Laboratory and field studies supporting the development of Heringia calcarata as a candidate biological control agent for Eriosoma lanigerum in New Zealand S. D. M. Gresham, J. G. Charles, M. W. R. Sandanayaka & J. C. Bergh
BioControl Journal of the International Organization for Biological Control ISSN 1386-6141 BioControl DOI 10.1007/s10526-013-9530-2
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Author's personal copy BioControl DOI 10.1007/s10526-013-9530-2
Laboratory and field studies supporting the development of Heringia calcarata as a candidate biological control agent for Eriosoma lanigerum in New Zealand S. D. M. Gresham • J. G. Charles • M. W. R. Sandanayaka • J. C. Bergh
Received: 1 February 2013 / Accepted: 12 June 2013 Ó International Organization for Biological Control (IOBC) 2013
Abstract Studies supporting a project seeking to introduce Heringia calcarata (Loew) (Diptera: Syrphidae) to New Zealand (NZ) to supplement biological control of woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) are reported. Annual surveys of H. calcarata presence and abundance in a Virginia, USA apple orchard revealed a bimodal distribution, with peaks in mid-June and mid-September. In the field, female H. calcarata oviposited on sentinel apple shoots infested with WAA, providing an efficient method for egg collection and larval production. Similarly, most fieldcollected females readily deposited viable eggs on WAA colonies in laboratory cages, demonstrating that mated females will oviposit in captivity. Survivorship of eggs and larvae transported to NZ was good, yielding adult flies in containment in Auckland. Adult, virgin female H. calcarata reared from eggs in
captivity developed mature oocytes, providing an important step toward future mating studies in containment. Oviposition and larval feeding studies examined aspects of the intraguild interactions between H. calcarata and Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae), the sole biological control agent of WAA in NZ. Field tests using paired sentinel apple shoots with a non-parasitized or parasitized WAA colony revealed that although H. calcarata deposited eggs on both parasitized and nonparasitized colonies, fewest eggs were deposited on heavily parasitized colonies. Feeding studies showed that larval H. calcarata consumed fewer mummified aphids or aphids in an earlier stage of parasitization than non-parasitized aphids.
Handling Editor: Stefano Colazza.
Introduction
S. D. M. Gresham (&) J. C. Bergh Alson H. Smith, Jr. Agricultural Research and Extension Center, Virginia Tech, Winchester, VA 22602, USA e-mail:
[email protected] J. G. Charles M. W. R. Sandanayaka The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Private Bag 92169, Auckland 1142, New Zealand
Keywords Heringia calcarata Syrphidae Eriosoma lanigerum Aphididae Aphelinus mali Biological control
In recent decades, introducing classical biological control agents to new systems has become more complex (Babendreier et al. 2005), requiring a better understanding of the biology and ecology of the target system to maximize efficacy and minimize risk (van Lenteren et al. 2006). Studies in the native range of candidate agents can greatly benefit this process
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(Charles 2012). The predaceous syrphid, Heringia calcarata (Loew), is native to the United States and an important member of a guild of natural enemies of woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) in Mid-Atlantic apple orchards (Bergh and Short 2008; Short and Bergh 2004). Heringia calcarata gained the attention of New Zealand (NZ) entomologists following recent, damaging outbreaks of WAA in NZ apple orchards that resulted from chemical and climatic disruption of populations of the introduced parasitoid, Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae). Since A. mali is effectively the sole biological control agent of WAA in NZ (Shaw and Walker 1996), the sensitivity of this system to disturbance highlighted the need for increased stability of WAA biological control. Given its apparent specialization on WAA in apple ecosystems in Virginia (Bergh and Short 2008; Short and Bergh 2004), H. calcarata was deemed the most suitable, potential candidate to supplement the effects of A. mali in NZ orchards. In 2010, importation of H. calcarata from Virginia to Plant and Food Research’s NZ quarantine and containment facility in Auckland was authorized by the NZ Environmental Risk Management Authority (now the Environmental Protection Authority). Future regulatory action to release H. calcarata from containment is contingent upon demonstrating that it will not negatively impact the NZ environment, its native arthropods, or the effectiveness of existing biological control. To address these requirements, respective studies in NZ and the USA will need to examine the potential effects of H. calcarata on selected native arthropods and its impact on A. mali populations through intraguild predation. In the short term, periodic shipments of H. calcarata larvae and eggs to NZ will be required to develop (1) practical procedures for transporting live specimens to NZ, and (2) techniques for maintaining larvae and adults in a containment laboratory. In the longer term, the success of this project will almost certainly require the development of sustained rearing capabilities in containment. In the first instance, risk-assessment logistics usually require a culture in containment so that the risk to native fauna can be assessed as these putative native hosts become available in the wild through one or more seasons. Subsequently, NZ regulatory authorities typically require imported natural enemies to be reared through at least one
