Subobjective 2.2.2. Development of BMSB-specific fungal Entomopathogens (St. Leger - U of Maryland & Lee – USDA Corvallis) Background

• Metarizhium species, a.k.a. green muscardine fungi, are recognized for their biological control potential against • Metarhizium infects and kills BMSB at high humidities • Poor performance of fungi at low humidities – Low humidities reduce germination – Increase susceptibility to volatiles • BMSB relies heavily on volatiles for defense – BUT haemolymph defenses are weak as shown by injecting spores

A “glow in the dark” BMSB produced by injecting 50 spores of Ma-GFP Metarhizium spray of nymphs (OR, Jana Lee)

• 66% of nymphs sprayed with * Met 52 EC died • Many nymphs (control or Met) still molted to adulthood • While Met can kill nymphs, this rate under optimal lab conditions is not promising enough to meet grower needs

Skillman, Lee -USDA Corvallis The Objective: (UMD, St. Leger) Genetically manipulate fungal strains (Metarhizium) to overcome this defense, leading to sustainable BMSB management tactics

The Challenge: Low virulence of fungi against BMSB

• Brown marmorated stink bug defensive compounds may be the cause of poor fungal performance • Two chemicals present in stink bug defensive secretions (trans-2- octenal and trans-2-decenal) found to strongly inhibit growth of entomopathogenic fungi

trans-2-octenal trans-2-decenal

http://pubchem.ncbi.nlm.nih.gov/compound/trans-2-Octenal http://www.sigmaaldrich.com/catalog/product/aldrich/w236608?lang=en®ion=US

Logical Next Steps – BMSB-specific fungal Entomopathogens

• Resistant mutants have fluffy white growth – this could be because BMSB volatiles resemble chemicals that fungi use for auto-inhibition of spores  select for mutants that do not auto inhibit  transgenic approaches to alter regulation of sporulation genes  Conduct mass spec analysis of volatile production by infected BMSB  Determine role of microbial community on cuticle  Employ field cages to test laboratory results that single are more susceptible than clustered insects  Protoplast fusion of BMSB-infecting Metarhizium strains with M. acridum (desert strain that infects at low humidities) .

 Use GE to convert volatile and/or RH resistant strain to hypervirulence Subobjective 2.2.5. Asian Natural Enemies Foreign exploration for Asian natural enemies of BMSB (2007-2015)

30+ parasitoid populations in culture at ARS BIIR for host range and efficacy testing tsp180

tsp181

tsp179

tsp175

tsp174

tsp173

Phylogenetic tree tsp172

tsp171

tsp169

tsp168

tsp166

tsp165

tsp164 based on tsp163 tsp157

TSP130 69 TSp125 Scelionidae recovered from Asian TSp118 TSP115 & TSp110 Maximum Likelihood, Tsp53 TSP98 Pentatomid eggs TSp102

TSp106

Tsp50

Tsp49

Tsp48 100 Tsp45 T. japonicus GTR+G+I model Tsp44 tsp185

TSP77

TSP78

TSP79

TSP88 99 59 TSP90 Scelionidae recovered from North (CO1) TSP91 TSP93

Tsp52

Tsp41

tsp154 100 American Pentatomid eggs tsp155

100 tsp167

tsp170

tsp182

85 TSP144 TSP141

100 TSP142

TSP143

TSP145 TSP146 T. spp. 58 100 TSP147

TSP148

Tsp51

100 tsp186

Tsp43

tsp161 22 100 tsp176 T. plautiae Tsp42

Tsp46 100 tsp162

Tsp54

TSP127

TSP128 100 TSp126

TSP96

TSP92 62 TSP87

TSP86 77 TSP84

TSP83

TSP82 TSP80 T. cultratus TSP81

TSP85

TSP89

TSP129

99 TSP131

TSP132

TSP133

TSP134

TSP135

TSP136

TSP137

TSP138

TSP139

TSP140 Tsp40 T. spp. 100 Tsp58

41 Tsp59 TSP94

100 TSP95

TSP152

TSP153 1 100 TSP151 TSP150 Contributors: TSP149 T. mitsukurii Tsp39 5 tsp189

99 tsp184 M.-C. Bon – ARS/EBCL 100 tsp188 26 tsp156

100 tsp177

tsp178 100 Telenomus spp. E. Talamas – ARS/SEL 73 tsp187

T. edessae 100 tsp190 tsp191

tsp183 76 M. Buffington – ARS/SEL Tsp55 83 Tsp57 61 tsp192 T. spp. 61 tsp193 100 C. Dieckhoff – ARS/BIIR tsp194

tsp159

tsp158 100 tsp160 Gryon sp. K. Hoelmer – ARS/BIIR 0.1 Trissolcus japonicus (Hymenoptera: Scelionidae) (first described as T. halyomorphae)

 solitary egg parasitoid Brian T. Cutting

 2 - 3 weeks/generation

 multiple generations/season

 female-biased sex ratio

 65 to 90% parasitism on BMSB reported in Asia

Yang et al. (2009) Annals of the Entomological Society of America . 102: 39-47 Trissolcus japonicus is oligophagous - it attacks several Asian pentatomid species

