BIOLOGY OF APHEUNUS CERTUS AND ITS SUSCEPTIBILITY TO INSECTICIDES USED FOR SOYBEAN
APHID MANAGEMENT
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
ANDREW JORDAN FREWIN
In partial fulfillment of requirements
for the degree
Master of Science
August, 2010
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¦+¦ Canada ABSTRACT
BIOLOGY OF APHEUNUS CERTUS AND SUSCEPTIBILITY TO INSECTICIDES FOR SOYBEAN APHID MANAGEMENT
Andrew J. Frewin Advisors: University of Guelph, 2010 Dr. Rebecca H. Halle« Dr. Arthur W. Schaafsma Dr. Jonathan Schmidt Dr. A. Bruce Broadbent
The soybean aphid (Aphis glycines Matsumura) is a serious pest of soybean (Glycine max L.) in
North America. Currently, soybean aphid management has focused on foliar applied insecticides. However, there is a growing body of evidence that natural enemies can regulate soybean aphid populations. Because soybean aphid has a high intrinsic rate of increase, natural enemies alone are not always a viable management option for soybean aphid. Integration of natural enemies and other management options will be necessary for reliable soybean aphid management. Determining the compatibility of natural enemies and insecticides is extremely important. In 2006, the exotic soybean aphid parasitoid Aphelinus certus Yanosh was discovered in Ontario. This parasitoid was found to be distributed throughout southern Ontario making it an ideal candidate for incorporation into soybean aphid management decisions. Temperature- dependent developmental parameters were investigated, as well as the susceptibility of this parasitoid to insecticides currently or potentially registered for soybean aphid management in
Canada. Acknowledgements I would like to thank my co-advisors Drs. Rebecca H. Hallett and Arthur W. Schaafsma for giving me the opportunity to take part in this project and providing me with such a wonderful learning experience. To the rest of my advisory committee, Drs. Jonathan
Schmidt and A. Bruce Broadbent, thank you for your advice and support.
Thank you to the funding agencies of this project: Agriculture and Agri-Food
Canada (AAFC), and the Ontario Soybean Growers. Thank you to Syngenta Crop
Protection Canada, United Agri Products Canada Inc., FMC Corporation, Bayer CropScience Canada, and BioWorks Inc. for generously providing test material.
Thank you to Dr. Keith Hopper for Aphelinus identification. Thank you to Cynthia and Mark for your support and guidance. A huge thank you to all my lab mates and colleagues: Angela, Lisa, Christie, Ziggy, Jamie, Erik, Yingen, and Kristen. Thank you to my parents, Andrea and Wayne, for always supporting my educational pursuits.
Finally, to Angela, thank you for your love, support, and understanding while I've been in thesis-town.
? Table of Contents
Acknowledgements i List of Figures ¡? List of Tables vi
1 Literature Review 1 1.1 Soybean 1 1.2 Soybean aphid 2 1.3 Soybean aphid management 5 1.3.1 Chemical control 6 1.3.2 Biopesticide 10 1.3.3 Host plant resistance 10 1.3.4 Conservation and classical biological control 11 1.4 Biology of Aphelinus certus 14 1.5 Non-target effects of insecticides 22 1.6 Conclusions 26 1.7 Objectives 27 2 Distribution and temperature-dependent development of a North American population of Aphelinus certus 28 2.1 Introduction 28 2.2 Material and methods 30 2.2.1 Survey 30 2.2.2 Rearing methods 30 2.2.3 Development rate experiment 30 2.2.4 Statistical analysis 31 2.3 Results 34 2.3.1 Survey 34 2.3.2 Development 34 2.4 Discussion 41
ii 3 Susceptibility of Aphelinus certus Yanosh to foliar-applied insecticides currently or potentially registered for soybean aphid control 50 3.1 Introduction 50 3.2 Materialsand methods 51 3.2.1 Insects 51 3.2.2 Insecticides 52 3.2.3 Residual contact LC50 52 3.2.4 Direct contact LC50 53 3.2.5 Screening bioassay 54 3.2.6 Statistical analyses 55 3.3 Results 57 3.3.1 Direct and residual contact LC50 57 3.3.2 Screening bioassay 59 3.4 Discussion 62 4 Susceptibility of Aphelinus certus Yanosh to two neonictotinoid seed treatments insecticides used for soybean aphid management 69 4.1 Introduction 69 4.2 Materials and methods 71 4.2.1 Insect rearing 71 4.2.2 Soybean plants 72 4.2.3 Sampling 73 4.2.4 Statistical analysis 73 4.3 Results 75 4.4 Discussion 81
5 General Conclusions and Recommendations 91 6 References 98
in List of Figures
Figure Page
1.1 Aphelinus certus adult emerging from a parasitized soybean aphid. 15 1.2 Scanning electron micrograph of a female Aphelinus certus. 16
2.1 Location of 56 commercial soybean fields surveyed during 35 July-August, 2007.
2.2 Mean temperature-dependent development rate of Aphelinus certus 36 from egg to mummy, and mummy to adult.
2.3 Mean proportion of Aphis glycines parasitized by Aphelinus certus 43 at six constant temperatures and linear regression of the proportion of parasitized A. glycines on temperature.
3.1 Mortality of A. certus 24h after direct contact with five insecticides 60 and one biopesticide currently or potentially used in soybean aphid management in Ontario. Insecticides were applied at ??, 1, and 2 times their recommended rate (RR), for soybean aphid or aphids on architecturally similar crops.
3.2 Mortality of A. certus 48h after direct contact with five insecticides 61 and one biopesticide currently or potentially used in soybean aphid management in Ontario. Insecticides were applied at Vi, 1, and 2 times their recommended rate (RR), for soybean aphid or aphids on architecturally similar crops.
4.1 Parasitism rates of Aphelinus certus on Aphis glycines feeding on 76 soybean plants that were untreated (control) or that received seed treatment with imidacloprid or thiamethoxam.
4.2 Parasitism rates of Aphelinus certus presented with Aphis glycines 77 feeding on soybean plants of different ages. Soybean plants were untreated (control) or receive seed treatments with imidacloprid or thiamethoxam.
4.3 Effect of plant age on net reproductive rate (R0) of the soybean aphid 82 exposed to excised soybean leaves from untreated plants or plants grown from seed treated with imidacloprid or thiamethoxam.
IV 4.4 The effect of plant age on the intrinsic rate of increase (r) of the 83 soybean aphid exposed to excised soybean leaves from untreated plants or plants grown from seed treated with imidacloprid or thiamethoxam.
? List of Tables
Table Page
2.1 Mean (± SE) development times and rate for egg to mummy and 37 mummy to adult lifestages of the parasitoid Aphelinus certus developing on Aphis glycines at 6 constant temperatures.
2.2 Variance analysis of the effect of temperature and sex on the 38 development rate of Aphelinus certus, from egg to mummy and from mummy to adult, developing within Aphis glycines at six constant temperatures.
2.3 Parameter estimates from non-linear iterative regression of 40 developmental data for Aphelinus certus developing within Aphis glycines at six constant temperatures
2.4 Variance analysis of the effect of temperature on parasitism rate 42 of Aphelinus certus, i.e. the proportion of available Aphis glycines parasitized by A certus in a 24h period at six constant temperatures.
3.1 Recommend rates for insecticides currently or potentially used for 56 soybean aphid control in Ontario.
3.2 Direct and residual contact toxicity of insecticides to Aphelinus 58 certus adults 24h following application.
4.1 Results of ANOVA, for the effects of treatment, plantage, and 79 parasitoid presence on the net reproductive rate of the soybean aphid Aphis glycines.
4.2 Results of ANOVA, for the effect of treatment, plant age, parasitoid 80 presence on the intrinsic rate of increase of the soybean aphid, Aphis glycines.
Vl 4.3 The R0 and r of the soybean aphid exposed to excised soybean leaves 84 from untreated plants or plants that received seed treatments of imidacloprid orthiamethoxam.
VII 1 Literature Review
1.1 Soybean Soybean, Glycines max L. (Fabaceae), is an economically important crop in North
America. In Canada, soybean is predominantly grown in Ontario and Quebec, with minor production in Manitoba and Prince Edward Island (Statistics Canada 2009a).
Currently, 1.5 million ha are seeded with soybean in Canada, and 1 million of this is planted in Ontario, Canada's largest producer (Statistics Canada 2009a). In 2009 the average price per tonne of soybean was CAD $375.44 and ca. 3.2 million tonnes were produced nationwide (Statistics Canada 2009b). Soybean is the most valuable crop in terms of gross revenues in Ontario and the fifth most valuable in Canada behind canola, wheat, potatoes, and corn (Statistics Canada 2007). Soybean is sold for human consumption, animal feed, and for use in industrial products including printing inks, biodiesel, waxes, solvents, adhesives, and plastics (Statistics Canada 2007). Soybean is also the largest source of protein feed and second largest source of vegetable oil in the world (USDA 2008). For agronomic reasons including nutrient cycling and pest management, soybean is predominantly grown in rotation with corn and winter wheat in Ontario.
A number of pests, including viruses, fungi, nematodes, and insects are capable of reducing soybean yields and require proactive and/or reactionary management. The principle arthropod pests of soybean in Ontario are bean leaf beetle (Cerotomo trifurcata Forster), green stink bug (Nezara viridula L), brown stink bug (Euschistus
1 servus Say), two-spotted spider mite (Tetranychus uricae Koch), and soybean aphid
(Aphis glycines Matsumura) (OMAFRA 2009a, b). Of these pests, the soybean aphid is the most damaging and challenging to control. Effective pest management is vital for maintaining high yields. Currently, insecticides are the primary tool used to deal with insect infestations of soybean pests (OMAFRA 2009a). However, pest management can be especially challenging for organically grown soybean where the use of synthetic pesticides is prohibited (Martin 2006). Typical yields for organically produced soybean are only 75% of those achieved in conventional systems (Martin 2006, Statistics Canada
2007). However, organically produced soybeans are typically sold for 50% more than conventionally produced beans which make this a valuable niche market (Martin 2006,
Statistics Canada 2007).
1.2 Soybean aphid The soybean aphid is native to southwest Asia (Blackman and Eastop 2000) and was first identified in North America in 2000 (Ragsdale et al. 2004). Soybean aphid is believed to have been accidentally introduced from China or Japan, carried by either an international air travel passenger or with horticultural cargo (Venette and Ragsdale
2004, Michel ef al. 2009). By 2001, it had spread to Ontario (Hunt et al. 2003) and 15
American states. Currently, soybean aphid has been reported in at least 23 American states and 3 Canadian provinces (DiFonzo 2009). Climate models predict that soybean aphid will ultimately spread through the entire soybean growing region of North
America (Venette and Ragsdale 2004). In North America, soybean aphid is heteroecious. The primary (winter) hosts are
Rhamnus cathartica L. and Rhamnus alnifolia L'Her, while its secondary (summer) host is soybean (Voegtlin ei al. 2004, Voegtlin et al. 2005). Soybean aphid are holocyclic: in the spring and summer they reproduce strictly by parthenogenic viviparae on the secondary host, while sexual morphs produce eggs that overwinter on the primary host (Dixon
1973). Olfactory cues are believed to play an important role in secondary host location in the spring. The soybean aphid is attracted to soybean volatiles, whereas it is repelled by non-host volatiles (Du ef al. 1994 as cited by Wu et al. 2004b).
The life cycle of the soybean aphid consists of four nymphal instars and an adult stage. Nymphs can be apterous or alatoid. However the wing pads only become visible at the third-instar if the individual is to develop into an alate adult (Hodgson ef al. 2005).
Adults can either be apterous or alates (Hodgson et al. 2005) and most aphid alates are obligatory migrants (Dixon 1985). Alates occur in response to photoperiod, temperature, host quality, and population density (Dixon 1973, 1985). There is evidence that the proportion of soybean aphid alates increases with decreasing photoperiod
(Hodgson ef al. 2005).
In North America, apterous females emerge from overwintering eggs in March when they can be observed on swelling buds and on the ventral sides of leaves
(Ragsdale ef al. 2004). After three generations on the primary host, alates are produced that migrate to soybean (Ragsdale ef al. 2004). The dispersal capabilities of the soybean aphid are not completely characterized, but their rapid range expansion has been
3 attributed ¡? part to their flight capabilities (Zhang ef al. 2009). in the laboratory, sustained flights of 4.1h duration have been observed under a variety of environmental conditions (Zhang et al. 2008). A reduction in fecundity can result from extended flights
(Zhang et al. 2009). In addition to active flight, many aphids are capable of utilizing low pressure systems for passive long distance migration (Zhu 2006). This mechanism has likely contributed to the rapid range expansion of the soybean aphid.
The developmental rate of soybean aphid is dependent on temperature. The optimal temperature for soybean aphid development is 27.8°C (McCornack et al. 2004).
In a controlled environment chamber the population doubling time at 25°C was ca. 1.5d
(McCornack et al. 2004). However, population doubling times between 2.7 to 13.4d have been observed in the field (Ragsdale et al. 2007).The fecundity of an individual soybean aphid has been estimated between 20.3-63.5 nymphs (McCornack et al. 2004,
Rutledge and O'Neil 2006). Due to its high intrinsic rate of increase, soybean aphid outbreaks can reach economically damaging levels very quickly.
After its initial establishment in Ontario the population cycle of soybean aphid followed an alternating pattern of one year of severe infestation followed by a year with low aphid populations (Bahlai and Sears 2009). However, in recent years there is evidence that this two year cycle is breaking down, resulting in sequential years of moderate levels of aphid infestations (Hager 2009).
The soybean aphid damages soybean by phloem-feeding, transmitting of plant viruses, and by depositing honeydew. Feeding damage has the capacity to reduce plant height, pod number, seed quality, photosynthetic efficiency, the amount of chlorophyll
4 ¡? leaves, and yield (Ostlie 2001, Macedo et al. 2003, Diaz-Montano et al. 2007,
Beckendorf et al. 2008). Honey dew, a sugary waste product of the soybean aphid, is deposited on the leaf surface during feeding and acts as a substrate for the growth of sooty mold, which in turn blocks sunlight and reduces photosynthesis (Nielsen and
Hajek 2005). Soybean aphid is a vector of at least 13 plant viruses. Most important of which is soybean mosaic virus which has the capacity to reduce yields and seed quality
(Hill et al. 2001, Wu ef al. 2004b, Davis and Radcliffe 2008, Gildow et al. 2008).
Yield loss associated with soybean aphid damage ranges from 12-70% (Hunt er al. 2003, Wu ef al. 2004b) and is most severe when infestations occur on younger plants
(Beckendorf ef al. 2008, Catangui ef al. 2009). In Ontario in 2001, the year of soybean aphid's introduction, aphid feeding damage contributed to a reduction in provincial soybean yields of ca. 50% (Hunt ef al. 2003). In addition to direct yield loss, feeding can reduce seed oil content below the economically desirable levels (Beckendorf ef al.
2008).
1.3 Soybean aphid management
Insect pest management in many agricultural systems has relied historically on the use of synthetic insecticides. However, the overuse and misuse of insecticides has had negative consequences on the environment, non-target organisms including humans, and the overall viability of agro-ecosystems. As a result more sustainable and environmentally friendly management strategies have been sought. The concept of integrated pest management (IPM), aims to combine a variety of complementary pest
5 management methods to achieve sustainable crop protection. Although the IPM
concept emerged in the 1960s, its adoption is far from complete (Dent 2000). IPM tactics may include biological, physical, cultural, and chemical control, which must be
facilitated by a thorough knowledge of pest biology and pest monitoring. In most
modern agricultural systems at least some IPM practices are implemented (e.g. crop
rotation, or economic action thresholds) (OMAFRA 2009b). Currently soybean aphid
control in Ontario is predominantly achieved by chemical control in response to
economic action thresholds. However, there is recognition that natural enemies play an
important role in soybean aphid population suppression (OMAFRA 2009b).