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generation (egg to egg) before release to ensure that individuals are free of unwanted parasitoids and entomopathogens. As initial steps toward achieving these goals, the following studies were conducted. Female H. calcarata oviposit on arboreal WAA colonies (Short and Bergh 2004) and in the soil above WAA colonies on apple roots (Bergh and Short 2008), but are most commonly and easily seen while foraging at or near the base of apple trees. Thus, the seasonal phenology and abundance of female H. calcarata was assessed via annual surveys of flies observed foraging in this manner. Oviposition on sentinel apple shoots infested with a WAA colony and deployed near the base of apple trees was evaluated, toward satisfying the need for an efficient and effective method to collect H. calcarata eggs. Logistical considerations associated with packaging and transporting H. calcarata eggs and larvae from the USA to NZ were addressed via several shipments in 2012. As a prelude to further studies on rearing H. calcarata in captivity, information on some fundamental aspects of their reproductive physiology and behavior associated with initiating and sustaining captive populations was needed. Female syrphids are synovigenic: they emerge with poorly developed ovaries and typically must obtain carbohydrates and amino acids to become sexually mature (Chambers 1988; Schneider 1969; Thomson 1999). Assuming that females having a complement of mature oocytes are in a physiological state of readiness to mate, we investigated egg development in laboratory-reared, virgin female H. calcarata. Given that the deposition of fertile eggs can be used as a proxy for successful mating but that some insect species that mate readily in captivity will not necessarily oviposit readily (Frank et al. 2010), the propensity of fieldcollected female H. calcarata to lay eggs in captivity, and the viability of those eggs, was examined. Finally, aspects of the intraguild interactions between H. calcarata and A. mali were examined in laboratory and field studies in which the effect of parasitization of WAA by A. mali on H. calcarata oviposition and larval feeding was assessed. Syrphids are known to prey on parasitized aphids, leading to coincidental intraguild predation (Almohamad et al. 2008; Kindlmann and Ruzicka 1992; Meyhofer and Klug 2002; Pineda et al. 2007), which can either have negative effects on parasitoid population dynamics and suppress the effectiveness of biological control (Erbilgin
Author's personal copy Laboratory and field studies supporting the development of H. calcarata
et al. 2004; Jazzar et al. 2008; Martinou et al. 2010; Putra et al. 2009) or stabilize parasitoid populations (Nakazawa and Yamamura 2006), and predator presence can enhance biological control (Colfer and Rosenheim 2001; Gontijo 2011; Kindlmann and Ruzicka 1992; Walker 1985).
Materials and methods Insects WAA colonies for rearing H. calcarata and for experimental trials were maintained on one- to fouryear-old apple trees, Malus domestica Borkhausen, of various cultivars growing in a 1:1 mix of peat moss and loamy soil in 10 l plastic pots. Trees were held in a greenhouse or outdoors in 3.6 9 1.8 9 1.8 m screened cages (BioQuip Products, Inc., Rancho Dominquez, CA, USA) at Virginia Tech’s Alson H. Smith, Jr. Agricultural Research and Extension Center (AHSAREC), Winchester, VA, USA. Spider mites and fungal pathogens were controlled as required, utilizing miticides and fungicides considered to have no effect on aphids or dipterans (Virginia Cooperative Extension Service 2012). To reduce A. mali populations in WAA colonies on some of the potted trees, spinetoram was applied on 25 April and 2 June, 2012. WAA colonies on some trees were allowed to become parasitized by A. mali. On other trees, parasitoid-free colonies were initiated by transferring 1st instar aphids from colonies with low levels of parasitization and holding the trees in isolation. Heringia calcarata larvae were reared from fieldcollected eggs (see below) and adults were reared from larvae collected from WAA colonies in orchards and from field-collected eggs. Eggs and larvae were held in 50 9 9 mm Petri dishes (BD-Falcon Tight-Fit Lid Dishes, Fisher Scientific, Pittsburg, PA, USA) in covered translucent plastic boxes (30 9 20 9 8 cm) in a growth chamber (Percival Scientific Inc., Perry, IA, USA) at 25 °C and under a 15 h photoperiod. Larvae fed ad libitum on WAA provided regularly during their development. H. calcarata seasonal phenology Bergh and Short (2008) reported that all adult H. calcarata captured while foraging near the base of apple trees were female and that most sightings
occurred between 13:00 and 16:00. The flight behavior of female H. calcarata near and at the base of apple trees is quite distinctive and cannot be mistaken for that of another species in Virginia orchards. Generally, their flight consists of rapid, small amplitude, lateral oscillations within larger amplitude lateral oscillations and occurs within about 1 cm from the soil surface. Unlike other syrphids, H. calcarata females foraging at the soil surface do not usually hover in place, although they may do so within the tree canopy. Between 2008 and 2012, annual surveys of H. calcarata presence and abundance were conducted from mid-April through mid-October in experimental apple blocks at the AHS-AREC. In 2008, a 0.65 ha block of six-year-old ‘Buckeye Gala’ and ‘Idared’ on M.26 rootstock was used, while a 1.1 ha block of ‘Smoothee Golden’ on M.26 rootstock (one-year-old in 2009) was used from 2009 to 2012. Using an airblast sprayer, routine disease management occurred in the block used in 2008 and experimental insect management programs were applied to 13 % of the trees. The block used from 2009 through 2012 was managed for diseases and treated with a minimal insect management program. Generally, surveys within each season were conducted