Halyomorpha halys Glaucias subpunctatus

Plautia crossota Dolycoris baccarum Erthesina fullo Host Range Evaluations– A team effort to fast-track the evaluation process

Funding for Host Range Evaluations: OR MI Farm Bill funding (APHIS PPQ) CA DE NIFA SCRI multi-institution BMSB grant

Collaborators: = active screening for 2+ yrs FL = started 2013 USDA-ARS (Newark, DE & EBCL, ) = started 2014 University of Delaware (D. Tallamy) Florida Dept. Agriculture & Consumer Services, Division of Plant Industry MSU – Michigan State University – Department of Entomology (E. Delfosse) Oregon Department of Agriculture Oregon State University – Department of Horticulture (V. Walton, P. Shearer) University of California, Riverside & CDFA (M. Hoddle, C. Pickett) Host Range Evaluations: Progress

62 species total tested nationwide

22 species 40 species in progress completed

0 17.5 35 52.5 70 Host range testing procedures

No Choice Test Choice Test

Exposure to non-target species egg mass Egg masses of non-target species and only – for 24h: If parasitism BMSB presented together for 24h: on non-target is recorded

Followed by a BMSB target egg mass as control for another 24h: No-Choice Test Outcome - Part 1 (as of 2015)

Agonoscelis puberula (N=12) Amaurochrous sp. (N=8) Antheminia remota (N=6) Bagrada hilaris (N=7) Brochymena quadripustulata (N=17) Chlorochroa ligata (N=25) Coenus delius (N=12) KOA (N=12) Cosmopepla intergressa (N=5) Edessa florida (N=28) Holcostethus abbreviatus (N=20) Murgantia histrionica (N=23) Oebalus pugnax (N=12) Perillus bioculatus (N=7) Podisus brevispinus (N=9) Stiretrus anchorago (N=6) Thyanta pallidovirens (N=16) Trichopepla semivittata (N=) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

live males live females unemerged parasitoids hatched nymphs unhatched nymphs No-Choice Test Outcome - Part 2 (as of 2015)

Banasa dimiata (MI, N=13) Banasa dimiata (OR, N=22) Chinavia hilaris (MI, N=17) Chinavia hilaris (OR, N=19) Chlorochroa uhleri (CA, N=8) Chlorochroa uhleri (OR, N=8) Cosmopepla lintneriana (DE, N=26) Cosmopepla lintneriana (OR, N=16) Euschistus servus (DE, N=20) Euschistus servus euschistoides (MI, N=13) Euschistus tristigmus (DE, N=26) Euschistus tristigmus luridus (MI, N=20) Euschistus variolarius (MI, N=17) Euschistus variolarius (DE, N=21) Holcostethus limbolarius (DE, N=20) Holcostethus limbolarius (MI, N=20) Podisus maculiventris (DE, N=34) Podisus maculiventris (MI, N=19) Thyanta custator (DE, N=23) Thyanta custator (OR, N=23) Thyanta custator accerra (DE, N=13) Thyanta custator accerra (MI, N=22) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% live males live females unemerged parasitoids hatched nymphs unhatched nymphs Choice Test Outcome - Part 1 (as of 2015)

BMSB Banasa dimiata (N=33) BMSB Chinavia hilaris (N=23) BMSB Chlorochroa ligata (N=18) BMSB Edessa florida (N=21) BMSB Euschistus servus (N=25) BMSB Murgantia histrionica (N=27) BMSB Podisus maculiventris (N=31) BMSB Stiretrus anchorago (N=6)

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% live males live females unemerged parasitoids hatched nymphs unhatched nymphs Choice Test Outcome - Part 2 (as of 2015) BMSB Cosmopepla lintneriana (MI, N=22) BMSB Cosmopepla lintneriana (DE, N=12) BMSB Euschistus tristigmus luridus (MI, N=8) BMSB Euschistus tristigmus (DE, N=25) BMSB Euschistus variolarius (MI, N=9) BMSB Euschistus variolarius (DE, N=17) BMSB Holcostethus limbolaris (MI, N=21) BMSB Holcostethus limbolaris (DE, N=10) BMSB Thyanta custator (OR, N=26) BMSB Thyanta custator (DE, N=35) BMSB Thyanta custator accerra (MI, N=28) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% live males live females unemerged parasitoids hatched nymphs unhatched nymphs Logical next steps A Closer Look at Host Choice Behavior in T. japonicus