1.3.1 Chemical control
Soybean aphid management involves weekly scouting to determine aphid densities and
population trends followed by application of insecticides (NCSRP 2009, OMAFRA 2009a,
b). Insecticide application is recommended when soybean aphid populations exceed 250
aphids per plant and are actively increasing on more than 80% of plants sampled (Baute
2007). Alternatively, seed-applied insecticides can be used in high risk areas to prevent aphid colonization up to 60 days after planting (OMAFRA 2009a), although, foliar-
applied insecticides may still be required in fields planted with seed-treated soybeans
(Johnson and O'Neal 2008).
Four foliar-applied insecticides are currently registered for soybean aphid control
in Ontario (OMAFRA 2009b): two formulations of the active ingredient ?-cyhalothrin
(Matador® 120EC, Syngenta Crop Protection Canada, and Silencer® 120EC, Makhteshim
6 Agan of North America, Inc.) and two of dimethoate (Cygon® 480EC, Cheminova Canada
Inc., and Lagon® 480EC, United Agri-Products Canada Inc.). Additionally, two seed- applied insecticides are available for early season aphid protection. Both are formulations of thiamethoxam (Cruiser® 5 FS, Syngenta Crop Protection Canada, Inc., and Cruiser Maxx Beans®, Syngenta Crop Protection Canada, Inc.). There are also a
number of insecticides that may have potential efficacy against soybean aphid, including
spirotetramat (Movento® 240EC, Bayer CropScience Canada), flonicamid (Beleaf® 50SG, FMC Corporation), mineral oil (Superior 70 Oil, United Agri-Products Canada Inc.) , and
an imidacloprid seed treatment (Gaucho®, Bayer CropScience Canada).
?-cyhalothrin, a second generation phyrethroid, is registered for use in Canada to
control numerous pests on corn, cereal, tree fruits, leafy and fruiting vegetables, and
oilseeds including soybean aphid on soybeans (OMAFRA 2009a). ?-Cyhalothrin is
extremely effective at controlling soybean aphid (Johnson and O'Neal 2008). Pyrethroids
are neurotoxins that cause hyper-excitability, discoordination, convulsions, paralysis,
and finally death in arthropods by binding to the alpha-subunit of voltage-gated sodium
channels on arthropod nerve cells (Zlotkin 2001). In general, pyrethroids have high
contact activity, are effective at low doses, are non-persistent, selective to arthropods,
and have low mammalian toxicity (Zlotkin 2001).
Dimethoate is used to control numerous pests on oilseeds, cereals, leafy and
fruiting vegetables, stone and pome fruit, bush berries, and numerous ornamentals,
including soybean aphid on soybeans (OMAFRA 2009a). Dimethoate is as effective as ?-
7 cyhalothrin at controlling soybean aphid (Johnson and O'Neal 2007, 2008). However, due to its high volatility dimethoate may be more effective at controlling soybean aphid in tall plant canopies or narrow-row soybeans (Rice et al. 2007). Dimethoate is an organophosphate insecticide. Organophosphates are acetylcholinesterase inhibitors and bind irreversibly to acetylcholinesterase preventing the breakdown of acetylcholine in the synaptic cleft (Pedigo 2002). This results in hyper-excitability, convulsions, paralysis, and death. Dimethoate is non-persistent and can be used effectively in warm weather
(Rice et al. 2007). Several formulations of dimethoate are registered for use in Canada.
Spirotetramat is registered in Canada to control aphids, thrips, scales, whiteflies on cucurbit, fruiting and leafy vegetables, grapes, hops, pome and stone fruit.
Spirotetramat is not yet registered for use on soybean in Canada (OMAFRA 2009a). Spirotetramat is a tetrameric acid insecticide that is both phloem and xylem mobile (Nauen et al. 2008). It is a lipid biosynthesis inhibitor and has more pronounced effects on younger insects, resulting in growth anomalies (Nauen ef al. 2008). Spirotetramat is especially efficacious against rapidly growing soft bodied insects, and, due to its two- way systemic action is ideal for managing sucking pests (Schnorbach et al. 2008). Initial field efficacy studies suggest that spirotetramat may be as effective at controlling soybean aphid as currently registered insecticides (Johnson and O'Neal 2008).
Flonicamid is a novel insecticide of potential use against soybean aphid in
Ontario. Flonicamid has an inhibitory effect on aphid and thrips feeding, but its mode of action is currently unknown (Morita ef al. 2007). Flonicamid is used to control aphids in
8 greenhouse and nursery plants and for sucking pests in field and greenhouse vegetables, and stone and pome fruit. Currently, flonicamid is not registered for use in Canada.
Mineral oil may be of potential use for soybean aphid control in organic soybean production. Mineral oil acts as an asphyxiant, blocking the arthropod respiratory system.
It also interacts with the waxy surface of the arthropod cuticle and is believed to impair the insect's ability to maintain homeostasis (Stephenson and Solomon 2007). Mineral oil is sold both as an adjuvant for herbicides and as an insecticide. Thirty-five formulations of mineral oil produced by several companies are registered for use in Canada on stone and pome fruit, ornamental trees and shrubs to control scale insects, mites, Psylla spp. and various Lepidoptera. Mineral oil is not registered for use on soybeans in Canada.
Imidacloprid and thiamethoxam are neonicotinoid insecticides that bind post- synaptic nicotinic acetylcholine receptors. This results in excitation of the post-synaptic nerve cells, causing hyper-excitability, convulsions, paralysis, and death. Imidacloprid and thiamethoxam are xylem mobile and can be taken up by the roots of a plant and transported to meristems. Both are toxic to soybean aphid and are able to reduce soybean aphid populations in the field when applied as seed treatments (Johnson and
O'Neal 2008, Magalhaes et al. 2009). Seed treatment with thiamethoxam is registered for soybean aphid control in Canada (OMAFRA 2009a). Imidacloprid seed treatments are registered for soybean aphid control in the United States, but have not yet been registered for soybean aphid control in Canada.
9 1.3.2 Biopesticide Beauveria bassiana (Hypocreales) is an entomopathogen with potential use as a
biopesticide in organic soybean production systems. Beauveria bassiana causes white
muscardine disease in insects (McCoy ef al. 1988). During infection, conidia attach to the cuticle of the insect and germinate, penetrating the haemocoel and proliferating.
Once in the haemocoel, B. bassiana competes with the host for resources, eventually
resulting in the death of the host (McCoy er al. 1988). Beauveria bassiana is a toxin-
producing entomopathogen, although the role of these toxins is unclear (McCoy et al.
1988). Two strains of B. bassiana are registered for use in Canada. Strain HF23 is used to control houseflies in poultry production houses, while strain GHA (BotaniGard®
22WP, Bioworks, Inc.) is used to control aphids, thrips, and whiteflies on greenhouse
vegetables and ornamentals. Beauveria bassiana is not yet registered for use on
soybeans in Canada. Despite successful use in closed environments (i.e. greenhouses),
Beauveria bassiana is challenging to use in a field setting, due to unfavorable
environmental conditions (e.g. extreme temperature and humidity), which limit the
spread of the pathogen.
1.3.3 Host plant resistance Host plant resistance is another method used to control soybean aphid. There are three
categories of host plant resistance: antixenosis, antibiosis, and tolerance (Dent 2000).
Antixenosis is a form of deterrence which influences an insect's behavior towards the
plant. A plant variety that exhibits antixenosis is expected to have a reduced
colonization rate compared to a susceptible variety. Antibiosis is a mechanism in which
10 the plant has adverse effects on physiology and survival of the herbivorous insect.
Insects feeding on a plant variety exhibiting antixenosis will have a greater mortality rate than those feeding on a susceptible variety. Tolerance is a mechanism where a certain variety can sustain feed damage with no negative effects to an extent that susceptible varieties cannot. Several soybean cultivars have been found with either antixenotic or antibiotic traits (Diaz-Montano et al. 2006, Mensah et al. 2008). For example, the soybean cultivars Jackson, Dowling and Pl 71506, exhibit antixenosis and experience reduced colonization by the soybean aphid (Hill et al. 2004). No soybean aphid tolerant cultivars of soybeans have been found.
1.3.4 Conservation and classical biological control Conservation biological control (CBC) is the "modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests" (Zehnder et al. 2007). This can be accomplished by providing natural enemy populations with refuge, resources, or by using pesticides which are ecologically or physiologically selective (Barbosa 1998). CBC utilizes natural enemies which are already present in the local agro-ecosystem and already recognize the pest as prey. There is a growing body of evidence that CBC is effective at regulating various pest populations in agriculture, including herbivorous beetles (Szendrei and Weber 2009), gall midges (Nwilene ef al. 2008), and aphids (Holland et al. 2008).
CBC is distinguished from classical biological control by the use of established natural enemies. Classical biological control aims to import exotic biological control
11 agents, usually originating from the same geographic region as the pest (Barbosa 1998).
The success of classical biological control is varied (Barbosa 1998, Dent 2000). In some situations, such as greenhouse vegetable and plant production, the introduction of imported biological control agents has proven highly effective (van Lenteren et al.
1997). However, in other situations the introduction of a biological control agent has lead to the extirpation of native species (Hesler and Petersen 2008). A major problem with classical biological control is the inability to successfully introduce and establish the biological control agent, which has lead to the failure of many classical biological control programs. This failure can be a result of insufficient size of the introduced populations
(i.e. Allee effect) (Fauvergue and Hopper 2009), inability of the biological control agent to recognize the target pest, preference for another host or prey item, and/or misidentification of the biological control agent.
In its native range soybean aphid is only an occasional pest, due to natural regulation by predators and parasitoids (Miao et al. 2007). A number of generalist predators including members of the Coccinellidae, Syrphidae, Chrysopidae, and
Hemiptera are commonly found feeding on soybean aphid (Miao et al. 2007). At least 14 wasps in the families Aphelinidae and Braconidae parasitize soybean aphid in China (Wu ef al. 2004b, Heraty er al. 2007) and are considered important contributors to soybean aphid population regulation (Miao et al. 2007). However, few soybean aphid parasitoids have been found in North America. Six species of Aphelinidae and Braconidae were found parasitizing soybean aphid in Michigan (Kaiser er al. 2007) and three unidentified
12 Aphidünae, one Aphidius sp., and two Praon sp., were found parasitizing soybean aphid in New York state (Nielsen and Hajek 2005).
In North America the predominant natural enemies of the soybean aphid are
Harmonía axyridis and Onus spp. These predators contribute to soybean aphid population suppression (Costamagna and Landis 2006, Costamagna et al. 2007). Based on field observations in China, it has been suggested that conservation and augmentation of native natural enemies is a viable management option for soybean aphid (Miao et al. 2007). Due to the lack of native parasitoids in North America, the prospect of importing exotic parasitoids has been investigated (Heimpel et al. 2004,
Wyckhuys et al. 2008, Wyckhuys et al. 2009). To date two parasitoid species have been released to control the soybean aphid: Binodoxys communis (Wyckhuys et al. 2009) and a 'Wyoming strain' of Aphelinus albipodus (Heimpel et al. 2004). Binodoxys communis was collected from soybean aphid in China and released at 32 sites in Iowa, Indiana,
Minnesota, Michigan, South Dakota, and Wisconsin in 2007 (Wyckhuys et al. 2008,
Anonymous. 2009, Wyckhuys et al. 2009). Aphelinus albipodus is native to Europe and
Central Asia and was released in mid-west United States the 1990s to control Diuraphis noxia, the Russian wheat aphid (Hopper ef al. 1998, Prokrym et al. 1998). Individuals from this release collected in Wyoming were found capable of parasitizing soybean aphid in the laboratory and subsequently released in Minnesota and Wisconsin in 2002
(Heimpel et al. 2004). The outcome of these classical biological control programs have not been evaluated.
13 In the summer of 2006, a parasitoid recognized as an Aphelinus sp. was found parasitizing soybean aphid in a number of commercial soybean fields in Chatham-Kent,
Ontario (Welsman 2007). This parasitoid was eventually identified as Aphelinus certus
Yanosh (Hymenoptera: Aphelinidae) (see Chapter 2) (Figs. 1.1 and 1.2). This parasitoid has a historical association with the soybean aphid (Heraty et al. 2007) and its range extends throughout the soybean growing region of Ontario (see Chapter 2), therefore it could be used in CBC of the soybean aphid.
1.4 Biology of Aphelinus certus
Data pertaining to the biology of A. certus are extremely limited. For instance, The
London Natural History Museum's Universal Chalcidoidea Database, which contains
48,689 references, has just one reference for A. certus (Natural History Museum London
2009). Therefore, biological and life-history data must be inferred from closely related species.
The genus Aphelinus contains ca. 60 species (Natural History Museum London
2009) with nearly worldwide distribution. Many have extended their range as a result of human activity (Howard 1929, Prokrym et al. 1998). Aphelinus certus belongs to the A. varipes species complex which includes, A. albipodus, A. atriplicis, A. hordei, A. kurdjumovi, A. varipes, and is native to the Palaearctic region and (Heraty et al. 2007).
Aphelinus spp. are parasitoids of the family Aphididae (Hagen and van Den Bosch 1968,
Hayat 1998). They have often been utilized as biological control agents (Prokrym et al.
1998). Records of Aphelinus spp. parasitizing members of Coccidae, Diaspididae, and
14 Figure 1.1: Aphelinus certus adult emerging from a parasitized soybean aphid.
15 i W
m.,,„¿*
Figure 1.2: Scanning electron micrograph of a female Aphelinus certus.
16 Chrysopidae exist; however, some authors believe these reports to be erroneous (Hagen and van Den Bosch 1968, Hayat 1998). Many Aphelinus spp. have a broad host range.
For example, A. albipodus and A. asychis parasitize at least six aphid species in India
(Hayat 1998), and under laboratory conditions both species are capable of parasitizing at least twelve aphid species each (Elliott et al. 1999). The host range of A. certus is unknown at this time. It was first collected from an unidentified aphid in Russia
(Yasnosh 1963), but has been since been found parasitizing A. glycines in China, Japan, and Korea (Heraty et al. 2007) and Ontario. It is also capable of parasitizing
Rhopalosiphum padi L. in the laboratory (Frewin unpublished data).
Aphelinus spp. are primary koinobiont endoparasitoids (van Alpehn and Vet
1986), meaning that host aphids will continue to feed and grow after being parasitized and that the parasitoid larvae develops within the host (Godfray 1994). Aphelinus spp. will deposit one egg per host, which develops into one adult (van Alpehn and Vet 1986).
Aphelinus are biparental, and rr\a\e Aphelinus use a female-produced trail sex pheromone to locate potential mates (Fauvergue et al. 1995). Females are capable of arrhenotokous parthenogenesis (Fauvergue et al. 1998, Honek et al. 1998, Perng and Liu
2002, Wu et al. 2004a). Haploid males develop from unfertilized eggs, and diploid females develop from fertilized eggs. Mated female Aphelinus are able to determine the sex of their offspring at the time of oviposition. Pre-mating interval (Fauvergue et al.
1998), host species (Yokomi and Tang 1995), and host size (Cate et al. 1977, Prinsloo
2000) are all important factors determining offspring sex allocation. Aphelinus
17 parasitoids normally deposit female eggs into larger hosts (Honek et al. 1998), as is generally observed for most parasitoids (Godfray 1994). This behaviour is believed to be a mechanism to provide female offspring with more resources, in order to increase their size and fecundity (Godfray 1994). The degree to which this holds true in Aphelinus is uncertain, but, in some species, size correlates directly with egg load (Wu and Heimpel
2007).
Aphelinus have a relatively long aphid handling time (Lester and Holtzer 2002). Some Aphelinus require more than thirty seconds to oviposit into a host aphid, whereas the typical time for a braconid parasitoid is a few seconds (Bai and Mackauer 1990, De
Farias and Hopper 1997, Lester and Holtzer 2002). This longer handling time is attributed to the fact that Aphelinus use internal cues to determine host suitability prior to oviposition.
Only one larval parasitoid is capable of completing its development in the host.
As a result the incidence of superparasitism is extremely low (Hartley 1922, Wu and
Heimpel 2007). Heterospecific superparasitism can occur with aphidiinine parasitoids, and the Aphelinus parasitoid seldom survives (Bai and Mackauer 1991). There is evidence that Aphelinus parasitoids are capable of detecting con- and heterospecific parasitism, whereas Aphidiinae parasitoids are only capable of detecting conspecific parasitism (Bai and Mackauer 1991). The occurrence of superparasitism has implications for biological control, as these two groups of parasitoids could interfere with each other, reducing overall biological control.
