann. Entomol. Soc. Am. 108, 117-129. doi: 10.1093/aesa/sau014. Mazzetto, F., Marchetti, E., Amiresmaeili, N., Sacco, D., Francati, S., Jucker, C., Dindo, M.L., Lupi, D., Tavella, L., 2016. Drosophila parasitoids in northern Italy and their potential to attack the exotic pest Drosophila suzukii. J. Pest Sci. 89, 837-850. doi: 10.1007/s10340-0160746-7. Melk, J.P., Govind, S., 1999. Developmental analysis of Ganaspis xanthopoda, a larval parasitoid of Drosophila melanogaster. J. Exp. Biol. 202, 1885-1896. Miller, B., Anfora, G., Buffington, M., Daane, K.M., Dalton, D.T., Hoelmer, K.M., Stacconi, M.V.R., Grassi, A., Ioriatti, C., Loni, A., Miller, J.C., Ouantar, M., Wang, X.G., Wiman, N.G., Walton, V.M., 2015. Seasonal occurrence of resident parasitoids associated with Drosophila suzukii in two small fruit production regions of Italy and the USA. Bull. Insectology 68, 255-263. Mitsui, H., Kimura, M.T., 2010. Distribution, abundance and host association of two parasitoid species attacking frugivorous drosophilid larvae in central Japan. Eur. J. Entomol. 107, 535540. Mitsui, H., Van Achterberg, K., Nordlander, G., Kimura, M.T., 2007. Geographical distributions and host associations of larval parasitoids of frugivorous Drosophilidae in Japan. J. Nat. Hist. 41, 1731-1738. doi: 10.1080/00222930701504797. Nomano, F.Y., Kasuya, N., Matsuura, A., Suwito, A., Mitsui, H., Buffington, M.L., Kimura, M.T., 2017. Genetic differentiation of Ganaspis brasiliensis (Hymenoptera: Figitidae) from

24

East and Southeast Asia. Appl. Entomol. Zool. 52, 429-437. doi: 10.1007/s13355-017-04930. Nomano, F.Y., Mitsui, H., Kimura, M.T., 2015. Capacity of Japanese Asobara species (Hymenoptera; Braconidae) to parasitize a fruit pest Drosophila suzukii (Diptera; Drosophilidae). J. Appl. Entomol. 139, 105-113. doi: 10.1111/jen.12141. Novkovic, B., Mitsui, H., Suwito, A., Kimura, M.T., 2011. Taxonomy and phylogeny of Leptopilina species (Hymenoptera: Cynipoidea: Figitidae) attacking frugivorous drosophilid flies in Japan, with description of three new species. Entomol. Sci. 14, 333-346. doi: 10.1111/j.1479-8298.2011.00459.x. Poyet, M., Havard, S., Prévost, G., Chabrerie, O., Doury, G., Gibert, P., Eslin, P., 2013. Resistance of Drosophila suzukii to the larval parasitoids Leptopilina heterotoma and Asobara japonica is related to haemocyte load. Physiol. Entomol. 38, 45-53. doi: 10.1111/phen.12002. Poyet, M., Le Roux, V., Gibert, P., Meirland, A., Prevost, G., Eslin, P., Chabrerie, O., 2015. The wide potential trophic niche of the Asiatic fruit fly Drosophila suzukii: The key of its invasion success in temperate Europe? PLoS One 10(11): e0142785. doi: 10.1371/journal.pone.0142785. Rossi Stacconi, M.V., Buffington, M., Daane, K.M., Dalton, D.T., Grassi, A., Kaçar, G., Miller, B., Miller, J.C., Baser, N., Ioriatti, C., Walton, V.M., Wiman, N.G., Wang, X., Anfora, G., 2015. Host stage preference, efficacy and fecundity of parasitoids attacking Drosophila suzukii in newly invaded areas. Biol. Control 84, 28-35. doi: 10.1016/j.biocontrol.2015.02.003.

25

Rossi Stacconi, M.V., Panel, A., Baser, N., Ioriatti, C., Pantezzi, T., Anfora, G., 2017. Comparative life history traits of indigenous Italian parasitoids of Drosophila suzukii and their effectiveness at different temperatures. Biol. Control 112, 20-27. doi: 10.1016/j.biocontrol.2017.06.003. Schilthuizen, M., Nordlander, G., Stouthamer, R., Van Alphen, J.J.M., 1998. Morphological and molecular phylogenetics in the genus Leptopilina (Hymenoptera : Cynipoidea : Eucoilidae). Syst. Entomol. 23, 253-264. doi: 10.1046/j.1365-3113.1998.00049.x. Stewart, T.J., Wang, X.-G., Molinar, A., Daane, K.M., 2014. Factors limiting peach as a potential host for Drosophila suzukii (Diptera: Drosophilidae). J. Econ. Entomol. 107, 17711779. doi: 10.1603/ec14197. Tochen, S., Dalton, D.T., Wiman, N., Hamm, C., Shearer, P.W., Walton, V.M., 2014. Temperature-related development and population parameters for Drosophila suzukii (Diptera: Drosophilidae) on cherry and blueberry. Environ. Entomol. 43, 501-510. doi: 10.1603/en13200. Van Timmeren, S., Horejsi, L., Larson, S., Spink, K., Fanning, P., Isaacs, R., 2017. Diurnal activity of Drosophila suzukii (Diptera: Drosophilidae) in highbush blueberry and behavioral response to irrigation and application of insecticides. Environ. Entomol. 46, 1106-1114. doi: 10.1093/ee/nvx131. Van Timmeren, S., Isaacs, R., 2013. Control of spotted wing drosophila, Drosophila suzukii, by specific insecticides and by conventional and organic crop protection programs. Crop Prot. 54, 126-133. doi: 10.1016/j.cropro.2013.08.003. Vinson, S.B., Iwantsch, G.F., 1980. Host suitability for insect parasitoids . Annu. Rev. Entomol. 25, 397-419. doi: 10.1146/annurev.en.25.010180.002145.