once per week, between 14:00 and 15:00, on days considered conducive to adult activity (i.e. warm and dry), although prolonged periods of inclement weather and other factors precluded surveys during a few, different weeks each season. Using an all-terrain vehicle, an observer drove slowly up and down each row of the orchard block and recorded the number of flies observed walking or flying at the base of each tree. Each survey was typically completed in about 20 min. To account for the possibility that an individual fly could be recorded at the base of more than one tree during each survey, data were reported as the total number of sightings per survey. Collecting H. calcarata eggs Sections of apple shoot (10–15 cm) with at least one WAA colony were pruned from the potted trees, placed individually in 50 ml glass vials with water, and secured to the vial with Parafilm M (Pechiney Plastic Packaging, Chicago, IL, USA) at the base of the shoot. Although the colonies used were not necessarily free of aphids parasitized by A. mali, those with the lowest levels of parasitization were selected on each deployment date. Between 8:00 and 9:00 on each of 19 days
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between 8 June and 21 September 2012, shoots were deployed (5–18 shoots per day; n = 233 shoots) by inserting the vials into holes created in the soil beneath the canopy and near the base of mature apple trees at the AHS-AREC, so that the shoot was essentially perpendicular to the ground. Periodic herbicide treatments around the base of trees maintained a soil surface that was mostly free of vegetation. Shoots were deployed on warm, relatively calm days considered optimal for adult H. calcarata foraging activity. Mean (±SE) daily maximum and minimum temperature and wind speed on days when shoots were deployed was 30.0 ± 0.75 °C, 15.8 ± 1.01 °C, and 3.3 ± 0.44 km h-1, respectively. After 8–12 h, shoots were collected and examined for H. calcarata eggs under a dissecting microscope at 209 magnification. Eggs were identified based on their characteristic exochorionic sculpturing (Short and Bergh 2005).
Transporting H. calcarata from Virginia to New Zealand Heringia calcarata eggs were collected and larvae were reared using methods described above. To transport eggs, small apple shoot sections (\2 cm), each with one or two eggs and a WAA colony in situ, were placed individually into 1.5 ml microcentrifuge tubes (Fisher Scientific, Pittsburg, PA, USA) containing a small plug of facial tissue at the tip and sealed with a tight-fitting lid that had been punctured several times for ventilation. Larger shoot sections ([2 cm) were wrapped in slightly damp facial tissue and placed individually in 30 ml plastic vials (Fisher Scientific, Pittsburg, PA, USA) with a tight-fitting, punctured lid. To transport larvae, specimens were placed individually in small, plastic Petri dishes (50 9 9 mm) with a tight-fitting lid containing a short section of apple shoot with a WAA colony. The microcentrifuge tubes, vials, and Petri dishes were packed in a polystyrene box (20 9 20 9 25 cm) containing an icepack and lined with a plastic bag. The box was taped closed and packed inside a sealed cardboard box. Unhatched eggs and larvae of H. calcarata were transported to NZ on four occasions between 8 June and 26 September 2012. Three shipments were sent via DHLTM International Express courier service and one was carried as hand luggage. Upon arrival in NZ, larvae were immediately transferred to Petri dishes with WAA
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from NZ, whereas unhatched eggs were held with their substrate for several days before being inspected, to allow for larval eclosion and development. Oviposition by H. calcarata in captivity In June, 2011(n = 8 flies) and June, July, and September 2012 (n = 14 flies), female H. calcarata observed at the base of apple trees were netted and placed individually in 1.7 l translucent plastic containers (100 mm high 9 150 mm diam.) with a screened lid. Containers were provisioned with white, granulated sugar (*1 g), crushed fresh bee pollen (*0.5 g) (High DesertÒ, CC Pollen Co., Phoenix, AZ, USA), a piece of cotton dental wick in a 30 ml vial of water, and a *10 cm section of WAA-infested apple shoot in a vial of water. The containers were held in a room with overhead florescent lights (450 lux) at 24–26 °C, 15 h photoperiod, and 50–80 % RH. After 24 h, eggs deposited on each shoot were counted in situ using a dissecting microscope. To determine egg viability, shoots exposed to 11 of the females from 2012 were held for five days in Petri dishes in a growth chamber at 25 °C and 15 h photoperiod. Under a microscope, each egg was prodded gently with a small, fine-bristled brush. Hatched eggs were empty of contents, had an exit slit on the anterior, ventral surface, and collapsed readily when probed, whereas unhatched eggs were turgid. Upon death, nine flies were dissected using methods described below and the dimensions of their remaining mature oocytes were measured using a Dinolite-X-scope digital microscope (BigC, Torrance, CA, USA). Ovary development in virgin female H. calcarata Upon emergence, adult female H. calcarata were placed individually in cylindrical plastic cages (16 9 7.5 cm diam.) with open bottom and screened top in a flower pot (10.2 cm diam.) containing 100 ml of moist, coarse sand. Cages were provisioned with the same food and water sources described above and a 5 cm section of apple shoot as a perch and held under overhead, florescent lighting (450 lux) in room at 24–26 °C, 50–80 % RH and 15 h photoperiod. The initial objectives of this study were to examine the rate of ovary development and the effects of access to pollen and male presence on ovary development in caged, laboratory-reared, virgin female H. calcarata.