18 Aphelinus are synviogenic, and females emerge from their pupae with few or no mature eggs (Bai and Mackauer 1990, Jervis ef al. 2001, Wu and Heimpel 2007). Eggs start to mature within 24 hours (Wu and Heimpel 2007). Aphelinus are also capable of reabsorbing their mature eggs in the absence of hosts. Egg reabsorption is common among synviogenic species (Jervis et al. 2001). The egg load of Aphelinus spp. are rather small compared to pro-ovigenic parasitoids. In contrast with braconid parasitoids having large egg loads Aphelinus spp. produce fewer larger nutrient-rich eggs (Le Ralee 1995). Although at any given time egg loads are low, over their life time Aphelinus are capable of producing hundreds of eggs. For example, A. gossypii parasitized an average of 598 aphids over their lifetime (Perng and Liu 2002).
Female Aphelinus host feed, although the extent to which they do so may vary by species (Cate et al. 1977, Perng and Liu 2002, Wu and Heimpel 2007). Most Aphelinus prefer to feed on early aphid instars (Cate et al. 1977, Wu and Heimpel 2007), although some species show no preference (Rohne 2002). Female Aphelinus host feed by piercing the aphid's integument with their ovipositor and then feeding on the exuded haemolymph. Aphids tend to die within hours or days of falling victim to Aphelinus host feeding (Cate er al. 1977). Although Aphelinus probe aphid hosts with their ovipositor before host feeding, no eggs are deposited. Parasitism and host feeding are mutually exclusive acts, and will have an additive effect on reducing host populations.
During development, an Aphelinus larva consumes the contents of the host aphid haemocoel, and then pupates within the hosts' cuticle (van Alpehn and Vet 1986).
19 At this stage of development, the aphid is often referred to as a mummy. During the final stages of parasitism the cuticle of the host aphid becomes black in colour,
presumably due to sclerotizing or melanizing agents released by the parasitoid. This colour change facilitates identification of Aphelinus mummies in the field during scouting, as the black aphid mummies are very conspicuous and are easily distinguished from the tan mummies produced by Aphidiinae parasitoids, such as Aphidius colemani
(Stary 1970).
Development times for Aphelinus vary by host species (Elliott et al. 1999,
Tatsumi and Takada 2005), host instar, and temperature (Rohne 2002). In general, the time required for development from egg to mummy and from mummy to emergence is
approximately equal (Rohne 2002, Tatsumi and Takada 2005). Adult Aphelinus exit the
mummy by chewing a circular emergence hole in the dorsal posterior region of the
aphid cuticle (Hagen and van Den Bosch 1968). Adult Aphelinus are long lived and ages
upwards of 20d are often reported (Hagen and van Den Bosch 1968, Perng and Liu
2002).
The lifecycle of Aphelinus is intimately tied to that of their hosts. In temperate
regions overwintering and diapause regimes of the Aphelinus are similar to that of their
hosts. Some species overwinter as last instar larvae within their hosts (Trimble et al.
1990, Yu 1992a, Bernal et al. 2001), while others overwinter as pupae (Prinsloo and du
Plessis 2000) or adults (Hamilton 1973, Tatsumi and Takada 2005). Both temperature
20 and photoperiod play a role in regulating Aphelinus dormancy and diapause (Tatsumi and Takada 2005).
Aphelinus parasitoids have been used as biological control agents in field, and fruit crops, and with greenhouse vegetable production. The best example of Aphelinus spp. biological control in field crops is the management the Russian wheat aphid, D. noxia. The Russian wheat aphid was first detected in the United States in March 1986 in
Texas and quickly spread through dry-land wheat fields of the mid-western USA
(Morrison and Peairs 1998). Between 1987 and 1993, direct losses and indirect losses associated with D. noxia exceeded US $893.1 million (Morrison and Peairs 1998). At least six species of Aphelinus were imported from Eurasia and released including A. abdominalis, A. albipodus, A. varipes, A. asychis, Aphelinus sp. nr. varipes and Aphelinus sp. (Hopper et al. 1998, Prokrym et al. 1998). There are no reported intentional releases of A. certus in North America from this program. Despite the high release numbers and presumed suitability of the chosen biotypes, only A albipodus, A. varipes, and A. asychis were detected in recovery surveys (Prokrym et al. 1998, Brewer et al. 2005). Of the three introduced species, A. albipodus was the only confirmed exotic, as native varieties of A varipes and A asychis were known to exist in the region (Prokrym ef al. 1998). No techniques were available at the time to distinguish introduced from native biotypes, and the origin of these populations is unclear. Resolving the origins of these parasitoid populations is further complicated by the scarcity of parasitoid fauna records for the area. From the limited evidence, a case can be made that the current natural enemy fauna consists primarily of endemic strains (Brewer et al. 2005). Regardless of its origin,
21 A. albipodus has become the dominant member of the D. noxia natural enemy guild in wheat, replacing Diaeretiella rapae in 1995 (Brewer et al. 2001). Uniquely, A. albipodus populations do not appear to be entirely dependent on host density (Brewer et al. 2001). The lack of density-dependent parasitism may result from the foraging strategy of A. albipodus, which relies heavily on patch intensive searching (Lester and Holtzer
2002) rather than attraction to host related volatiles (De Farias and Hopper 1997, Rao ef al. 1999). Since the implementation of this biological control program, the frequency of serious outbreaks of Russian wheat aphid has been reduced (Prokrym et al. 1998,
Brewer et al. 2005).
1.5 Non-target effects of insecticides Insecticides can either be selective or specific to the target pest. A selective insecticide is one that is more toxic to the pest species than to beneficial species, whereas a specific insecticide is one that causes little or no mortality in beneficial species (Johnson and Tabashnik 1999). Selectivity can either be ecological or physiological. Ecological selectivity is achieved by applying the insecticide in a manner that minimizes contact with natural enemies (Ruberson et al. 1998). For example, a systemic insecticide applied as a seed treatment will only affect tissues- or phloem- feeding insects, whereas a foliar-applied insecticide will affect any insect residing on the treated material regardless of whether it is consuming the treated plant tissue or not. Physiological selectivity is based on properties of the insecticide itself and the physiology of the pest and natural enemy species (Ruberson er al. 1998).
22 The goal of using selective insecticide is to increase the natural enemy to pest ratio after application (van Emden 2003). Death or injury of natural enemies can severely reduce their ability to control primary and secondary pest populations (Stark et al. 2004b, 2007), and can result in secondary pest outbreaks or pest resurgence. As soybean aphid is primarily controlled with insecticides this component of CBC using A. certus is very important. Soybean is extremely vulnerable to soybean aphid damage prior to the R5 (Beginning Seed) plant stage, and the soybean aphid has an extremely high intrinsic rate of increase (McCornack et al. 2004, Ragsdale et al. 2007, Catangui et al. 2009). If soybean aphid populations reach the economic threshold the application of insecticides is vital to prevent crop loss. However, these insecticides have the capacity to harm beneficial insects, including A. certus, through acute or chronic lethal effects on parasitoids (Johnson and Tabashnik 1999). Sub-lethal exposures to pesticides can affect parasitoid development, longevity, fecundity, immune function, offspring sex ratio, mobility, orientation, feeding behavior, reproductive behavior, and learning performance (Desneux et al. 2007).
According to the International Organization for Biological Control (IOBC), most broad-spectrum insecticides such as organophosphates and second generation pyrethroids are highly toxic to non-target arthropods (Hassan et al. 1983, Hassan ef al. 1991, Hassan et al. 1994, Longley 1999). For example, the organophosphate dimethoate was harmful to 13 of 15 beneficial arthropods in laboratory assays, and harmful to 6 of 7 in semi-field and field assays (Hassan ef al. 1988). The second generation pyrethroid ?- cyhalothrin was harmful to 14 of 15 beneficial arthropods in laboratory assays and 5 out
23 of 5 ¡? semi-field and field assays (Sterk et al. 1999). Similar results have been reported elsewhere. For instance, application of organophosphate and pyrethroid insecticides reduce natural enemy-related pest mortality in cotton (Kerns and Gaylor 1993) and drift of pyrethroid applications into field margins can result in mortality of predators and parasitoids (Langhof et al. 2005).
In general, data on the effect of seed- and soil-applied insecticides on natural enemies are limited. Seed- and soil-applied insecticides are usually considered safe and compatible with natural enemies due to ecological selectivity. However, some natural enemies feed on plant material including pollen, nectar, and guttation fluid (Jonsson ef al. 2009) and this material can be contaminated with systemic insecticide from seed treatments. Parasitoids feeding on floral and extra-floral nectars of plants treated with imidacloprid suffer reduced host foraging ability and longevity (Stapel et al. 2000, Krischik ef al. 2007). Moreover, natural enemies may consume hosts/prey that are contaminated with these insecticides. Similarly, predators feeding on imidacloprid- contaminated hosts suffer reduced life span and survival of immature stages (Walker et al. 2007, Papachristos and Milonas 2008).
Although Beauveria bassiana has a broad host range (Devi et al. 2008), it is considered compatible with many biological control agents including Aphidius matricariae (Rashki er al. 2009), Dicyphus hesperus and Encarsia formosa (Labbe er al.
2009). The predatory lady beetles Harmonía axyridis and Coccinella septempunctata are not harmed by Beauveria bassiana strain GHA (Cottrell and Shapiro-Man 2008).
24 Mineral oil is toxic to some natural enemies and compatible with others. In general, mineral oil appears to be more toxic to immature stages than adults and more toxic when applied directly than as a residue (Stansly and Liu 1997). For example, mineral oil is harmless to the parasitoids Encarsia perniciosi, and Aphytis spp which are used as BCAs in fruit tree production(Badenes-Perez et al. 2002), but mineral oil is harmful to immature and adult Encarsia pergandiella, a natural enemy of whiteflies
(Stansly and Liu 1997). Mineral oil is also toxic to first- and third- instar Harmonía axyridis, whereas it has no effect on pupae and adults (Kraiss and Cullen 2008).
Non-target data for novel insecticides is limited. For example, few studies on non-target effects of spirotetramat have been conducted. Spirotetramat appears to have a degree of selectivity towards Hemipterans (Nauen et al. 2008, Schnorbach et al.
2008), and is considered harmless to a number of non-target insects (Schnorbach et al.
2008). Its mode of action as a lipid biosynthesis inhibitor may be detrimental to insects which require synthesis of lipids for reproduction and homeostasis. However, some adult parasitoids lack a functional lipid biosynthesis pathway (Visser and Ellers 2008), and may therefore be resistant to spirotetramat. Flonicamid is reported to be selective to aphids and thrips (Morita ef al. 2007). Flonicamid is harmless to pupae and adults of the parasitoid Leptomastix dactylopii Howard, and the mealybug destroyer
Cryptolaemus montrouzieri Mulsant (Cloyd and Dickinson 2006).
There are numerous non-target effects of insecticides on beneficial insects, and these effects vary with species and insecticide. The proper selection and use of
25 insecticides can mitigate their damage on beneficiáis, which can enhance CBC based pest management.
1.6 Conclusions
Soybean is an economically significant field crop in Canada, the soybean aphid is a major pest of soybean that has the potential to cause severe crop damage and loss. An effective pest management is critical for minimizing yield losses. Currently, insecticides are the primary control method for soybean aphid. CBC using native natural enemies such as A. certus provides an alternative pest management technique which may reduce reliance on insecticides for soybean aphid pest management. In many crop protection programs, there has been a movement towards the practice of IPM in an effort to reduce chemical pesticide use and mitigate against potentially damaging effects. CBC is an IPM tactic for field crops that aims to improve or enhance favorable environmental conditions for natural enemies. In its native range, soybean aphid populations are primarily regulated by parasitoids and predators. Little is known about what role natural enemies play in regulating soybean aphid populations in Ontario. However, the discovery of A. certus in Ontario indicates that this parasitoid is contributing to aphid population control. CBC using A. certus may be a low-cost and viable option as part of future IPM programs for soybean aphid. In order to successfully employ CBC, it is necessary to have an extensive understanding of the life history traits of A. certus.
Any insecticide used for aphid management has the potential to harm natural enemies. The loss or reduction of natural enemy populations can result in pest
26 resurgence or secondary pest outbreak, which will then require further insecticide application. Successful CBC using A. certus as part of soybean IPM requires the use of insecticides with a low toxicity to this parasitoid. Therefore, it is important to evaluate insecticides developed for use against soybean aphid for their potential impact on A. certus.
1.7 Objectives
The objectives of this research program were to: 1. determine the developmental rate of A. certus; 2. determine the effects of foliar-applied insecticides on A. certus; and 3. determine the effects of seed-applied insecticides on A. certus. The results of this research will be useful in the development of a soybean aphid IPM program that integrates the use of insecticides and conservation biological control with A. certus.
27 2 Distribution and temperature-dependent development of a North American population of Aphelinus certus.
2.1 Introduction
Soybean aphid (Aphis glycines Matsumura), is an economically important pest of soybean (Glycine max L) in North America (McCornack et al. 2004). Since its introduction, the soybean aphid has spread throughout the entire soybean growing region (DiFonzo 2009). Outbreaks of soybean aphid can dramatically reduce yields and plant quality (Diaz-Montano et al. 2007, Beckendorf et al. 2008). Currently, growers rely exclusively on insecticides for soybean aphid management (Baute 2007, NCSRP 2009).
However, insecticide use can have negative effects on non-target organisms, farm workers, and the environment. Overreliance on insecticides can also result in the development of resistance within the target pest population (Devonshire and Moores 1982). Therefore the development of novel and supplemental management techniques is essential for sustainable management of soybean aphid.
The development and implementation of an integrated pest management (IPM) system for soybean aphid is the most practical and sustainable management solution. Integrated pest management is a pest control strategy that uses a combination of complementary control methods to reduce pest populations below an economic threshold. A potential and cost effective tool for soybean aphid IPM is biological control by natural enemies. In its native range soybean aphid populations are regulated by a
natural enemy guild consisting of various predators and parasitoids (Miao er al. 2007). In the summer of 2006 a large number of soybean aphids parasitized by an unidentified
28 Aphelinus spp. were found ¡? Chatham-Kent, Ontario (Welsman 2007), and subsequently identified as Aphelinus certus (Yanosh). While other soybean aphid parasitoids are present in Ontario, to date none are known to be found so frequently at high densities.
Therefore this parasitoid may be a candidate for incorporation into soybean aphid IPM using conservation biological control (CBC) techniques.
Detailed life history information is critical for using a biological control agent in any IPM system (Dent 2000). Such life history traits may include functional response, host range, life-time fecundity, and temperature-dependent developmental rate. Knowledge of the temperature-dependent developmental rate allows the estimation of seasonal occurrence, developmental time, and the number of generations per year of the natural enemy (Bernal and Gonzalez 1993a). This information can be used in an IPM system to make informed management decisions to minimize contact between the natural enemy and pesticides and to reduce or avoid pesticide use when robust naturally enemy populations are present.
The objective of this study was to determine range of A. certus in Ontario, and to determine parameters characterizing its development. This research will provide valuable information to incorporate this parasitoid into soybean aphid IPM in Southern
Ontario.
29 2.2 Material and methods
2.2.1 Survey In July and August of 2007, a survey of soybean aphid parasitoids was conducted in
Ontario. Soybeans infested with A. glycines were collected from 56 locations across
Ontario. At least 10 soybean plants were sampled at each location and the presence of aphid mummies was recorded. Soybean trifoliates with aphids and aphid mummies were held in growth chambers maintained at 25±1°C, 16:8h lightidark photoperiod, and
70% RH for two weeks. All parasitoids emerging from mummified aphids were collected.
Individuals from four sampling locations were subsequently sent to Dr. Keith R. Hopper at the USDA Beneficial Insect Introduction Research Unit, Newark, DE for species identification using genetic and morphological characters.
2.2.2 Rearing methods
Emerged parasitoids from the collected soybeans were used to establish laboratory colonies. Colonies were provided with soybean plants infested with soybean aphid ca. once per week and were maintained at 26±2°C, 65-75% RH, and a 16:8h light:dark photoperiod. Individuals from the laboratory colony were also sent to Dr. Hopper for species identification.