Influence of arena size and complexity Role of parasitoid physiology & experience

 Size (Finished) o Parental experience o 10 dram o Parental physiology o 100 dram o Effect of host choice on o 500 dram offspring physiology & o 1000 dram behavior o 2000 dram

 Complexity (Started) o Choice tests on plants Behavioral observations

Influence of time of exposure Arena  Ongoing Egg mass o Searching behavior Oviposition behavior  Finished o o Host choice o 1 h Olfactometer Studies o … o 4 h (FL, MI) - ongoing o 6 h o 24 h

www.ars-fla.com Ecological Host Range of T. japonicus in Asia Recovery of Trissolcus japonicus in Maryland in 2014

Photo: E. Talamas ARS/SEL a potential game changer?

Origin of the adventive T. japonicus populations

Principal coordinates analysis of genetic diversity among 23 microsatellite markers in T. japonicus Take home message: Japan DC area recoveries 1) These populations are South Korea China adventive – they were not released nor did they escape quarantine! Coord. 2 Coord. 2) DC area populations genetically similar to Beijing populations sampled in Japan and S. Korea Vancouver WA recovery Coord. 1 3) WA population genetically similar to populations Genetic distance between populations Axis 1 & axis 2 explain, 22% and 20% respectively, of the distribution sampled in S. Korea Logical next steps – Asian Natural Enemies

Quarantine Host Range Evaluations: • Continue laboratory host range research (pending evidence of establishment and dispersal of adventive populations) towards a Petition to Release (APHIS requires a Petition to Release for each state)

Adventive Trissolcus japonicus: • Expand surveys initiated in 2015 to determine the extent of establishment, incl. an increased focus on wooded habitats and a widened survey area to see how quickly populations spread

• Analyze recovered parasitoid microsatellite DNA to determine heterogeneity of the adventive populations

• Increase monitoring of parasitism of BMSB & non-target pentatomid egg masses in the field Subobjective 2.2.6. Native Natural Enemies Survey area and Collaborators

Delaware – USDA-ARS: K. Hoelmer, K. Tatman & C. Dieckhoff

Maryland – University of Maryland: P. Shrewsbury & A. Jones

North Carolina – NC State University: J. Walgenbach & E. Ogburn

Oregon – USDA-ARS Corvallis: Jana Lee

Pennsylvania – Penn State University: D. Biddinger & N. Joshi

Virginia – Virginia Tech: T. Kuhar, D. Pfeiffer & T. Trope Native Biocontrol of BMSB In PA D. Biddinger & N. Joshi Penn State University Entomology BMSB Adult Parasitism by pennipes (Diptera: )

Date No BMSB No. No. % No. Eggs Av. # Eggs/ Collected & Locations Parasitized Parasitized Found Host Examined (range by (range of location) eggs/host) 2012 4,595 7 115 2.44 157 1.37 (0-10%) (up to 9)

2013 3,087 8 57 1.81 75 1.32 (0-7%) (up to 5)

Date % Males % % of % Eggs % Eggs % Eggs % Eggs on % Eggs Females Eggs on on on Head on Abdomen on Dorsum Venter Thorax Legs

2012 59.62 40.38 39.49 61.15 1.91 56.69 39.49 0.64

2013 49.12 50.88 36.00 64.00 1.33 65.33 33.33 0.00

Biddinger 2014 Objective: Survey and Identify native natural enemies of BMSB A. Colavecchio

Naturally laid (wild) BMSB egg masses – DE, MD, NC, VA

Sentinel BMSB egg mass placement (lab-reared) - DE, NC Habitats surveyed

Egg masses on plant hosts in non- managed habitats (wild and sentinel) – DE, NC & VA

By Arthur Haines. Copyright © 2014 Naturally laid egg masses in 3 MD nurseries - MD only Tree genera – Maple (Acer), Cherry (Prunus), Other species and Elm (Ulmus) http://www.treesandhedging.co.uk/ fruits-berries-wildlife/f353 http://www.utopiasprings.com/ptree.htm Tree of heaven http://www.carolinan ature.com/trees/pato.h tml – Ailanthus sp. Paulownia

Photo by Steve Black, Raemelton Farm Photo by Ruppert Nurseries D e l Fate of sentinel and naturally laid BMSB eggs a w 12724 9538 17207 9780 14260 7599 1847 2512 2950 9784 N 756 849 2462 16928 5118 2014 1168 1395 567 N a 100% 100% r 90% 90% e 80% 80%