Author's personal copy Laboratory and field studies supporting the development of H. calcarata
However, due to premature mortality of some flies compared with that expected, based on results from Short (2003) under similar conditions, sample sizes were small and unbalanced and treatment factors were confounded. Consequently, a simplified, descriptive approach was taken. The caged flies were observed daily and collected if dead or euthanized if alive at 5–15 days post-emergence. Flies were placed individually in 1.5 ml plastic microcentrifuge tubes with 70 % ethanol and stored at -20 °C (n = 27) for 1–20 days, after which they were thawed, softened, and rehydrated for 10–30 min in a 10 % potassium hydroxide solution, then rinsed and soaked for 30–60 min in 60 % phosphate buffered saline (Ungureanu 1972). Flies were dissected in the saline solution on a glass microscope slide under a microscope at 15–309 magnification. The abdomen was removed from the thorax using fine-tipped forceps, and the abdominal sternites were removed using number 0 insect pins in a pin clamp tool and fine forceps. The ovaries were teased apart from the alimentary tract and surrounding tissues and stained with drops of 10 % methylene blue for 10 min, rinsed in 60 % phosphate buffered saline. Ovaries were photographed using a Dinolite-X-scope digital microscope at 50–2009 magnification and measurements were made using the digital measurement tool.
Effect of parasitism by A. mali on oviposition by H. calcarata Apple shoot sections (5–15 cm) with a WAA colony were pruned from potted trees and deployed beneath mature apple trees as described above. The dimension of each colony was estimated and showed a surface area of *1–2 cm2. Two shoots, one with a parasitized and one with a non-parasitized colony of approximately equal size were placed 30–50 cm from the trunk of each tree and separated by 50–100 cm. On each of eight days between 31 May and 6 July 2012, five pairs of shoots were deployed at 9:00 h. Mean (±SE) daily maximum and minimum temperature and wind speed was 32.3 ± 1.7 °C, 18.3 ± 1.4 °C, and 3.5 ± 0.6 km h-1, respectively. At 17:00 h, the shoots were retrieved and the number of H. calcarata eggs, live and mummified aphids were counted and colony dimensions were measured under a dissecting microscope.