2.2.3 Development rate experiment
All experiments were conducted in controlled environment chambers held at 15.3, 18.3,
20.6, 25.3, 26.6, or 30.2±1°C, 70% RH, and 16:8h light:dark photoperiod. Soybean leaves were collected from 3-5 week old plants naive to soybean aphid. Each leaf was placed
30 adaxial slide down on damp cotton batting within a 10 cm Petri dish bottom. Twenty- five 3rd or 4th instar soybean aphid were then transferred onto each leaf. The aphids were allowed to settle for 4h, after which one mated female A. certus was introduced into the dish. Petri dishes were then sealed with Parafilm® and assigned randomly to a controlled environment chamber. After 24h the parasitoids were removed. This was replicated five times at 30.20C, and ten times at all other temperatures . Aphid mummies were removed daily from the leaves, placed in a 0.5mL Eppendorf tube and returned to their respective controlled environment chambers. The total number of mummies per dish, days to emergence and sex of adult parasitoids were assessed.
2.2.4 Statistical analysis Statistical analysis was conducted using SAS ? 9.1 (SAS Institute Cary, NC). Developmental rate data were subjected to ANOVA using the MIXED procedure (SAS Institute Inc. 2004). Variance was partitioned into the random effect block, and the fixed effects temperature, sex of wasp, and the temperature X sex interaction. Linear and lack-of-fit regression partitions were also included for temperature and the temperature X sex interaction where significant. The lack-of-fit partition was included to confirm the existence of a higher order relationship between development and temperature within the temperature range tested. F-tests were used to test for significant effects.
Differences between treatment means were determined using Tukey's procedure. The
lack-of-fit regression partition was significant and developmental rate data was fitted to a non-linear temperature-dependent growth model (Equation 1) (Briere ef al. 1999).
31 O if T < T0 R(T) aT(T - T0)JT1- T ifT0
Institute Inc. 2004). Briere's model was chosen for this study as it provides estimates of upper and lower threshold of development with the least number of terms in the equation (Roy et al. 2003). Optimal growth rate was calculated using equation 2 (Briere et al. 1999):
4TL + 3T0+ ¡16T£ + 9Tg-16T0TL Topt = ^10 [2]
The coefficient of determination was calculated using equation 3 (Lamb et al. 1984): ft¿?2_?= l- S}=1(?G9})'-"-' J" [3] zy=1(yy-y)2 where y¡ is the jth observed mean development rate, y^is the expected, and y is the mean observed developmental rate.
The linear component of the developmental data was subjected to an ANOVA using the GLM procedure. Variance was partitioned into the fixed effect temperature and linear and lack-of-fit regression partitions. The regression was used to estimate the
32 lower threshold of development (LT0) and the degree-days (0D) requirements for each life stage (Campbell et al. 1974). The linear estimate of the lower threshold of development is defined as the x-intercept of the regression of developmental rate on temperature and the degree-days ("D) is the reciprocal of the slope. Standard error of
¿70 was calculated by equation 4: "<*»¦.= f^m' '4I where s2 is the residual mean square of developmental rate R, and b is the slope (Campbell et al. 1974). Standard error of 0D was calculated by equation 5 (Campbell ef al. 1974):
S.Eof°D = S-^f± [5]
The effect of temperature on pupal morality, parasitism rate, and Fl sex ratio was examined by ANOVA using the MIXED procedure (SAS Institute Inc. 2004). Variance was partitioned into the random effect block, and the fixed effect temperature. Linear, quadratic and lack-of-fit regression partitions were also included for temperature.
Parasitism rate was calculated as proportion of mummified aphids per Petri dish.
Parasitism rate was subjected to an arcsine square-root transformation to better meet the assumptions of ANOVA.
All tests were conducted at a significance level of 0.05. To ensure all data conformed to the assumptions of analysis of variance and regression the residuals were examined for homoscedasticity, independence, and randomness using plots of residuals
33 by predicted values, block, temperature, and sex. Normality was tested using Shapior-
WiIk test. Lund's test of studentized residuals was performed to identify outliers in the data (Bowley 1999). F-tests were used to test for significant effects. Differences between treatment means were determined using Tukey's procedure. All tests were conducted at a significance level of 0.05.
2.3 Results
2.3.1 Survey Soybean aphids were present at all sites surveyed. The vast majority (>99%) of aphid mummies collected were Aphelinus-Wke, while the remaining were Aphidiinae-like. Only
Aphelinus-like mummies were reared to emergence because of their predominance in the survey. The black /Aprte/Z/it/s-aphid mummies were observed at 52 of 56 (93%) sites
(Fig. 2.1). At 22 of these sites, parasitoids reared from aphids were identified as belonging to the genus Aphelinus Dalman. Individuals from four sites and the laboratory colony were identified as A. certus using genetic and morphological characters (K.
Hopper, personal communication).
2.3.2 Development Aphelinus certus completed development at all temperatures tested. The development rate increased over the entire temperature range tested for both life-stages (Fig. 2.2,
Table 2.1). Both temperature and sex had a significant (P <.0001) effect on development rate for the egg-pupae and pupaeghghj-adult development periods (Table 2.2).
However, a
34 IO CU
\ 52 a> ?3 C ? 3 E ? ?? 3 CO CU E ? t Q. CO O C CD Q.- ^ CM ? G 0) =3 ^. Oi -CCD -COJ -CCU "-^ •ÏÏ Q. Q. Q. O •C o G» o O O (N 3 o QO ? 3 QO _C '&- 3 ?3 ¦s CU > L- 3 10 10 ey**^ CU u« C to CU >¦ O (? ~ñ '? SK L- .-.y CU E ^" E ? ?. W ? co m e +?g OJ ? O cu 3 00 0.20 re ¦s L- F 0.15 Q. O) <0 C 0.10 (U e Q. _? (U > (U Q 0.05 0.00 20 Temperaturen 0.20 (O TJ ?_ (U 0.15 CL (U (C C 0.10 (U E Q. ? > ? O 0.05 H 0.00 15 20 25 Temperature 0C Figure 2.2. Mean temperature-dependent development rate of Aphelinus certus from (A) egg to mummy, and (B) mummy to adult. Calculated physiological growth curve, solid line; linear regression, dotted line. Only data represented by solid circles were used in linear regression. 36 IO 3 QO >- Si (D (D -Q -Q -Q IU ID (D D ? C ? io 3 G·» nT ro m lo Ln co (NI (T) O O O O o o o o o .C O O ? ? ? o "d O O O ö ? ö ri ¦c +1 +1 +1 +1 +1 +? oo Q. C in m ?? in o LD cn o ¦a pp O 'd ö ö ö ö ö ri o o o o o o U (D LT) RI Q. O > ^- m r~- r~~ ri ?) LD O V CO ID l/i l/i CL. Q XS C t? O m <* t? o m «* cu <î N U3 G? *j- r-» LD (ni ?3 C op >· (O C OJ (D (O (U ?] ro ?- (D (D U in no m cm id (NI (NI (NJ Ñl- M-- to p? o o o o o O O ? o o O 'c > O O- o- ? o- - O O ? ? O ?? O +-? QO 4-» ö O o ri Ö ri ri ?" ri ri 'io >· +1 +1 +? +1 +1 +1 +1 +? +? +1 ^l CU e (N m 00 CTI t-\ <-l ? t_ LD o Is» 00 ? Ln en O "(D (D e O «? ? ? ? (Nl +¿ O OO Ö O O o o o o o ri Q. e (? tj e (D r-N m >¦ G^ t-? LD (Yl <* (NI e re ai od ?/i uS LD in e ? (NI 3 S- e Xl (NI T-I LD TJ m (? Egg - Mummy Cov. Param. Estimate Standard error Z value PrZ Block 0 Residual 0.000496 0.000031 16.2 <0.0001 Effect NumDF Den DF F value Pr>F Sex 1 502 24.18 <0.0001 Temperature 5 502 342.74 <0.0001 Linear (1) 502 1701.23 <0.0001 Lack of fit (4) 502 19.84 <0.0001 Temperature X sex 5 504 2.06 0.069 Mummy -Adult Cov. Param. Estimate Standard error Z value PrZ Block 1.44E-06 3.98E-06 0.36 0.3586 Residual 0.000243 0.000015 15.88 <0.0001 Effect NumDF Den DF F value Pr>F Sex 1 502 16.64 <0.0001 Temperature 5 502 1024.55 <0.0001 Linear (1) 502 5143.94 <0.0001 Lack of fit (4) 502 53.86 <0.0001 Temperature X sex 5 502 3.37 0.0053 Linear (1) 502 35.84 <0.0001 Lack of fit (4) 502 0.48 0.7489 38 significant temperature X sex interaction was only found for the pupae-adult life stage (P =0.0053; Table 2.2). The linear and lack-of-fit regression partitions for the effect of temperature on egg-mummy and mummy-adult development rate were significant (P <.0001). The linear and lack-of fit regression partitions for the temperature X sex interaction were omitted because the term was not significant. The linear regression partition of the temperature X sex interaction for the mummy-adult development rate was significant (P «c.OOOl; Table 2.2). The non-linear growth model provided an excellent fit for both egg-pupae (R2=0.99) and pupae-adult (/?2=0.99) developmental data sets. Parameter estimates from the non-linear iterative regression are presented in Table 2.3. Optimal growth rates for egg-pupae and pupae-adult life stages were achieved at 29.5°C and 31.00C respectively. Estimates of T0 and TL were 7.8°C, 35.7°C and 11.6°C, 36.9°C for egg-pupae and pupae- adult respectively. The linear, quadratic and lack-of-fit regression partitions for growth rate were all significant. To obtain the linear component of the developmental curve, developmental data collected at 26.6 and 30.20C were dropped from the analysis (as per Campbell et al 1974). After removal, only the linear regression partition remained significant. The regression provided LT0 and °D estimates of 9.1 ± 0.26, 96 ± 2.2 and 11.6 ± 0.14, 90 ± 1.4 for egg-pupae and pupae-adult respectively. Temperature had no effect on pupal mortality (average 12%) (F:1.59, DF: 5,39, P=0.1860) or Fl sex ratio (57% Female: 43% Male) (F: 1.74, DF: 5,508, P=0.1243). 39 Table 2.3. Parameter estimates from non-linear iterative regression of developmental data for Aphelinus certus developing within Aphis glycines at six constant temperatures: a is a constant, T0 is the lower threshold of development; T1 is lethal temperature or upper threshold of development. Life stage a (xlO 4) T2 7j. r2 Egg -mummy 1.19 ±0.063 7.8 ±0.59 35.7 ±0.48 0.99 Mummy -adult 1.37 ±0.060 11.6 ±0.31 36.9 ±0.53 0.99 40 Temperature had a significant effect (P=O. 0004) on parasitism rate, with parasitism increasing with temperature (Table 2.4; Fig. 2.3). The linear regression partition for the effect of temperature on parasitism rate was significant (P<0.0001) (Fig. 2.3). 2.4 Discussion In cropping systems, natural enemies are capable of regulating pest populations (Costamagna et al. 2007, 2008). When natural enemies are present, CBC techniques can enhance their activity. Supplementing food/nectar (Banks et al. 2008), enhancing landscape diversity (Gardiner et al. 2009, Isaacs et al. 2009), use of reduced risk insecticides, or timing insecticide application (Johnson and Tabashnik 1999) to minimize non-target impact all have the ability to increase natural enemy populations and pest control. A thorough understanding of the natural enemies' biology such as seasonal occurrence, intrinsic rate of increase, and thermal requirements of development is crucial to the success of CBC. Knowing these parameters allows one to make management decisions to avoid disrupting, or alternatively to enhance natural enemies. This study provides the first record of A. certus in Ontario. The presence of A. certus was only confirmed at four sites on the periphery of the sampling range. However, the presence of unidentified Aphelinus spp. and black Aphelinus-\\ke aphid mummies in other samples, strongly suggests A. certus occurs throughout the sampling range. The sites examined in this survey do not reflect the extent of the Canadian soybean growing region and, it is possible the range of A. certus extends 41 Table 2.4. Variance analysis of the effect of temperature on parasitism rate of Aphelinus certus, i.e. the proportion of available Aphis glycines parasitized by A. certus in a 24h period at six constant temperatures. Cov. Param. Estimate SJE Z value PrZ Block 0.02623 0.01568 1.67 0.0473 Residual 0.05513 0.01218 4.53 <0.0001 Effect Num DF Den DF F value Pr>F Temperature 5 43 5.63 0.0004 Linear (1) 43 24.76 <0.0001 Quadratic (1) 43 0.00 0.9738 Lack of fit (3) 43 1.14 0.343 42 15 20 25 35 Temperature (0C) Figure 2.3. Mean proportion of Aphis glycines parasitized by Aphelinus certus at six constant temperatures and linear regression of the proportion of parasitized A. glycines against temperature (solid line). 43 beyond that of the current survey. Further work will be required to confirm the range and distribution of A. certus in North America. Aphelinus certus is native to Asia, first collected and described from unidentified aphids in the region of the former U.S.S.R (Yasnosh 1963). More recently it has been found parasitizing soybean aphid in China, Korea, and Japan (Heraty et al. 2007). It is unclear when A. certus was introduced into North America, but it was likely present in Ontario in 2006 when an unidentified Aphelinus spp. was found parasitizing soybean aphid in Chatham-Kent Ontario (Welsman 2007). Due to the sudden appearance and extent of its range, it is likely that A. certus is capable of long range dispersal either as an adult or larva within an alate host. Members of the Aphelinidae are regarded as poor fliers and are often found walking and jumping in search of hosts rather than flying (Viggiani 1984, DeFarias and Hopper 1997). Species of Aphelinidae vary in dispersal capacity from 1.6 to 100km per year (DeBach and Argyriou 1967, Onillon 1990). At the highest rate it would have taken A. certus a minimum of 6 years to travel the 580km between the furthest confirmed sites within its range. However, some aphid parasitoids have the capacity to be transported long distances as an egg-larva within an alate host during aphid dispersal events (Feng et al. 2007, Huang et al. 2008). Since soybean aphids are strong fliers (Zhang et al. 2008), and are dispersed in low pressure weather systems it is possible that A. certus could have been distributed more rapidly this way. To confirm this, parasitism of migratory aphids should be assessed. 44 Aphids are often caught in suction traps during migration and dispersal events (Teulon and Scott 2006, Kasprowicz et al. 2008). An extensive suction trap network in the soybean growing region of North America is currently being used to determine the spread of soybean aphid throughout the growing season (Voegtlin 2009). In addition to monitoring aphids, these suctions traps could be used to detect parasitoids. The presence of adult or larval parasitoids in these traps would indicate an influx of parasitoids into local agro-ecosystems. This information could then be used in local soybean aphid management decisions by adding parasitoid incidence to already established aphid scouting programs. This is particularly important as more native parasitoids use soybean aphids as hosts (Brewer et al. 2005, Kaiser et al. 2007). If transportation in alate aphids is a common trend in soybean aphid parasitoids, aphid suction trap networks technique could help verify the spread of novel parasitoids. In addition to aiding in natural control of aphids, this work would also provide information on parasitoid dispersal of which little is known (Godfray 1994). Temperature is the most important abiotic factor contributing to the development time of arthropods (Campbell et al. 1974, Logan et al. 1976, Lactin et al. 1995, Briere et al. 1999). As anticipated the development rate of A. certus varied significantly with temperature. The development pattern of A. certus is similar to that of other Aphelinus spp. In other studies an increase in development rate was seen for temperatures up to ca. 300C (Asante and Danthanarayana 1992, Lajeunesse and Johnson 1992, Bernal and Gonzalez 1993b, Bernal and Gonzalez 1996, Lee and Elliott 1998, Prinsloo and du Plessis 2000, Rohne 2002, Schirmer et al. 2008). The lower threshold of 45 development (7 - 100C) ¡s also consistent with that reported for other Aphelinus spp. (Asante and Danthanarayana 1992, Lajeunesse and Johnson 1992, Yu 1992b, Bernal and Gonzalez 1993b, Bernal and Gonzalez 1996, Lee and Elliott 1998, Prinsloo and du Plessis 2000). The numerically high, lower threshold of development and the ability to develop successfully at temperatures of ca. 300C indicate that in general Aphelinus spp. are tolerant of high temperatures. This ability is in contrast with Aphidiidae parasitoids that typically show signs of physiological stress at temperatures in excess of 26°C. In a study by Bernal & Gonzalez (1993) Aphidius matricariae (Haliday) was not able to complete development at 29.4°C. At the same temperature, Diaeretiella rapae showed a reduction in developmental rate compared to 25°C. However, in the same experiment, development rates of two Aphelinus spp. increased over the same temperature range tested (Bernal and Gonzalez 1993b). The lower threshold of development of Aphidiidae parasitoids is generally lower than that of Aphelinus spp. and values less than 7°C are often reported (Bernal and Gonzalez 1993b, Sigsgaard 2000). Gender also had a significant effect on development rate. In general male Aphelinidae develop faster than females (Viggiani 1984); however in the present study the opposite was true for A. certus. In this experiment, female A. certus developed more quickly than males during both life-stages at all temperatures tested. However, the difference was only significant at the two highest temperatures. This is consistent with other work on A. albipodus and A. spiraecolae (Tang and Yokomi 1995, Bernal and Gonzalez 1996). In the current study, host aphid age and instar were mixed. Therefore, it is possible that the differences in development time were a result of differences in 46 host age and parasitoid behaviour. Parasitoids, including Aphelinus spp., often deposit female eggs into larger hosts (Honek et al. 1998). Generally, larger hosts support faster development in parasitoids (Rohne 2002, Mironidis and Savopoulou-Soultani 2009). In this study, it was possible that female eggs were preferentially deposited into the larger 4th instar aphids, while 3rd instar aphids were being reserved for males, and thus the females developed faster than the males. Additionally, the rate of development for the females increased with temperature a greater rate than that of the males. Similarly, this could be a result of the underlying physiological differences between the sexes oran artifact of host size Temperature had no effect on pupal mortality which is consistent with previous observations for other Aphelinus spp. For example, temperatures between 15.6 and 29.4°C have no effect on pupal mortality of A. albipodus (Bernal and Gonzalez 1996). In contrast, Aphidiinae parasitoids (Bernal and Gonzalez 1993b, Sigsgaard 2000) and other Braconids (Krugner et al. 2007) often exhibit temperature-dependent larval and pupal mortality. Lack of temperature dependent mortality at the pupal stage suggests that during development A. certus is tolerant to temperatures within the range tested. It would be expected that as temperatures approach the TL that pupal mortality would increase. However, the effect of exposure to low temperature is less clear. Aphelinus subjected to clines exhibit a number of diapause regimes (Yu 1992b, Bernal et al. 2001, Tatsumi and Takada 2006b, a). It is likely that A. certus is capable of entering diapauses at similar temperature as it appears to be overwintering in Ontario. 