26

Walsh, D.B., Bolda, M.P., Goodhue, R.E., Dreves, A.J., Lee, J., Bruck, D.J., Walton, V.M., D., O.N.S., Zalom, F.G., 2011. Drosophila suzukii (Diptera: Drosophilidae): invasive pest of ripening soft fruit expanding its geographic range and damage potential. J. Integ. Pest Manage. 2, G1–G7. doi: doi.org/10.1603/IPM10010. Wang, X.G., Johnson, M.W., Daane, K.M., Yokoyama, V.Y., 2009. Larger olive fruit size reduces the efficiency of Psyttalia concolor, as a parasitoid of the olive fruit fly. Biol. Control 49, 45-51. doi: 10.1016/j.biocontrol.2009.01.004. Wang, X.G., Kaçar, G., Biondi, A., Daane, K.M., 2016a. Foraging efficiency and outcomes of interactions of two pupal parasitoids attacking the invasive spotted wing drosophila. Biol. Control 96, 64-71. doi: 10.1016/j.biocontrol.2016.02.004. Wang, X.G., Kaçar, G., Biondi, A., Daane, K.M., 2016b. Life-history and host preference of Trichopria drosophilae, a pupal parasitoid of spotted wing drosophila. Biocontrol 61, 387397. doi: 10.1007/s10526-016-9720-9. Wang, X.G., Messing, R.H., 2002. Newly imported larval parasitoids pose minimal competitive risk to extant egg-larval parasitoid of tephritid fruit flies in Hawaii. Bull. Entomol. Res. 92, 423-429. doi: 10.1079/ber2002181. Wang, X.G., Stewart, T.J., Biondi, A., Chavez, B.A., Ingels, C., Caprile, J., Grant, J.A., Walton, V.M., Daane, K.M., 2016c. Population dynamics and ecology of Drosophila suzukii in Central California. J. Pest Sci. 89, 701-712. doi: 10.1007/s10340-016-0747-6. Woltz, J.M., Lee, J.C., 2017. Pupation behavior and larval and pupal biocontrol of Drosophila suzukii in the field. Biol. Control 110, 62-69. doi: 10.1016/j.biocontrol.2017.04.007.

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Figure legends

Fig. 1. Egg maturation dynamics of differently aged female G. brasiliensis and L. japonica when deprived of hosts but provided with food. Values are mean  SE, and within each species bars bearing different letters are significantly different (Tukey's HSD, P < 0.05).

Fig. 2. Effect of host larval age of D. suzukii on the parasitism by G. brasiliensis and L. japonica in a no-choice test using a short (6 h) exposure period. Values are mean  SE, and within each species bars bearing different letters are significantly different (Tukey's HSD, P < 0.05).

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Fig. 3. Effects of two different host larval ages (1–2 day vs. 3–4 day old) of D. suzukii on percentage parasitism over a 6 h exposure period for (A) G. brasiliensis and (B) L. japonica, as determined both by dissecting exposed host immediately after exposure and rearing exposed hosts to adult flies or parasitoids. Values are mean  SE, and within each species bars bearing different letters are significantly different (Tukey's HSD, P < 0.05).

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Fig. 4. Effects of two different host larval ages (1–2 day vs. 3–4 day old) of D. suzukii on offspring development time from egg to adult emergence for (A) G. brasiliensis and (B) L. japonica. Values are mean  SE, and within each species bars bearing different letters are significantly different (Tukey's HSD, P < 0.05).

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Fig. 5. Host age preference by (A) G. brasiliensis and (B) L. japonica between two different ages (1 day vs. 4 day old) of host D. suzukii larvae in a choice test. Values are mean  SE, and within each sub-figure bars bearing different letters are significantly different (Tukey's HSD, P < 0.05).

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Fig. 6. Lifetime reproduction and offspring sex ratio by G. brasiliensis (A) and L. japonica (B) when parasitizing D. suzukii. Values (means ± SE) are number of progeny produced daily and percentage of female offspring (n = 25 females).

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Wang et al. Aspects of the biology and reproductive strategy of two Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii

Highlights  Two figitid parasitoids, Ganaspis brasiliensis and Leptopilina japonica, were evaluated as potential parasitoids for classical biological control of Drosophila suzukii.  G. brasiliensis and L. japonica adult females eclosed with a high mature egg-load.  Both adult parasitoid species preferentially attacked younger host larvae, but host age did not affect their fitness (survival, developmental time, and body size) or sex ratio.  Demographic parameters, such as lifetime fecundity, were determined for both parasitoid species and showed strong similarity in their performance as parasitoids of D. suzukii.

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