Effect of parasitism by A. mali on feeding by H. calcarata larvae Late instar and adult WAA were collected from potted trees and sorted under a dissecting microscope according to their status of parasitism by A. mali. Nonparasitized aphids were purplish-pink and actively extruding waxy filaments. Aphids containing a developing A. mali larva were pale yellow and often showed a dark spot internally, which was the gut contents of the developing parasite (Bodenheimer 1947). Black, mummified aphids in the final stage of parasitism were very apparent (Viggiani 1984). In choice feeding studies, second or third instar H. calcarata (five- to sixdays-old) that had been reared from eggs on WAA and then starved for 24 h were offered 15 non-parasitized aphids and 15 aphids containing a developing parasite or 15 non-parasitized and 15 mummified aphids, with 25 replicates of each treatment. Aphids and larvae were placed together in glass dishes (1.5 cm diameter 9 0.5 cm deep) sealed with Parafilm M and held in a growth chamber (Percival Scientific Inc., Perry, IA, USA) at 25 °C and 15 h photoperiod for 24 h, after which the aphids in each dish that had not been preyed upon were counted. Thirty-six larvae from the choice test were used in a subsequent no-choice trial. Following the choice test, each larva was starved for 8–12 h, then placed in a glass dish with 30 non-parasitized aphids, 30 aphids containing a developing parasite, or 30 mummified aphids (n = 12 larvae per treatment). Based on their prior experience in the choice trial, larvae were assigned to treatments in a balanced completely randomized 2 9 3 factorial design with six replications. The numbers of aphids in each dish that had not been preyed upon were counted after 24 h. Data analysis Descriptive statistics were used for results from fly surveys, egg collections in the field, captive female oviposition and egg viability, and ovary development in captive females. Dimensions of the oocytes in laboratory-reared and field-collected females were compared using the Wilcoxon signed-rank test. All statistical analyses used JMP statistical software (SAS Institute 2010) and were based on a significance level of a \ 0.05. The effect of parasitism by A. mali on oviposition by female H. calcarata on field-deployed
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shoots was analyzed using ANCOVA, with number of eggs as the response variable, parasitism as the treatment factor, and number of live WAA per colony as a covariate. Spatio-temporal variation was accounted for by deploying treatments in pairs with WAA colonies of approximately equal size. Although colony size (i.e. number of live WAA) was found to differ among replicates (F1,78 = 13.96, P = 0.0004), an initial assessment revealed that there was not a significant interaction between parasitism and the number of live WAA (F1, 76 = 0.342; P = 0.560) on the number of eggs laid and that variance appeared to be homogenous between treatment levels. The frequency of shoots with C1 egg was compared between those with parasitized and non-parasitized colonies using a 2 9 2 v2 contingency table, as the number of live WAA did not have a significant effect on presence/ absence of H. calcarata eggs and was therefore omitted. A post-hoc analysis of egg numbers from shoots with parasitized colonies was conducted using logistic regression analysis to assess the effect of percentage parasitization on the presence or absence of H. calcarata eggs. The numbers of parasitized and nonparasitized aphids consumed in each choice feeding trial were compared using the paired t test. The effect of prior choice-test allocation (i.e. non-parasitized aphids vs those with a developing parasite or non-parasitized aphids vs mummified aphids) and aphid stage of parasitism (non-parasitized, with developing parasite, mummified) on the number of aphids consumed in nochoice feeding trials was assessed as a 2 9 3 factorial design using a two-way ANOVA. Means were separated using the Tukey–Kramer test.
Results
Collecting H. calcarata eggs Female H. calcarata deposited C1 egg on 29 % of the 233 WAA-infested apple shoots deployed in orchards for 8–12 h per day between June and September, 2012. Across all shoots, 0.78 ± 0.01 SE eggs per shoot were deposited, and among the 68 shoots with C1 egg, females laid 2.4 ± 0.21 SE eggs per shoot (range = 1–9). There were pronounced temporal differences in egg deposition rates: shoots with C1 egg were recorded most frequently in June (45 %; n = 132 shoots) and September (26 %; n = 35 shoots) and least frequently in July (0 %; n = 20 shoots) and August (6 %; n = 46 shoots). Transporting H. calcarata from Virginia to New Zealand In total, 178 eggs and larvae were sent from Virginia to a quarantine facility in Auckland, NZ (Table 1). Transport time via courier ranged from three to ten days, with variation due to delays associated with both the courier and border agencies in the US and NZ. Delayed delivery of the 16 June shipment resulted in higher mortality than with others (Table 1). In total, 124 adult flies were generated in NZ, representing 79 % of the number of eggs and larvae recovered upon delivery to quarantine. Sex ratio (including those that died in their puparium as sexable adults) was skewed toward males (53$ and 76#). Highest mortality was the failure to emerge from puparia, or parasitoidism by Phthorima bidens (Davis) (Hymenoptera: Ichneumonidae) which was not detected until adults emerged from H. calcarata puparia (Table 1). Parasitization of larvae by P. bidens was recorded only from the 24 June shipment, due to the inclusion of field-collected larvae.
H. calcarata seasonal phenology Oviposition by H. calcarata in captivity Across all years, the earliest H. calcarata sighting was during the week of 23–29 April (Julian week 17), although in most years activity began in mid-May (Fig. 1). Seasonal fly activity and abundance was bimodal, with peaks in mid-June and mid-September. The fewest sightings occurred between mid-July and mid-August. Flies were observed as late as 20 October (Julian week 42), although activity typically diminished rapidly after the first week of October.
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In 2011 and 2012, respectively, 5 of 8 and 12 of 15 field-collected, caged female H. calcarata laid C1 egg on a WAA colony within 24 h. Among the females that oviposited, the mean (±SE) number of eggs laid in 2011 and 2012 was 3.6 ± 1.05 and 13.8 ± 3.5, respectively (range = 1–40). Assessments of the viability of eggs from 11 females in 2012 revealed that 98 % ± 1.0 SE hatched.