47 The lower threshold of development for the soybean aphid is 8.60C (McCornack et al. 2004). The lower threshold of development for pupal A. certus was estimated to be 11.6°C. This indicates that soybean aphid will be able to complete its development earlier in the season than A. certus. However, throughout Ontario, outbreaks of soybean aphid do not normally occur until July, at which point the average temperatures are well above 11. 60C and therefore differences in development parameters may not affect the biological control potential of A. certus. Additionally, it is unknown how A certus overwinter. Other Aphelinus spp. overwinter as last instar larvae (Yu 1992b, Bernal et al. 2001, Tatsumi and Takada 2005), mummies (Prinsloo and du Plessis 2000), or as free living adults (Hamilton 1973). The consequences of different overwintering strategies are the ability of the parasitoid to develop early in the season. More work will need to be completed to resolve how A. certus overwinters. Based on this study, the optimal temperature of development for A certus was 29.5°C and 31.0°C for the egg-mummy and mummy-adult life stages, respectively. These temperatures are both higher than the optimal temperature for development of the soybean aphid (27. 80C) (McCornack et al. 2004) suggesting that A certus is more tolerant to higher temperatures. In a study by McCornack ef al. (2004) it was found that the population doubling time, intrinsic rate of increase, and gross fecundity of the soybean aphid was lower at 300C compared to 25°C. Considering that A certus does not experience temperature-dependent mortality below 30°C and parasitism rates increase with temperature, A certus biological control should be enhanced by warm weather. In a simulation study on the oleander scale system it was shown that the parasitoid 48 Apyhtis chilensis contributed more to the suppression of this insect during warm weather than a coccinellid predator (Gutierrez and Pizzamiglio 2007), presumably due to their tolerance for warm temperatures. Similar analyses could be completed on the soybean aphid system once more data is collected on A. certus and other soybean aphid natural enemies (Donaldson et al. 2007, Costamagna et al. 2008, Xue et al. 2009). This would provide information on the relative impact of natural enemies and would aid management decisions. For example, temperature trends favoring one natural enemy over another could be used to select compatible chemical controls to promote its population growth. Due to the extent of its range and its historical relationship with soybean aphid, A. certus appears to be valuable asset for soybean IPM in North America. However, more work is needed to determine the range of A. certus and its impact on soybean aphid in a field setting. The data collected here are an important first step in determining the utility of A. certus in soybean aphid IPM. 49 3 Susceptibility of Aphelinus certus Yanosh to foliar-applied insecticides currently or potentially registered for soybean aphid control. 3.1 Introduction Soybean aphid (Aphis glycines Matsumura) was first identifed in North America in 2000 (Hunt ef al. 2003, Ragsdale et al. 2004). Since then it has become a serious economic pest of soybean (Glycines max L) throughout North America (Ragsdale et al. 2007). Soybean aphid feeding can reduce plant yield (Beckendorf et al. 2008), reduce photosynthetic capacity (Clark and Perry 2002, Diaz-Montano ef al. 2007), and vector viruses (Macedo et al. 2003). Currently, application of insecticides is the only recommended control method (Baute 2007, NCSRP 2009, OMAFRA 2009). As a result, the soybean acreage treated with insecticide has increased 9-40% in aphid affected areas (National Agriculture Statistics Service 2000, 2006). The use of natural enemies in IPM can reduce pesticide applications and in turn, the negative consequences associated with pesticide use. There is growing evidence that natural enemies can control soybean aphid (Costamagna and Landis 2006, Desneux ef al. 2006, Costamagna ef al. 2007, Donaldson ef al. 2007, Miao ef al. 2007, Costamagna ef al. 2008) and it may be possible to incorporate these natural enemies into an integrated pest management (IPM) program for soybean (Zhang and Swinton 2009). However, the use of insecticides for soybean aphid control remains necessary due to the occurrence of unfavorable climatic conditions for natural enemies, biotic interference between natural enemies (Chacon ef al. 2008), the presence of secondary pests (Johnson and Tabashnik 1999), 50 and/or insufficient populations of natural enemies (Dent 2000). As a result, natural enemies may be exposed to insecticides targeting soybean aphid, increasing the risk of aphid resurgence due to loss of biological control (Johnson and Tabashnik 1999). Natural enemies can encounter foliar-applied insecticides at the time of application, as dried residues on plant or soil surfaces, and on or within contaminated prey. Pesticides can have lethal and sub-lethal effects on natural enemies. Sub-lethal effects of pesticides can also reduce natural enemy efficiency by affecting searching behavior, sex-ratio, and fecundity (Johnson and Tabashnik 1999). Therefore pesticides with minimal impact on natural enemies should be used whenever possible. Aphelinus certus Yanosh is an exotic parasitoid of soybean aphid in Ontario and is distributed throughout soybean producing regions. Aphelinus certus has the potential to regulate soybean aphid populations. The presence and population densities of A. certus should be evaluated in the context of soybean aphid IPM when deciding if and when to apply pesticides. No toxicological information currently exists for this beneficial species. The objective of this study was to assess the susceptibility of A. certus to various registered and novel foliar-applied insecticides. 3.2 Materials and methods 3.2.1 Insects Aphelinus certus were obtained from laboratory colonies (see chapter 2). Aphelinus certus mummies were collected from rearing chambers and placed in emergence chambers. Emergence chambers consisted of 1 L Mason jars, with cone traps attached 51 to the lids. An inverted plastic funnel was placed in a 1 cm diameter hole cut in the lid of the Mason jar. A 1cm hole was cut into the lid of a Fisherbrand® snap-top vial size so that the vial would be placed over the inverted funnel. Emergence chambers were kept in a growth room held at 26±2°C, 65-75% RH, and a 16:8h light:dark photoperiod. Every two days all adult wasps in the emergence chamber were removed to ensure that no individual older than 48h was used for testing. 3.2.2 Insecticides Insecticides tested included ?-cyhalothrin ([A. I.] 120g/L; Matador® 120EC, Syngenta Crop Protection Canada, Guelph, ON), dimethoate ([A.l.]480g/L; Lagon® 480E, United Agri Products Canada Inc., Dorchester, ON), flonicamid ([A.I.] 50%wt Beleaf® 50SG, FMC Corporation, Philadelphia, PA), mineral oil ([A.I.] 99%; Superior 70 Oil, United Agri Products Canada Inc., Dorchester, ON), spirotetramat ([A.I.] 240g/L; Movento® 240EC, Bayer CropScience Canada, Toronto, ON). In addition the foliar-applied biopesticide Beauveria bassiana strain GHA was evaluated ([A.I.] 22%wt; BotaniGard® 22WP, BioWorks, Inc., Victor, NY). ?-cyhalothrin and dimethoate are currently registered for control of soybean aphid in Canada, and the remaining products are being evaluated for potential registration (Hallett and Schaafsma, unpublished data). 3.2.3 Residual contact LC50 The 24h residual contact LC50 for the two insecticides currently used for soybean aphid control in Ontario were estimated for A. certus. Adult A. certus were exposed to dried residues of 0, 30, 60, 120, 240, 480 and 0, 80, 95, 110, 130, 150 ppm, of ?-cyhalothrin 52 and dimethoate, respectively. Both treatments regimes were replicated eight times. Formulated insecticides were dissolved in water and water was used as a control. Bioassay containers consisted of a 10cm Petri dish with two entry holes cut in opposite sides of the wall. Filter paper (P8, Fisherbrand) was affixed inside the lid and base of the Petri dish using LePage® Bondfast® White Glue (Henkel Consumer Adhesives, Inc., Avon, OH) to prevent wasps from crawling underneath. One mL of treatment solution was applied to both pieces of filter paper with a micropipette. Bioassay dishes were allowed to dry for one hour. After drying, four equally spaced 2mm wide drops of undiluted honey were placed on the inside wall of the Petri dish bottom as a food source. The bioassay container was assembled and held together with an elastic band. Approximately 10 A certus adults were randomly selected from emergence chambers and aspirated into a 25mL glass vial. The mouth of the glass vial was inserted in an opening on the side of the bioassay container. Wasps were encouraged to enter the bioassay container by tapping the vial. After the wasps had been transferred, small cotton balls ca. 1cm in diameter were used to plug the openings. The cotton balls were then saturated with distilled water using an eye-dropper to provide wasps with water. Bioassay containers were stored in a growth chamber maintained at 24±1°C, 16:8h lightrdark, and 70% RH. Mortality was assessed 24h after application. 3.2.4 Direct contact LC50 The direct contact LC50 for the two insecticides currently used for soybean aphid control in Ontario were estimated for A. certus. Adult A. certus were directly exposed to 25, 40, 60, 75, 100 and 50, 75, 95, 110, 150, 200 ppm of ?-cyhalothrin and dimethoate, 53 respectively. Each treatment was replicated eight times. Formulated insecticides were dissolved in water and water was used as a control. Direct contact LC50bioassays were conducted using a calibrated airbrush spray tower (Conroy 2008). Fifteen A. certus were randomly selected from the emergence chambers and transferred to a pretreatment container, consisting of a 5cm Petri dish with two holes in the lid; a 1.5cm diameter hole covered in mesh to allow ventilation and a 0.5cm hole with a removable cork. A filter paper (P8, Fisherbrand) was glued inside the Petri dish base with glue (as above). Wasps were anaesthetized with CO2 for ca. 8 sec. The lid of the pretreatment container was removed and the base containing the anesthetized wasps was placed in the mini-tower, and 0.5mL of insecticide solution was applied. Wasps were then transferred to a post-treatment container consisting of a 10cm Petri dish with a filter paper glued to the bottom. A 1cm diameter cotton ball saturated with water was added to the post treatment container along with four equally spaced 2mm wide drops of undiluted honey on the inside wall of the bottom. Post treatment containers were sealed with Parafilm® and stored in a growth chamber maintained at 24±1°C, 16:8h light:dark, and 70% RH. Mortality was assessed 24h after application. 3.2.5 Screening bioassay To assess the toxicity of unregistered insecticides a screening bioassay was used. The following insecticidal agents where applied at 1A, 1, and 2 times their recommended field rates for soybean/or crops with similar architectures (Hallett and Schaafsma, 54 unpublished data): flonicamid, spirotetramat, mineral oil, Beauveria bassiana, ?- cyhalothrin and diamethoate (Table 3.1). Each treatment was replicated six times. The rate used for ?-cyhalothrin was 66.4ppm, which was the highest rate recommended rate at the time the experiment was conducted (OMAFRA 2008). Formulated insecticides were dissolved in water and water was used as a control. Insecticides were applied using methods as for the direct contact assay, except mortality was assessed 24 and 48h after application. Afterwards, insecticidal agents were ranked according to the standards of the International Organization for Biological Control (IOBC): harmless (<30% mortaility of test population), slightly harmful (30-79%), moderately harmful (80- 99%), and harmful (>99%) (Hassan et al. 1988, Sterk et al. 1999). 3.2.6 Statistical analyses 3.2.6.1 LC50 bioassays All analyses were conducted with SAS v9.1.3 (SAS Institute Inc. 2004). The Probit procedure was used to estimate the median lethal concentration (LC50), fiducial limits, and the ?2 goodness of fit for all established insecticides. Prior to analysis, Henderson- Tiltons' formula was used to correct for control mortality (Henderson and Tilton 1955). Mortality in the controls never exceeded 10% (range 0-8.3%). Slopes were compared using paired t-tests at a significance level a=0.05. Significant differences between LC50's were determined by lack of overlap between fiducial limits. A hazard quotient was calculated for each insecticide by dividing the highest recommended rate (Table 3.1) by the direct and residual contact LC50 determined for A certus (Stephenson and Solomon 2007). 55 Table 3.1. Recommend rates for insecticides currently or potentially used for soybean aphid control in Ontario. Recommended rate Trade Name of Product Spray volume [A.I.] Insecticide/Biopesticide A.I. /ha (L/ha) (ppm) Matador®120EC ?-cyhalothrin 83 ml.1 1501 66.4 Lagon® 480E Dimethoate 1000 mL1 2501 1920 Movento® 240EC Spirotetramat 250 mL2 2502 240 BotaniGard® 22WP Beauveria bassiana 1350 g2 2502 1188 Beleaf® 50SG Flonicamid 267 g2 2502 533 Superior 70OiI Mineral oil 14000 mL2 2502 58636 '(OMAFRA 2009) 2Rates used to control aphids on crops of similar plant arctitechure as per conversation with the each products respective manufacturer (Hallett and Schaafsma, unpublished data) 56 3.2.6.2 Screening bioassay All analyses were conducted with SAS ? 9.1.3. Prior to analysis Henderson-Tilton's formula was used to correct for control mortality (Henderson and Tilton 1955). Mortaility in the controls never exceeded 10% (range 0-7.8%) Percentage mortality was then arcsine square-root transformed and subjected to ANOVA using the MIXED procedure. Variance was partitioned into the random effect, replication, and the fixed effects, treatment, time of observation (24 and 48h after application), and the time of observation by treatment interaction. Time of observationwas considered a repeated measure. F-tests were used to test for significant effects. Differences between treatment means were determined using Tukey's procedure. Assumptions of the ANOVA were verified by plotting the residuals by predicted values, by blocks, and by treatments. The mean of residuals was equal to zero, and the Shapiro-Wilk test confirmed the residuals were normally distributed. A Type 1 error rate of a=0.05 was used for all statistical tests. 3.3 Results 3.3.1 Direct and residual contact LC50 The LC50 of ?-cyhalothrin was numerically lower but not significantly different than dimethoate for both exposure methods (Table 3.2). The LC50 of ?-cyhalothrin was 101.9 ppm (95% FL, 78.2 - 130.4ppm) and 87.9ppm (95% FL, 73.2 - 123.6ppm) for direct and residual contact, respectively (Table3.2). 57 TJ C ir, no go 5 ? in oo . ro O ^ _; «? 1 3- TJ (U 1 E (U CL «a; o ^t ^: o /r! us «^ E S OO CT) E S 2 2 o U ce m m OO IN O 3 oo ro TJ CTl «o y I m LT) 3 IN OO q LO .C LT) Ö "d -C Q. (/) 00 «t IT) +1 no O 4-» Ö O Q. +1 (U +1 U rsi TJ LTl TJ *-< ni C ID ro OO +1 Ó O +1 +1 Q. _o (O +¦» C O U ro TJ G-- O g (U O "ifô *?- ?? en 3 ro TJ O Ol TJ C IO Ol (U ?