Author's personal copy Laboratory and field studies supporting the development of H. calcarata
Fig. 1 Mean (±SE) weekly number of sightings of female Heringia calcarata during annual surveys (2008–2012) of flies foraging at or near the base of apple trees in an experimental orchard in Virginia, USA
Table 1 Details of shipments of Heringia calcarata eggs and larvae sent from Virginia to a quarantine containment facility in Auckland, New Zealand in 2012 Senta
Receiveda
Transport method
# shipped Eggs
Larvae
# larvae arrived (% survival)b
# lost in culturec
# died in puparia
# adults
09 Jun
12 Jun
DHL
22
15
31 (84)
0
0
31
16 Jun
26 Jun
DHL
20
6
15 (58)
0
1
14
24 Jun
25 Jun
Carry on
27
30
57 (100)
3
7 ? 11d
36
27 Sep
02 Oct
DHL
21
37
54 (93)
3
8
43
a
New Zealand standard time
b
Percentage of H. calcarata recovered as live larvae or puparia at Plant and Food Research, NZ
c
Mostly as very young larvae
d
Parasitised by Phthorima bidens; attacked in field as larvae and yielded adult parasitoids from puparia
Ovary development in virgin female H. calcarata Female H. calcarata emerged with a pair of partially developed ovaries. The ovaries continued to develop after emergence, with the follicles increasing in number, elongating, increasing in size, and ultimately producing apparently mature eggs. Oocyte development rate varied greatly among individuals. Seemingly mature oocytes showing the characteristic exochorionic sculpturing reported by Short and Bergh (2005) were observed in five-days-old individuals, although some individuals [15-days-old did not contain mature oocytes. In general, mature oocytes were most commonly observed in seven- to ten-days-
old individuals. Mature oocytes (n = 20) were 0.20 ± 0.007 SE mm wide 9 0.623 ± 0.006 SE mm long. Oocytes from the ovaries of gravid, field-collected females (n = 9) were 0.256 ± 0.010 SE mm wide 9 0.646 ± 0.010 SE mm long and were significantly wider (Z = 3.56; P = 0.0004) and longer (Z = 2.1; P = 0.034) than oocytes of laboratory-reared females.
Effect of parasitism by A. mali on oviposition by H. calcarata At least one egg was deposited on 30 of the 40 pairs of shoots deployed. The mean (±SE) numbers of eggs on
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shoots with parasitized (1.5 ± 0.34) and non-parasitized (1.75 ± 0.42) WAA colonies were not significantly different (F1,77 = 0.28; P = 0.595), nor were the number of parasitized and non-parasitized shoots with C1 egg (v2 = 0.05; df = 1; P = 0.82). A posthoc analysis of shoots with parasitized colonies revealed a significant negative effect of percentage parasitization on the presence of H. calcarata eggs (v2 = 4.207; df = 1; P = 0.040). For each one percent increase in parasitization, the likelihood of finding an egg decreased by 4.5 % (likelihood odds ratio = 0.96). Effect of parasitism by A. mali on feeding by H. calcarata larvae In choice tests, larvae consumed significantly more non-parasitized aphids than mummified aphids (t = 30.7; df = 24; P \ 0.0001). Similarly, larvae consumed significantly more non-parasitized aphids than aphids containing a developing parasite (t = 6.4; df = 24; P \ 0.0001) (Fig. 2a). Prior larval experience in the choice tests did not significantly affect the results of the no-choice trial (F1,29 = 0.54; P = 0.47). There was a significant treatment effect on the number of aphids consumed in the no-choice feeding trial (F2,29 = 56.9; P \ 0.0001): larvae consumed significantly more non-parasitized aphids than aphids with a developing parasite or mummified aphids, and significantly more aphids with a developing parasite than mummified aphids (Fig. 2b).
Discussion Deploying WAA-infested apple shoot sections in orchards was comparatively efficient and effective for collecting H. calcarata eggs. Bergh and Short (2008) collected eggs by destructively sampling WAA colonies from young, potted apple trees deployed in orchards for 48-h intervals. While this method yielded eggs, it was very labor and plant resource intensive and severely limited the number of possible deployment locations per collection interval. Deploying individual shoot sections enabled much better accommodation of temporal and spatial variations in fly presence and relative abundance. Despite the relatively short daily deployment interval (8–12 h), H. calcarata eggs were collected on 15 of 19 days
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(a)
(b)
***
***
a b
c
Fig. 2 Mean (?SE) number of aphids consumed by second instar H. calcarata larvae during 24-h a choice and b no-choice laboratory feeding trials in which woolly apple aphids that were not parasitized by Aphelinus mali, contained a developing A. mali larva, or had been mummified by A. mali were offered alone or in combination. Asterisks above pairs of bars in (a) indicate significant difference (P \ 0.0001) by paired t test. Letters above bars in (b) indicate a significant difference (P \ 0.05) according the Tukey–Kramer test
during which shoots were deployed. On one occasion a fly was observed alighting on a shoot within 15 min. We also showed that WAA colonies were readily cultivated in screened, sleeve cages on shoots of mature apple trees in an orchard: in future, the use of colonies propagated on shoot sections on orchard trees could reduce the need to maintain potted trees. Seasonal differences in the percentage of sentinel shoots upon which eggs were laid conformed well to the results of our survey of H. calcarata presence and abundance and to data from previous studies of its seasonal patterns of oviposition on WAA colonies on potted, sentinel trees (Bergh and Short 2008). Survey data from five consecutive seasons showed that foraging activity at the base of apple trees was most prevalent in June and September, coinciding with the periods during which eggs were most frequently laid on sentinel shoots. The numerous and quite regularly
Author's personal copy Laboratory and field studies supporting the development of H. calcarata
spaced peaks in the number of sightings recorded in spring and late summer during our annual surveys may provide some, albeit speculative, insights into the voltinism of H. calcarata in the field. Bergh and Short (2008) reported a mean (±SE) generation time (egg– adult) of 19.3 ± 0.74 days at constant 25 °C in the laboratory. Given that ambient temperatures in the field throughout much of the activity period of H. calcarata often exceed 25 °C and would accelerate developmental rates, it may be that each peak represents a generation. The low activity levels recorded in mid-summer may suggest aestivation of flies during the hottest portion of the year. If these activity patterns do reflect multivoltinism, this should be considered a positive biological attribute for its intended role in NZ orchards. Overall, a high percentage of H. calcarata eggs and larvae survived transport to NZ by commercial courier. Bergh (unpublished data) showed that larval H. calcarata can withstand food deprivation for at least one week without adverse effects on their resumption of feeding and development to the adult stage. This suggested that H. calcarata should be amenable to transport over multiple days, even if aphids are depleted, which has been confirmed by the success of our shipments and in particular by the shipment that was delayed by more than one week. Ultimately, the success of this project will require an ability to sustain H. calcarata in captivity over multiple generations. With some exceptions, syrphids are considered difficult to mate in captivity (Schneider 1969). Successful reproduction with captive-reared syrphids has been achieved with Pseudodorus clavatus (Belliure and Michaud 2001), Episyrphus balteatus de Geer (Branquart and Hemptinne 2000; Hart and Bale 1997), Syrphus luniger (Meigen) (Medvey 1988), and Syrphus corollae (Fabricus) (Barlow 1961; Frazer 1972). Research with these syrphid species has shown that high light conditions, access to flowers or floral substitutes, sufficient cage size, and presence of host aphids are important factors associated with successful reproduction in captivity. The main impediment to sustained rearing of H. calcarata is our poor understanding of its mating behavior in the field or in captivity. Indeed, we have never captured or observed adult male H. calcarata in the field. However, we have demonstrated two key factors that will influence future research on this goal. First, field-collected females readily laid viable eggs on WAA-infested apple shoots
in the laboratory and thus provide an effective alternative for collecting eggs. This suggests strongly that the deposition of viable eggs can serve as indication of successful mating in captivity. To date, preliminary trials in which virgin H. calcarata females were paired with males in various kinds of cages provisioned with food and a WAA-colony as an oviposition stimulus resulted, on some occasions, in the deposition of only non-fertile eggs (Charles unpublished; Short 2003). Second, caged, virgin H. calcarata females developed oocytes that appeared mature, suggesting that females can reach a state of physiological readiness to mate in captivity. Clearly, the effects of exogenous conditions and cues on mating by H. calcarata need to be examined systematically. Our results provide new insights into the intraguild interactions between A. mali and H. calcarata that are relevant to future deliberations regarding the potential impact of releasing Heringia in NZ. In absolute terms, female H. calcarata did not appear to discriminate between WAA colonies with and without parasitized aphid mummies in the field, conforming to results from other studies showing that unhatched H. calcarata eggs were found in WAA colonies parasitized by A. mali (Bergh unpublished data). In relative terms however, the percentage of mummified aphids in individual colonies appeared to influence oviposition. Eggs were deposited less frequently in colonies with a higher percentage of mummies, suggesting that heavily parasitized colonies are less likely to be exposed to intraguild predation by H. calcarata. Similar apparent discrimination has been shown for adults of other syrphid species. Female E. balteatus laid fewer eggs on Acyrthosiphon pisum Harris colonies with a large proportion of aphid mummies parasitized by Aphidius ervi Haliday than on non-parasitized colonies (Almohamad et al. 2008). E. balteatus also laid fewer eggs on colonies of Aphis fabae Scopoli that were in late stages of parasitization by Lysiphlebus fabarum Marshall compared with non-parasitized colonies, but this difference was not detected during the early stages of colony parasitization (Meyhofer and Klug 2002). Pineda et al. (2007) demonstrated that E. balteatus avoided laying eggs near green peach aphid, Myzus persicae Sulzer, colonies parasitized by Aphidius colemani Viereck. Oviposition site selection was mediated by a number of cues, including exudates and honeydew, which were not produced by mummified aphids.