- c o o 'a ? C c 03 O (N W O oi E 3 -M TO ro >- "?? Q. ai (U _o b CC LO The LC50 of dimethoate was 115.8ppm (95% FL, 108.4 - 124.6ppm) and 122.7ppm (95% FL , 80.9 - 373.17) for direct and residual contact, respectively (Table 3.2). The slope of the dimethoate dose response curve for the residual contact bioassay was significantly greater than all other treatments (Table 3.2). The hazard quotients for ?-cyhalothrin were 2.1 and 1.8 for direct and residual contact, respectively. The hazard quotients for dimethoate were 15.7, and 16.6 for direct and residual contact, respectively (Table 3.2). However, because of recommended rate differences, the hazard quotient for dimethoate was 7-25 times higher than that of ?-cyhalothrin (Table 3.2.) 3.3.2 Screening bioassay The fixed effect of insecticide on wasp mortality was significant, while the effects of time of observation and time of observation X insecticide interaction were not (insecticide: df=17,137 ,F=100.40 , P<0.0001; day: df-1,137, F=2.76, P=0.0989; day by insecticide: df=17,137, F=0.55, P=0.9232) The descending order of toxicity for 24h mortality for the recommended rates was dimethoate > ?-cyhalothrin > flonicamid > Beauveria bassiana > mineral oil > spirotetramat (Fig. 3.1). The descending order of toxicity for 48h mortality for the recommended rates was dimethoate > ?-cyhalothrin > flonicamid > mineral oil > Beauveria bassiana > spirotetramat (Fig. 3.2). All wasps treated with dimethoate died in all replicates at all treatment rates. According to the IOBC ranking system, the recommended rate of dimethoate was harmful, ?-cyhalothrin was slightly harmful while flonicamid, Beauveria bassiana, mineral oil, and spirotetramat were harmless. 59 AAA 1.00 SZ CN ^. 0.80 (D ¡tí CO T3 0.60 H (D B V) ? a. 04Ojy y V) § 0.20 / 0.16 - e O ¦eg 0.12 o Q. o 0.08 0.04 c c p c c Ç £, 0.00 ????^??? c c c c ? ?-?-?-?-?-?-?-?-?-?-?-?-?-?-?-?-?-?- ¦fV'fv{V/'V{V/rv{V/'V{V'fv{V'fv /^C < V ? 'Vi» iV^ 'Ni ' » jar3> I j¿ è i * // * iI tt > / * / / -f ¦& ß & vp§ Treatment Figure 3.1: Corrected mortality of A. certus 24h after direct contact with five insecticides and one biopesticide currently or potentially used in soybean aphid management in Ontario. Insecticides were applied at Vi, 1, and 2 times their recommended rate (RR), for soybean aphid or aphids on architecturally similar crops. Columns with the same letter are not significally different (Tukey's a = 0.05). 60 AAA 1.00 SZ co i 0.80 f ¡e co ¦s 0.60 f BC 0.40 - V) ? O. y y V) CD co 0.20/ ? 5 0 0.16 H 1O 0.12 H ? 0.08 H ? 0.04 A D DDn D D G? 0.00 Iti ?> s>* ^ ^ # ·£¦ ¦?^ ¡S? Jt ·# iM #â ß& à s>$ /y£ ^&& <§> £~£> Jf # gf ..?- # ^ s> Q * -? "^ Treatment Figure 3.2: Corrected mortality of A. certus 48h after direct contact with five insecticides and one biopesticide currently or potentially used in soybean aphid management in Ontario. Insecticides were applied at VS, 1, and 2 times their recommended rate (RR), for soybean aphid or aphids on architecturally similar crops. Columns with the same letter are not significally different (Tukey's a = 0.05). 61 3.4 Discussion Dimethoate and ?-cyhalothrin were both toxic to A. certus by direct and residual contact. However, the hazard quotients for dimethoate are ca. 7-25 times greater than ?-cyhalothrin for both exposure methods. Because there is no evidence of yield differences when dimethoate or ?-cyhalothrin are used for soybean aphid control (Johnson and O'Neal 2006, 2007, 2008), dimethoate use should be avoided when A. certus is present in order to minimize parasitoid mortality in soybean fields. The most current recommended rate for ?-cyhalothrin is 66-186ppm (OMAFRA 2009). The hazard quotients of the highest rate for direct and residual contact are 2.1 and 1.8, respectively. Application of this rate (186ppm) is recommended when conditions favor soybean aphid population growth or when the soybean canopy is dense (OMAFRA 2009). However, the use of the high rate is potentially more damaging to A. certus populations, which may contribute to aphid resurgence and necessitate the further use of insecticides (Johnson and Tabashnik 1999). The hazard quotients for the lowest recommended rate are 0.8 and 0.6 for direct and residual contact, respectively. This low rate (66 ppm) is less toxic to A. certus and if possible should be used when A. certus is present. Insecticides vary in persistence and the amount of pesticide residue present on plant material after an application will vary (Pedigo 2002). The safe re-entry time for a natural enemy will vary depending on the persistence of, the insecticide and its susceptibility. Many pyrethroids are repellent to insects, which may affect parasitism 62 rates for aphid parasitoids (Longley and Jepson 1996). Additionally, ?-cyhalothrin is more persistent than dimethoate on plant surfaces. In a test of six insecticides applied to lettuce including dimethoate, ?-Cyhalothrin provided the longest residual efficacy against the lettuce aphid Nasonovia ribisnigri Mosley (Stufkens and Wallace 2004). Dimethoate disappears rapidly from the surface of soybean leaves, and after eight days residues are undetectable (Beck et al. 1966). Furthermore in insecticide efficacy trials carried out in the summer of 2006, soybean plants treated with ?-cyhalothrin accumulated fewer aphid days than those treated with dimethoate (Johnson and O'Neal 2007). This result suggests that dimethoate treated plants were re-colonized more quickly than plants treated with ?-cyhalothrin. The probability that a natural enemy will be exposed to a pesticide residue increases with the persistence of the pesticide. As dimethoate dissipates more quickly than ?-cyhalothrin, A. certus may be less likely to contact dimethoate than ?-cyhalothrin residues. However, the recommended rate for dimethoate is 16 times the LC50 of A. certus and, therefore even if dimethoate dissipates quickly, it is likely to cause mortality of A. certus. Further study is needed to determine the fate of these pesticides residues in the field and their impact on A. certus. In this study direct application of ?-cyhalothrin to A. certus at 133 ppm resulted in 61 and 75% mortality after 24 and 48h, respectively which correspond to an IOBC ranking of slightly harmful. In 2009, the recommended rate range for ?-cyhalothrin was changed to 66.4 - 186.4ppm (OMAFRA 2008, 2009). ?-cyhalothrin applied at a rate of 186.4ppm, would be at least slightly harmful, and likely moderately harmful or harmful. 63 The IOBC ranking obtained for dimethoate on A. certus is similar with results obtained for the parasitoid Encarsia formosa Gahan, and the aphid parasitoid Aphidius matricariae Haliday (Hassan et al. 1987, Hassan et al. 1988). Dimethoate is harmful to adult and pupal A. matricariae and to adult E. formosa (Hassan et al. 1988). This ranking is similar to the organophosphates fenithrothion, which is harmful to adult and pupal E. formosa, and vamidothion, which is harmful to adults but harmless to pupae (Hassan et al. 1987). Pyrethroids are generally harmful to many parasitoids (Sterk et al. 1999). For example, fluvaliate, cyfluthrin, deltamethrin, Cypermethrin, and fenpropathrin are all considered harmful to adult A. matricariae and E. formosa (Hassan et al. 1987, Hassan et al. 1988, Sterk et al. 1999). Deltamethrin is harmful to pupal E. formosa, while fluvaliate and cyfluthrin are moderately harmful to pupal A. matricariae and slightly harmful to pupal E. formosa (Hassan ef al. 1987, Hassan et al. 1988, Sterk er al. 1999). ?-cyhalothrin is considered harmful to adult A matricariae, but only slightly harmful to E. formosa (Sterk er al. 1999). This is similar to the current results for A certus. However, ?- cyhalothrin is considered harmful to the pupae of E. formosa (Sterk er al. 1999). Future work should determine how susecptible A certus pupae are to ?-cyhalothrin, to determine if the pupae are an insecticide tolerant lifestage. There is evidence that entomopathogens are compatible with some parasitoids (Mesquita et al. 1997, Mesquita and Lacey 2001, Labbe er al. 2009, Rashki et al. 2009). In some cases an entomopathogen and a parasitoid can have an additive effect on pest 64 suppression. For example, Aphelinus osychis Walker was able to detect aphids which had been infected by the entomopathogen Paecilomyces fumosoroseus for 72h and preferentially parasitize non-infected aphids (Mesquita and Lacey 2001). If A. certus is capable of detecting infected soybean aphids in this manner, Beauveria tassiana and A. certus could provide complementary pest control. The ability to detect fungal infection is especially important for egg-limited synvitogenic parasitoids, such as A. certus, since each egg they oviposit represents a substantial investment of time and energy compared to pro-ovigenic parasitoids (Bernal er al. 1994, Godfray 1994). In the current study Beauveria bassiana was harmless to A. certus, even though it has a large host range which includes Hymenoptera (McCoy et al. 1988, Devi ef. al. 2008. It is possible that A. certus possesses some resistance to Beauveria bassiana Strain GHA. However it is more likely that the time required for A. certus to succumb to Beauveria bassiana infection is longer than 48h. For example, the median time-to-death of the pea aphid, Aphis craccivora Koch, exposed to 29 different strains of Beauveria bassiana was 4.1d and ranged from 1.6 to 7.4d (Devi et al. 2008). Fungal infection results in a number of pathological symptoms including restlessness, reduced appetite, and loss of coordination (McCoy et al. 1988). It is possible that wasps in this study did experience early symptoms of fungal infection, however, only mortality was assessed. If observed, these symptoms would help to determine the infection rate. Alternatively, the environmental conditions in the bioassay container may not have been conducive to conidia germination; for example the relative humidity may 65 have been too low, or the lights may have been too bright (McCoy et al. 1988). In the future, mortality should be assessed for longer periods of time to confirm the susceptibility of A certus to Beauveria tassiana. Spirotetramat was harmless to A. certus. In other studies, one day old residues of spirotetramat were harmless to the parasitoids Coccidoxenoides perminutus Planopar and Trichogrammatoidea cryptophlebia Nagaraja which suffered <1% mortality after 24h exposure (Schnorbach et al. 2008). One and seven day old residues were slightly harmful (38.3% mortality) and harmless (<1% mortality), respectively, to Aphytis lingnanensis Compere after 24h exposure (Schnorbach et al. 2008). These results suggest that spirotetramat is harmless to parasitoids when encountered as a dry residue and this study suggest that direct contact is harmless. However, spirotetramat is a fully systemic insecticide which is phloem and xylem mobile and has activity in target pests when ingested (Nauen et al. 2008). Additionally spirotetramat activity is more pronounced in younger insects (Nauen et al. 2008). It is possible that adult or larval parasitoids which feed on contaminated plant or host tissue may be affected. However, parasitism rates of Aphelinus mali Haldeman varied only marginally when treated with spirotetramat in the field (Schnorbach et al. 2008). Flonicamid is considered specific to hemipteran and thysanopteran pests (Morita ef al. 2007). Flonicamid is harmless to a number of natural enemies including the mealybug destroyer Cryptolaemus montrouzieri Mulsant and the parasitoid Leptomastix dactylopii (Cloyd and Dickinson 2006). This is consistent with results for A. 66 certus, in which the majority (ca. 74% ) of flonicamid related mortality occurred after 24h and the mortality after 48h after application was <10%. Flonicamid inhibits feeding in aphids (Morita et al. 2007). IfA certus is also affected in this way, lack of feeding could explain why the majority of mortality occurred 24h after application. Further work should determine whether flonicamid has an inhibitory effect on A. certus feeding. The toxicity of mineral oil to parasitoids is variable. Mineral oil is moderately harmful to the parasitoid Trichogramma cacoeciae Marchai (Grutzmacher et al. 2004), but harmless to Coccidocenoides peregrines (Wakgari and Giliomee 2003), and A. certus. However, in the field mineral oil application can reduce the number of parasitoids within the sprayed areas (Badenes-Perez et al. 2002). Future work should determine the effect of mineral oil application on A certus in the field. In conclusion, A certus and dimethoate are non-compatible agents for the control of soybean aphid. However, dimethoate is currently under re-evaluation and may be deregistered for use in Canada (Health Canada Pest Management Regulatory Agency 2009). Aphelinus certus and ?-cyhalothrin are moderately compatible. Flonicamid, spirotetramat, Beauveria bassiana, and mineral oil were harmless to A certus compared to the currently registered insecticides dimethoate and ?-cyhalothrin, which were harmful and moderately harmful, respectively. Of these novel compounds registration is currently being sought for Spirotetramat on soybean to control soybean aphids. Use of novel products would help to conserve existing populations of A certus, 67 making them ¡deal candidates for use in soybean aphid IPM programs, assuming that efficacy against soybean aphid is demonstrated. 4 Susceptibility of Aphelinus certus Yanosh to two neonictotinoid seed treatments insecticides used for soybean aphid management. 4.1 Introduction Since soybean aphid (Aphis glycines Matsumura) was first observed in North America in 2000 (Hunt et al. 2003, Ragsdale et al. 2004), it has become a serious economic pest of soybean (Glycine max L) (Ragsdale et al. 2007). Soybean aphid feeding damage has the capacity to reduce soybean seed oil content and yields (Beckendorf et al. 2008, Catangui et al. 2009). Intensive management is required to control the soybean aphid to protect yields. Currently, the predominant management tools for soybean aphid are foliar and seed-applied insecticides (OMAFRA 2009). There is a growing body of evidence that natural enemies can play a role in regulating soybean aphid populations (Costamagna et al. 2007, 2008). It may be possible to incorporate conservation of these natural enemies into existing soybean aphid management practices (Zhang and Swinton 2009). This practice will not only reduce the need for pesticides, but also create a more sustainable pest management system. One such natural enemy is the soybean aphid parasitoid Aphelinus certus. Use of insecticides will likely remain a component of soybean aphid management, because of the aphid's high intrinsic rate of increase and its ability to damage plants during critical plant growth stages. Any natural enemies present in the field will be exposed to these insecticides. Insecticides can have lethal or sub-lethal effects on natural enemies, including reduced longevity, fecundity, and impaired feeding 69 or searching behavior (Desneux et al. 2007). All of these effects have the potential to reduce the ability of natural enemies to control pest populations (Stark et al. 2004a, 2007). Therefore, insecticides with minimal impact on natural enemies should be used wherever possible. One tactic to minimize non-target effects is to use insecticides which are selective. Insecticides can be selective on the basis of chemistry, toxicity or differential exposure (Ruberson et al. 1998). Insecticides which are differentially selective are applied in such a way as to minimize contact with non-target species (Johnson and Tabashnik 1999). For example, systemic seed-applied insecticides are differentially selective. These insecticides are translocated via roots to the rapidly growing parts of the plant. Therefore, only insects feeding directly on plant material should be exposed to these insecticide residues. However, this is not always the case and natural enemies may encounter residues by feeding on contaminated plant tissue (Stapel et al. 2000, Krischik et al. 2007) or contaminated hosts (Walker et al. 2007, Papachristos and Milonas 2008). Currently, two neonicotinoid seed treatments, imidacloprid and thiamethoxam, show promise for soybean aphid control (Magalhaes et al. 2009). Thiamethoxam is currently registered to control soybean aphid on soybean in Ontario, whereas imidacloprid is only registered in the United States (NCSRP 2009, OMAFRA 2009). To date no research has been reported on the effect of seed treatments on soybean aphid 70 parasitoids. To successfully incorporate these products into soybean aphid management systems, their effect on natural enemies must be investigated. This study examines the effects of imidacloprid- and thiamethoxam- contaminated soybean aphid hosts on the developmental success of the parasitoid Aphelinus certus. The purpose of this research was to determine the compatibility of neonicotinoid seed treatments with A. certus, by exposing the parasitoids to aphids feeding on neonicotinoid-treated soybean plants. 