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Feeding studies using choice and no-choice assays showed that H. calcarata larvae discriminated between parasitized and non-parasitized aphids and that the extent to which this occurred depended on the stage of parasitism. A. mali developing within mummified aphids seem to be at negligible risk from predation by H. calcarata. Akinlosotu (1978) reported similar results for E. balteatus larvae, which consumed fewer cabbage aphid, Brevicoryne brassicae (L.) mummies parasitized by Diaeretiella rapae (McIntosh) than nonparasitized aphids but did not appear to discriminate between non-parasitized aphids and those in earlier stages of parasitism. Almohamad et al. (2008) found that pupal weight and survival of E. balteatus larvae were lower on diet of A. pisum parasitized by A. ervi than on a diet of non-parasitized aphids. In combination, the results from our oviposition and feeding studies suggest that although intraguild predation on A. mali by H. calcarata likely occurs in apple orchards, adverse effects on A. mali populations and its role in WAA biological control are mitigated by some degree of oviposition site selection behavior by females and discriminate feeding behavior by larvae. These effects would be further mitigated by the much higher population density and reproductive capacity of A. mali than H. calcarata. Finally, there is ample evidence to suggest that A. mali is compatible with a broad range of predators and contributes importantly to WAA biological control in concert with them. Asante (1997) reported that many predatory arthropods have been documented in association with WAA colonies in numerous countries where A. mali is established. Nicholas et al. (2005) found that the combination of predators and A. mali regulated WAA populations in Australian orchards in most years. In West Virginia, USA apple orchards, Brown and Schmitt (1994) showed that syrphids and A. mali were the predominant natural enemies of WAA. Bergh (unpublished data) confirmed this for orchards in Virginia, USA and revealed that H. calcarata was the most abundant predator. In Washington, USA orchards, Gontijo et al. (2012) reported that generalist syrphid species were the most common WAA predators. H. calcarata was also found within WAA colonies. Walker (1985) and Gontijo (2011) reported that predator exclusion inhibited the suppression of WAA colonies and that A. mali alone did not often suppress WAA populations, suggesting that the generalist predators had an additive effect on biological control in orchards treated with selective pesticides.
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In summary, A. mali is spatially and temporally sympatric with a range of arthropod predators of WAA in many of the world’s apple-producing regions. Field and laboratory studies in the native range of H. calcarata have shown that both species contribute significantly to biological control of WAA. Consequently it is expected that introducing H. calcarata to NZ would enhance the efficacy of this system. In combination, the results of these studies significantly advance the goals and needs of our project and will help guide further development of H. calcarata as a classical biological control agent for WAA in NZ. However, to realize the full potential of this project, further research is needed to better understand the reproductive biology and ecology of H. calcarata. Identification of the conditions necessary for H. calcarata mating is of special importance to sustained rearing and potentially for non-target impact assessments studies in NZ, given that the most thorough non-target assessments would involve both larval feeding and oviposition studies. Information about the floral resources used by adult H. calcarata in Virginia will be important to determining whether appropriate resources are available in NZ. Understanding the geographic range of H. calcarata in North America and its phenology and developmental rate at various temperatures will enable accurate climate matching with conditions in NZ and other countries that have also expressed interest in this species for WAA biological control. Acknowledgments We thank Jean Engelman and the summer interns in Virginia, and Vicky Davis in New Zealand, who provided technical support. This research was supported by a scholarship from Pipfruit New Zealand Ltd.
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Author Biographies S. D. M. Gresham This research was part of a master’s project of Sean Gresham focused on ecological and logistical considerations for the development of Heringia calcarata as a candidate biological control agent for Eriosoma lanigerum. J. G. Charles is a senior scientist in applied entomology, with current programmes including classical biological control of codling moth and woolly apple aphid, and assessing the risks to New Zealand’s native insect fauna posed by exotic natural enemies.
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M. W. R. Sandanayaka is a scientist studying the host selection and oviposition behavior of insect natural enemies and the feeding behavior of sucking insects using EPG. J. C. Bergh is a professor of High Value Horticultural Crops Entomology, emphasizing the development and validation of new and improved monitoring and management tactics for orchard and vineyard pests using the principals of applied insect ecology and behavior. Bergh served as major advisor to senior author, Sean Gresham.