4.2 Materials and methods 4.2.1 Insect rearing Soybean aphid colonies were established from individuals collected on soybean from several locations in southern Ontario in the fall of 2007. Soybean aphid colonies were maintained on soybean plants cv. Colby (Hyland Seeds, Thompsons Limited, Blenheim, Ontario, Canada) grown in Pro-Mix® BX/Mycorise® (Premier Horticulture Inc. Quakertown, PA, USA) in a growth room at 27±2°C, 65-75% RH, and a 16:8h light:dark photoperiod. Prior to planting, seeds were soaked in Cell-Tech Soybean® Bradyrhizobium japonicum inoculant (EMD Crop Bioscience Canada, Belgrave, Ontario, Canada). Plants were watered as necessary and fertilized once weekly with Plant-Prod® 20-20-20 All Purpose Fertilizer (Plant Products Co. Ltd., Brampton, Ontario, Canada) Colonies of A. certus were established by collecting black soybean aphid mummies from soybean plants at several locations in southern Ontario during the fall of 2007. Aphelinus certus colonies were maintained in a growth room at 25±2°C, 65-75% 71 RH, and a 16:8h light:dark photoperiod. A. certus were reared on soybean aphid on soybean for three generations before use in any experiment. Aphid mummies were collected daily from colonies, placed individually in 1.5mL Eppendorf centrifuge tubes and supplied with a droplet of undiluted honey placed on the inside wall. Aphid mummies were monitored daily for emergence of wasps. To obtain mated females, a naive female and male wasp less than 24h old were paired and introduced into a 1.5mL Eppendorf tube and observed until mating occurred. Mated pairs were then left for at least one hour before use in experiments, at which time the male was removed. 4.2.2 Soybean plants Soybean seeds cv. Colby (Hyland Seeds, Thompsons Limited, Blenheim, Ontario, Canada) were treated with either Cruiser® 5FS (a.i. thiamethoxam) or Gaucho® (a. i. imidacloprid) at the rates of 83mL and 26OmL per 100 kg seed, respectively. Two kilograms of soybean seeds were placed in a heavy plastic bag and treated with equal parts seed treatment and distilled water. The bag was inflated, tied shut and then rotated until the seeds received an even coating of treatment solution. Treated seeds were left to dry in a fume hood for three days prior to planting. Soybean seeds were planted individually in Pro-Mix® BX/Mycorise® (Premier Horticulture Inc. Quakertown, PA, USA) in 12.5 cm diam. pots and 40 seeds of each treatment were planted. Plants were maintained in a greenhouse on the University of Guelph campus under summer conditions between 18 and 300C. Two plants from each treatment were randomly placed in a planting tray with drainage holes. Planting trays where randomly placed on a greenhouse planting bench and the tray and plant position within a tray were re- 72 randomized weekly. Plants were watered three times weekly and fertilized weekly with Plant-Prod® 20-20-20 All Purpose Fertilizer. The experiment was arranged in a completely randomized design, and the experimental unit was the individual plant. 4.2.3 Sampling Starting four weeks after planting at weekly intervals for seven weeks, five plants (replicates) from each treatment were randomly selected for a bioassay, for a total of 35 plants per treatment. Plants were sampled by removing the youngest fully expanded trifoliate. Two leaflets from each trifoliate were each placed individually in 10cm Petri dishes lined with damp cotton. Leaflets were placed adaxial side down on the cotton batting and approximately 25 fourth instar soybean aphids were transferred onto each. Twenty-four hours after instruduction of the aphids, a mated female A. certus was placed in one of two paired the Petri dishes from each plant for each treatment. Petri dishes were then sealed with Parafilm and maintained in a growth room at 25±2°C, 65- 75% relative humidity, and a 16:8h photoperiod. The parasitoid was removed after 24h. Petri dishes were monitored daily for 8 d and the number of alive, dead and parasitized aphids were recorded. The number of juvenile aphids was also recorded in the final 5 weeks of the experiment. Dead aphids, juvenile aphids, and A. certus mummies were removed daily after observations were complete. 4.2.4 Statistical analysis The proportion of initial aphids parasitized after 8 days was subjected to ANOVA using PROC MIXED in SAS v9.1. Variance was partitioned into the fixed effects 73 treatment, plant age (¡? weeks), treatment by plant age, number of aphids at Oh, and number of aphids after 24h, and the random effect, replication. For all analyses, F-tests were used to test for significant effects, and mean estimates were compared using Tukey's procedure. For all analyses the assumptions of the variance analysis were verified by plotting the residuals by predicted and by all fixed effects. The mean of residuals was equal to zero, and the Shapiro-Wilk test confirmed that the residuals were normally distributed. A Type 1 error rate a=0.05 was used for all statistical tests. Aphid mummies were only produced on 10 and 17 of the 35 imidacloprid- and thiamethoxam-treated plants, respectively. Aphid mummies were produced on 33 of the 35 control plants. Due to the low number of aphid mummies produced in the insecticide treatments, emergence data did not meet the necessary criteria for an ANOVA. To compare emergence, mean emergence across all weeks and replications for each treatment was calculated, and the overlap of standard deviation of the means were used as a basis of comparison. To determine the effect of insecticide and parasitoid presence on the soybean aphid two life table parameters were calculated: intrinsic rate of increase and the net reproductive rate. Intrinsic rate of increase (r) was calculated using the formula [1] (Wyatt and White 1977): r = 0.74(logeMd)/d [1] 74 where Md is the number of progeny produced in 24h and d is the pre-reproductive time in days. Pre-reproductive time at 25°C was determined to be 4.9d (McCornack et al. 2004). Net reproductive rate (R0) was calculated using formula [2]: R0=ZlxTnx [2] where Ix is the age-specific survivorship, and mxis the age-specific fecundity. Life-table parameters were calculated for each replication within each treatment. R0 and r data were subjected to an ANOVA using PROC MIXED in SAS v9.1. Variance was partitioned into the fixed effects treatment, plant age, presence of a parasitoid, plant age by treatment, parasitoid by treatment, parasitoid by plant age and, the random effect, replication. When significant, linear and lack-of-fit regression partitions were included for the effect of plant age for each treatment. 4.3 Results The effect of insecticide on parasitism rate was significant, whereas week, week by treatment, the initial number of aphids, and the number of aphids alive after 24h were not (insecticide: df=2,82, F= 26.75, P<0.0001; week: df=6,82, F=0.86, P>0.53; week by treatment: df=12,82, F=0.47, P>0.92; initial aphid number: df=l,82, F=2.27, P>0.13; the number aphids alive after 24h: df=l,82, F=I.09, P>0.30). Parasitism rate was highest in the controls and not significantly different between the two insecticide treatments (Fig. 4.1). The first aphid mummies in the thiamethoxam and imidacloprid treatments were found during weeks 5 and 6, respectively (Fig. 4.2). Emergence rates were 97.1±6.5%, 84.7±31.9% and 77.1±34.5%, for the control, imidacloprid, and thiamethoxam 75 0.18 H » 0.14-1 I 0.10 H S 0.08 H ceS 0.06 H 0.04 H 0.02 H Control Thiamethoxam lmidacloprid Treatment Figure 4.1. Parasitism rates of Aphelinus certus on Aphis glycines feeding on soybean plants that were untreated (control) or that received seed treatment with imidacloprid or thiamethoxam. Bars with the same letter are not significantly different (Tukey's a = 0.05), n=35 for each treatment. 76 (D 0.1 6 7 8 Plant age (weeks) I I Control Thiamethoxam ? ? lmidacloprid Figure 4.2: Parasitism rates of Aphelinus certus presented with Aphis glycines feeding on soybean plants of different ages. Soybean plants were untreated (control) or received seed treatments with either imidacloprid or thiamethoxam, n=35 for each treatment. 77 treatments, respectively. These emergence rates were not significantly different determined by overlap of the standard deviations. The effect of treatment, plant age, plant age by treatment, and parasitoid on the soybean aphid net reproductive rate, R0, were all significant, whereas the effect of treatment by parasitoid and parasitoid by plant age was not (Table 4.1). The linear regression partition for the effect of plant age on R0 in the imidacloprid treatment was also significant (df=l,42, F= 20.62, P<0.0001), whereas the lack-of-fit partition was not (df=3,42, F= 1.74, P>0.17). The linear regression partitions for the effect of R0 in the control and thiamethoxam treatments were not significant (control: df=l,42, F= 0.16, P=0.6869; thiamethoxam: df=l,42, F= 3.63, P>0.06). The effect of treatment, plant age by treatment, and parasitoid on the soybean aphid intrinsic rate of increase, r, were significant, but the effects of plant age, treatment by parasitoid, and plant age by parasitoid were not (Table 4.2). The linear regression partition for the effect of plant age on r in the imidacloprid treatment was significant (df=l,42, F= 13.17 P= 0.0008), whereas the lack-of-fit partition was not (df=3,42, F=O. 91, P>0.44). The linear regression partitions for the effect of plant age on r in the control and thiamethoxam treatments were not significant (control: df=l,42, F= 0.37, P=0.5459; thiamethoxam: df=l,42, F= 0.01, P>0.99). 78 Table 4.1: Results of ANOVA tables, for the effects of treatment, plant age, and parasitoid presence on the net reproductive rate of the soybean aphid Aphis glycines. Net Reproductive rate (R0) Cov. Param. Estimate S.E. Z Value PrZ Block 0.2223 0.3808 0.58 0.2794 Residual 13.9765 1.7539 7.97 <0.0001 Effect NumDF Den DF F Value Pr>F Plant Age 4 127 5.34 0.0005 Treatment 2 127 22.89 <0.0001 Plant Age by Treatment 8 127 2.12 0.0386 Parasitoid 1 127 18.29 <0.0001 Parasitoid by Treatment 2 127 1.19 0.3081 Plant Age by Parasitoid 4 127 1.07 0.3723 79 Table 4.2 Results of ANOVA, for the effect of treatment, plant age, parasitoid intrinsic rate of increase of the soybean aphid, Aphis glycines. Intrinsic rate of Increase (r) Cov. Param. Estimate S1E. Z Value PrZ Block 0.000027 0.000171 0.16 0.4382 Residual 0.02818 0.003536 7.97 <0.0001 Effect Num DF Den DF F Value Pr>F Plant Age 4 127 1.25 0.2940 Treatment 2 127 19.91 <0.0001 Plant Age by Treatment 8 127 2.20 0.0316 Parasitoid 1 127 4.25 0.0414 Treatment by Parasitoid 2 127 0.01 0.9945 Plant Age by Parasitoid 4 127 0.09 0.9842 80 The R0 of soybean aphids in the ¡midacloprid treatment increased linearly over the course of the experiment (Fig. 4.3.). There was no change in Ro over time in the control and thiamethoxam treatments. The R0 of the soybean aphid was numerically lower in both insecticide treatments compared to the control. Similarly, the r of the soybean aphids in the ¡midacloprid treatment increased linearly over the course of the experiment (Fig. 4.4.), but there was no change in r in the control and thiamethoxam treatments. The r of the soybean aphid was numerically lower in both insecticide treatments compared to the control. The Ro of soybean aphid not exposed to parasitoids in the control treatment was significantly greater than all other treatment combinations. The R0 of soybean aphids in all treatments was numerically lower with parasitoid exposure than without; however, the difference was only significant in the control treatment (Table 4.3). The R0 of soybean aphid exposed to A. certus in the control treatment was significantly greater than that of soybean aphids exposed to A. certus in the thiamethoxam treatment. The R0 was lowest in soybean aphids exposed to A. certus in the thiamethoxam treatment. The r of the soybean aphid was highest in the no-parasitoid control treatment and was significantly different from all other treatments, except the parasitoid control treatment. 4.4 Discussion This experiment shows that A. certus was able to successfully parasitize soybean aphid feeding on excised leaves from neonicotinoid-treated plants. An aphid mummy 81 16 Control UJ 14 W 12 10 ce ¦? 8 Q. a? QL S 4 0 16 UJ Thiamethoxam W 14 H 12 H ?? H è 8 A (1) a: is ? a> 2? 16 UJ W 14 lmidacloprid -?> 12 AB (? 10 ?: ABC Ë. 6 BC ?: ÎÎ 4 8 10 Plant Age (weeks) Figure 4.3: Effect of plant age on net reproductive rate (R0) of the soybean aphid exposed to excised soybean leaves from untreated plants or plants grown from seed treated with imidacloprid or thiamethoxam. Within a treatment, means with different letters are significantly different (Tukey's a=0.05), n=50 for each treatment. 82 0.5 lij Control 05 i. 0.4 b 03 02 ¦£ 0.1 0.0 0.5 UJ cri Thiamethoxam a, 0.4 b 0.3 0.0 0.5 lmidacloprid Ul cri ? 0.3 (D b 0.2 7 8 9 Plant Age (weeks) Figure 4.4: The effect of plant age on the intrinsic rate of increase (r) of the soybean aphid exposed to excised soybean leaves from untreated plants or plants grown from seed treated with imidacloprid or thiamethoxam. Within a treatment means with different letters are significantly different (Tukey's =0.05), n=50 for each treatment. 83 Table 4.3: The R0 and r of the soybean aphid exposed to excised soybean leaves from untreated plants or plants that received seed treatments of imidacloprid or thiamethoxam. Net reproductive rate(R0) Parasitoid No Parasitoid Control 7.73 ± 0.952b 11.66 ± 0.952a Thiamethoxam 4.03 ± 0.952c 5.80±0.952bc Imidacloprid 4.79±0.952bc 6.92±0.952bc Intrinsic Rate of Increase (r) Parasitoid No Parasitoid Control 0.26±0.036ab 0.37 ± 0.036a Thiamethoxam 0.07 ± 0.036c 0.12 ± 0.036c Imidacloprid 0.09 ± 0.036c 0.15±0.037bc Means followed by different letters are significantly different (a=0.05) for each life table parameter. 84 was first found on a leaf excised from a thiamethoxam treated plant 5 weeks after planting. The first aphid mummy found on a leaf from an imidacloprid treated plant was 6 weeks after planting. Even though parasitoids were capable of completing development on aphids exposed to neonicotinoid treated material parasitism rate was reduced by 69 - 88%. This reduction in parasitism rate has the potential to disrupt A. certus biological control of soybean aphid. There are three likely explanations for the reduction in parasitism observed in insecticide treatments. First, the reduction in parasitism could be a result of the functional response of A. certus. Aphelinus certus exhibits a type Il functional response to the soybean aphid (Frewin et al. in press). Therefore, if insecticide exposure reduced the number of aphids available to the parasitoid, then the parasitism rate may change as well. However, this scenario seems unlikely, as the initial number of aphids available to the parasitoid and the numbers of aphids alive when the parasitoid was removed from the experimental arena were not found to be significant effects in the analysis. Second, the insecticide may have caused mortality of the parasitoid larvae and/or the host aphids. If larval parasitoids died due to exposure to insecticide residues within the host aphids, or if parasitism increased the lethal effect of insecticide residues on the host aphid, then a reduction in parasitism rate as well as a reduction in successful parasitoid emergence in insecticide treatments would be expected. If insecticide exposure impacted larval parasitoid development, then insecticide contamination could also have interfered with successful pupal development. Alternatively, parasitism and insecticide exposure could have an additive effect on aphid mortality. Both of these 85 effects would explain the results seen here. They could be distinguished by additional experiments. Live aphids could be dissected at a fixed time after parasitoid exposure to determine whether parasitoid larvae are dying from insecticide exposure. For example, if mummy formation normally occurs 6d after parasitism, it could be assumed that any parasitoid egg or larvae found in an aphid 6d after parasitism died or was developmentally inhibited as a result of pesticide exposure. In order to determine whether parasitized aphids are more likely to die from insecticide exposure, dead aphids should be examined for the presence of parasitoid eggs or larvae. Regardless of the mechanism, the death of immature parasitoids would result in a reduction of the next generation. If parasitoid larvae are dying within the host, or parasitized aphids are more likely to die from insecticide exposure, then the seed treatments are interfering with biological control by A. certus. Finally, the parasitoids may avoid insecticide-contaminated aphids. Aphelinus parasitoids use internal rather than extra-cuticular cues to assess host suitability and are capable of detecting con- and hetero-specific parasitism as well as fungal infection within potential hosts (Bai and Mackauer 1991, Mesquita et al. 1997, Mesquita and Lacey 2001). It is possible that these parasitoids are using stress-related or immune- related aphid-produced chemicals in assessing aphid quality. If this hypothesis is true, Aphelinus parasitoids should be able to reject a host which is stressed or injured for a variety of reasons, including insecticide poisoning. The reduced parasitism rate observed in the insecticide treatments may be due to the rejection of hosts by A. certus as a result of the hosts' exposure to the insecticides. If this is the case then these two control 86 mechanisms (i.e. parasitoids and insecticide seed treatments) are highly complementary as the parasitoid is only using the healthiest hosts within the exposed populations and enhancing overall soybean aphid mortality. To test this hypothesis, parasitoids would need to be simultaneously presented with aphids exposed to insecticide residues and aphids not exposed to insecticide residues. The duration of ovipositor probing can be used to determine if an Aphelinus has oviposited in a given host (Bai and Mackauer 1990, 1991). Long (>3sec) ovipositor probes are associated with host acceptance and short (<1 sec) ovipositor probes are associated with host rejection. If A. certus preferentially selects healthy aphids, then the majority of long ovipositor probes will be in untreated aphids, whereas insecticide contaminated aphids would receive the majority of short ovipositor probes. There is little information available on the ability of aphid parasitoids to develop within hosts contaminated with sub-lethal concentrations of neonicotinoids. Imidacloprid and thiamethoxam seed treatments reduced the parasitism rate and emergence of Aphidius colemani hosting on soybean aphid (Welsman 2007). Imidacloprid contamination also reduced the emergence of Aphidius ervi hosting on Acyrthosiphon pisum (Kramarz and Stark 2003). In general, neonicotinoids are toxic to parasitoids. The parasitoid Microplitis croceipes Cresson feeding on neonicotinoid contaminated material suffers from reduced foraging efficacy and longevity (Stapel et al. 2000). The parasitoid Anagyrus pseudococci can die from feeding on nectar from imidacloprid-treated plants (Krischik et al. 2007). Similarly, imidacloprid is highly toxic 87 to the aphid parasitoid Aphidius gifuensis when consumed (Kobori and Amano 2004), and to Aphidius mali through residual contact (Cohen et al. 1996). The analysis of life table parameters show that imidacloprid and thiamethoxam had both lethal and sub-lethal effects on soybean aphid. Across all treatments, soybean aphid exposed to contaminated material suffered a reduction in intrinsic rate of increase (r) and net reproductive rate (R0), and this is consistent with other work (Magalhaes et al. 2008). In additional to effect of insecticide, parasitism numerically reduced R0, and r of the soybean aphid in all treatments. However, there is no additive effect of parasitism and insecticide on the reduction of life table parameters of the soybean aphid. This is likely due to the extremely low parasitism rate in the insecticide treatments. If the reduction in parasitism is due to insecticide related mortality of A. certus larvae, then as the insecticide titre in the plant diminishes over the growing season an additive effect of parasitism and insecticide may become apparent. The estimated intrinsic rate of increase for soybean aphid in this experiment was 0.37 per day and this is almost identical to that reported elsewhere for soybean aphid under comparable laboratory conditions and temperature of 0.374 per day (Magalhaes et al. 2008). Estimates of r obtained from field populations of soybean aphid range from 0.20 - 0.26 per day and may be low due to diurnal fluctuations and daily variation in temperature and humidity (Rutledge and O'Neil 2006). Other estimates of r for aphids in controlled environment chambers are higher than our estimate, for example 0.474 per day at 25°C (McCornack et al. 2004) and 0.415 d"1 at 22°C (Myers et al. 2005). Variability 88 in r may be due to differences in test conditions, including temperature, humidity, light, and enclosure size. Additionally, the origin of the soybean aphid populations used by McCornack et al. (2004) and Myers et al. (2005) to generate these values are different from those in my study and included Nebraska, Minnesota, Indiana, Wisconsin, and Ontario. Although the population genetic structure of soybean aphid in North America indicates a single population, populations from Michigan and Ontario are more similar to each other genetically than to populations in the southern and mid-western USA (Michel et al. 2009). The intrinsic rate of increase and net reproductive rate of soybean aphids increased in a linear fashion in the imidacloprid treatments over the course of the experiment, possibly due to a decline in insecticide residues due to natural degradation and plant metabolism. However, there was no corresponding increase in parasitism rate, which may indicate that A. certus is more susceptible to thiamethoxam than the soybean aphids are. This result also indicates that thiamethoxam may have longer residual activity than imidacloprid at the rates tested. An increasing parasitoid population early in the season is considered extremely important to the success of biological control in field crops, as natural enemies often impact pest populations most dramatically at low densities before serious outbreaks occur (Barbosa 1998). Although imidacloprid and thiamethoxam slowed aphid population growth, they also dramatically reduced the parasitism rate of A. certus. This reduction in parasitism rate will likely impair the ability of A. certus to control soybean 89 aphid, if the parasitoid is unable to establish itself early in the soybean growing season. Furthermore, it is unknown if there are any further sub-lethal effects of these insecticides on the parasitoid, and it is possible that A. certus emerging from contaminated hosts will suffer behaviorally and physiological abnormalities similar to other parasitoids consuming neonicotinoids as adults (Stapel et al. 2000). In this experiment A. certus was able to successfully develop on aphids feeding on plants treated with neonictonioids, however the parasitism rate of these aphids was greatly reduced. This indicates that A certus biological control is not compatible with imidacloprid and thiamethoxam seed treatments up to 10 wks after planting. In order to determine the impact of seed treatments on A. certus more completely, future work should focus on how long after planting the soybean aphid life table parameters return to normal and when the parasitism rate approaches that of the controls. 90 5 General Conclusions and Recommendations The soybean aphid is a serious economic pest of soybean in North America (DiFonzo 2009). Soybean aphid has the capacity to reduce plant yields by vectoring viruses and reducing photosynthetic efficiency, and excessive soybean aphid feeding can reduce soybean seed oil content below desirable levels (Clark and Perry 2002, Catangui et al. 2009). Soybean aphid can be difficult to manage due to its dispersal capacity and high intrinsic rate of increase. Because of this, a variety of management tactics have been employed including the use of foliar- and seed-applied insecticides, the use of resistant plant varieties, and, more recently the use of classical and indigenous biological control agents. The most promising long term management strategy for soybean aphid is to incorporate a number of these management tactics together in a complementary manner. The incorporation of multiple complementary management tactics is the basis for integrated pest management (IPM). Careful and frequent scouting is important to determine aphid population numbers and growth. Management guidelines for the soybean aphid in Canada and the United States recommend weekly scouting for an action threshold of 250 aphids per plant with actively increasing populations (NCSRP 2009, OMAFRA 2009a). These management guidelines are recommended for the R1-R5 plant stage (i.e. beginning of flower to beginning of seed formation) (Ragsdale et al. 2007). Soybean aphid has little effect on yield at later reproductive plant stages R5-R7 (i.e. beginning of seed formation to maturity), and insecticide application is not recommended (Johnson and O'Neal 2008, Catangui ef al. 2009). The 250 aphids per plant action threshold was determined by 91 consensus ¡? 2001 and was widely promoted by extension workers. In 2007, an economic action level of 273 aphids per plant was determined experimentally (Ragsdale ef al. 2007). However the 95% CL of this new estimate overlaps 250. Since the 250 aphids per plant action threshold had already been widely publicized, subsequent management recommendations have not changed (Ragsdale ef al. 2007). With 273 soybean aphids per plant, populations will reach the economic injury level of 674 aphids per plant in about 7 days (Ragsdale et al. 2007). Determination of the stage-specific economic injury levels confirms that younger soybean plants are more vulnerable to soybean aphid infestation (Catangui et al. 2009). For natural enemies to be effectively utilized in soybean aphid management, data on their biology, life history, and susceptibility to various pesticides is required. Current management recommendations in Canada and the United States recommend scouting for soybean aphid natural enemies and suggest delaying pesticide application, if populations are large (NCSRP 2009, OMAFRA 2009a). However, no indication of what constitutes a "large population" is given in current extension publications. A bioeconomic model could be used to create a natural enemy-adjusted economic threshold (Zhang and Swinton 2009) but more work is needed before this information can be extended to producers. In its native range the soybean aphid is only an occasional pest and is controlled by a number of biotic factors including natural enemies (Wu et al. 2004b). The soybean aphid natural enemy guild consists of numerious predators, pathogens and parasitoids. Parasitoids play an important role in suppressing aphid populations (Wu ef al. 2004b). Aphelinus certus is native to Asia and Russia where it is known to parasitize soybean 92 aphid (Heraty et al. 2007). Until recently the natural enemy guild in North America consisted predominantly of generalist predators (Nielsen and Hajek 2005, Kaiser ef al. 2007). However, in 2006 the exotic soybean aphid parasitoid Aphelinus certus was found in Ontario and now has been documented naturally parasitizing soybean aphid throughout the soybean growing region of Ontario. Further work is needed to determine the complete geographical range of A. certus in Ontario and in neighbouring provinces and states. Aphelinus certus appears to be the dominant member of the soybean aphid parasitoid guild in Ontario. Aphelinus certus is a welcome and needed addition to the soybean aphid natural enemy guild in Ontario, and an ideal candidate for inclusion in soybean aphid management decisions. To properly incorporate A. certus into soybean aphid management practices, an in-depth knowledge of its biology and physiological growth parameters is required. This thesis reports the first detailed examination of the biology of A. certus. The data collected here indicate that A. certus has a favourable temperature-dependent growth rate in comparison to the soybean aphid and is capable of growth and development concurrently with the soybean aphid. Aphelinus certus is also capable of normal growth and development at temperatures that slow soybean aphid population growth. Therefore, current and predicted temperatures should be incorporated into soybean aphid management decisions whenever A. certus is present. It is likely that warm temperatures and parasitoid presence will have an additive effect on soybean aphid control. 93 The data collected herein provided a basis for estimating some critical developmental parameters, such as R0, r, LT0 and Topt. These can be used to model and predict seasonal occurrence and population growth of A. certus. Knowledge of seasonal occurrence and population trends will enable growers and extension workers to make soybean aphid management decisions that incorporate A. certus, such as postponding insecticide application. For example, once soybean plants reach the R5 growth stage soybean aphid feeding will not reduce yields. Therefore if insecticide application can be postponded until the R5 growth stage, no insecticide application will be required. The most common control method for soybean aphid is the application of foliar insecticides (OMAFRA 2009a). In Ontario two foliar-applied insecticides are registered to control soybean aphid, dimethoate and ?-cyhalothrin. Both are acutely toxic to A. certus as foliar residues or through direct contact. However, the recommended rate (a.i./ha) and hazard quotient of dimethoate are much higher than those of ?-cyhalothrin. The recommended rate of dimethoate is approximately 16 times greater than the LC50 for A. certus, whereas the highest recommended rate of ?-cyhalothrin is only 2.75 times the LC50 for A. certus. Therefore, ?-cyhalothrin is preferable to dimethoate to control soybean aphid. A rate range allowing application between 83mL/ha and 235mL/ha ??G?- cyhalothrin was approved for control of soybean aphid in 2009 (Syngenta Crop Protection Canada, Ine). The higher end of this range is recommended when soybean aphid populations are actively increasing or for dense soybean canopies (OMAFRA 94 2009). The hazard quotients for the 83 mL/ha and 235 mL/ha rates by direct contact are 0.75 and 2.75 respectively. Therefore, the lower rate is much safer for A certus than the high rate. Management guidelines should stipulate that the lower rate is preferred when natural enemies are present. However, at this time no information on the impact of pesticides on A. certus in the field, or the sub-lethal and/or long term effects of these insecticides on A. certus is available. In addition to foliar sprays, two seed-applied insecticides, imidacloprid and thiamethoxam, are available for the control of soybean aphid. Thiamethoxam is currently registered for use on soybean in Ontario, whereas imidacloprid is only registered in the United States. Aphelinus certus is capable of completing development on soybean aphids fed on soybean plants grown from imidacloprid- or thiamethoxam- treated seed. However, the parasitism rate of A certus on soybean aphid exposed to this plant material was drastically reduced over controls. A reduction in parasitism rate was observed up to 10 weeks after planting. Over the ten weeks of the experiment, life- table parameters of the soybean aphids exposed to imidacloprid increased in a linear fashion, approaching that of the controls, indicating diminishing sub-lethal effects as the concentration of imidacloprid decreased. However, there was no corresponding increase in parasitism rates by A certus. A possible explanation is that A. certus is more susceptible to imidacloprid than is the soybean aphid. These results suggest that neonicotinoid seed treatments are not compatible with A. certus. Future work should investigate the mechanism underlying the reduction in parasitism rate, in combination with choice assays. IfA certus intentionally avoids pesticide contaminated hosts, it may 95 be possible to use neonicotinoid seed treatments to complement biological control by A. certus in the field. All of the novel insecticidal agents tested have a promising selectivity ratio, and mortality was below 20% in all bioassays completed. In addition to the currently registered insecticidal agents, there are a number of products with efficacy against the soybean aphid that may be registered for use in Ontario in the future, including the insecticides spirotetramat, flonicamid, mineral oil, and the biopesticide Beauveria bassiana. All of these insecticidical agents are less toxic to A. certus than ?-cyhalothrin and dimethoate at relevant field rates when applied topically. Contact toxicity to A. certus in descending order was flonicamid > mineral oil > Beauveria bassiana > spirotetramat. However, the duration of this experiment (48h) was likely too short to detect B. bassiana-induced mortality, which may require a longer incubation time to exert its effect (Devi et al. 2008). Further work will be required to determine the susceptibility of A. certus to B. bassiana. The mortality of A. certus exposed to spirotetramat was the lowest of all insecticides tested and deserves further investigation because of potential sub-lethal effects of its mode of action, including a potential reduction in fecundity due to interference with lipid synthesis. Spirotetramat may need to be ingested for maximum effect (Maus 2008) and oral exposure of spirotetramat to A. certus should be investigated. Some adult parasitoids lack a functional lipid biosynthesis pathway, and this may provide A. certus with protection from spirotetramat (Visser and Ellers 2008). Further work is needed to examine sub- lethal effects of these novel insecticidal agents on A. certus in the laboratory and field. 96 In conclusion, A. certus appears to be a useful and valuable soybean aphid natural enemy, with the potential for inclusion in soybean aphid management intregated pest management using conservation biological control. Aphelinus certus has an evolutionary relationship with the soybean aphid and is already established in the soybean growing region of Ontario. It is capable of developing under the same environmental conditions as the soybean aphid, and is more tolerant to high temperatures than the aphid. A number of insecticides that are currently and potentially registered for soybean aphid management are selectively favorable to A. certus. Use of these insecticides will help to maintain A. certus populations, which in turn will reduce the likelihood of pest resurgence. 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