70% 70% parasitized 60% 60% Predation - Chew

50% Predation - Suck 50%

Egg Fate Nymph - unhatched 40% Nymph - hatched 40% missing 30% 30%

20% 20%

10% 10%

0% 0% 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2007 2008 2009 2010 2011 2012 2013 2014 2015 A . C Sentinel BMSB eggs Naturally laid BMSB eggs DD ee ll Fate of naturally laid non-BMSB eggs aa ww 210 389 876 1238 855 510 881 761 655 N aa 100% Brian T. Cutting rr ee 90% 80% parasitized 70% Predation - Chew 60% Predation - Suck 50% Nymph - unhatched Egg Fate 40% Nymph - hatched 30%

20%

10%

0% 2007 2008 2009 2010 2011 2012 2013 2014 2015 DD ee ll Species composition of parasitized sentinel BMSB eggs aa *2015 (N=870) ww aa 2014 (N=38) rr 2013 (N=30) ee 2012 (N=75) 2011 (N=143) 2010 (N=173) 2009 (N=36) 2008 (N=484) 2007 (N=128) 2006 (N=39)

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Anastatus spp. - male Anastatus mirabilis Anastatus pearsalli Anastatus reduvii Trissolcus brochymenae Trissolcus edessae Trissolcus euschisti Telenomus podisi Ooencyrtus spp. Unknown (undeveloped, missing ID) DD ee ll Species composition parasitized naturally laid BMSB eggs aa 2015 (N=108) ww aa 2014 (N=148) rr 2013 (N=210) ee 2012 (N=263) 2011 (N=196) 2010 (N=275) 2009 (N=69) 2008 (N=51) 2007 (N=16) 2006

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Anastatus spp. - male Anastatus mirabilis Anastatus pearsalli Anastatus reduvii Trissolcus brochymenae Trissolcus edessae Trissolcus euschisti Telenomus podisi Ooencyrtus spp. Unknown (undeveloped, missing ID) V A Parasitism of sentinel and naturally laid eggs in NC sites, T e 2013-2014 c A h Parasitism rates of sentinel and naturally laid eggs recovered in 2013 and 2014 ranged from 0.26% to 27.54%

In 2013, 150 naturally laid eggs were found with 57 (27.54%) successfully emerged parasitoids.

Also in that season, 749 sentinel eggs were placed in the wild for 5 days, none of which were parasitized.

In 2014, 772 naturally laid eggs were found with 55 (6.65%) successfully emerged parasitoids.

And 7545 sentinel eggs were placed in the wild for 5 days and 20 (0.26%) were parasitized, and 17 that were dissected for identification. Fate of Sentinel and Naturally Laid BMSB Eggs on Trees in Non-Managed NC Habitats – 2013-2014

100 100

Unemerged 80 parasitoid 80 Parasitoid emerged Missing 60 60 Sucked

40 Chewed 40

Percentage of total eggs oftotal Percentage Unhatched 20 20 Hatched

0 0 sentinel natural sentinel natural Cherry Catalpa Paulownia TOH Walnut Yellowwood 2013 2014 2014 only V A Species composition of parasitized sentinel and naturally laid BMSB eggs in NC sites T e c % Parasitoids on sentinel BMSB eggs in 2014 h % Parasitoids on naturally laid BMSB eggs in 2013-2014

Trissolcus brochymenae V A Parasitism of naturally laid eggs in VA sites, 2011 - 2015 T e c Parasitism rates of naturally laid eggs recovered between 2011 and 2015 h ranged from 1.97% to 27.57%

 In 2011, 2896 BMSB eggs were found with 106 (3.53%) successfully emerged parasitoids.  In 2012, 4687 eggs were found with 94 (1.97%) successfully emerged parasitoids.  In 2013, 2120 eggs were found with 166 (7.26%) parasitized and successful emergence of 87 parasitoids.  In 2014, 2590 eggs were found with 81 (3.03%) parasitized and successful emergence of 46 parasitoids and 35 that were undeveloped and could not be identified.  And in 2015, 394 eggs were found with 150 (27.57%) parasitized, successful emergence of 120 parasitoids and 30 that were either dissected for identification or undeveloped. V A Species composition of parasitized naturally laid BMSB eggs in Virginia

T from 2011-2015 e A c h M a Biological Control of naturally laid BMSB r y egg masses in MD Nurseries l a n • Sampled naturally laid eggs at 3 MD nurseries d • late May through September in 2012 and 2013 • Tree genera – Maple (Acer), Cherry (Prunus), and Elm (Ulmus) • Eggs returned to lab and monitored

Photo by Steve Black, Raemelton Farm Photo by Ruppert Nurseries M a r y BMSB Egg Mortality Factors l Total Mortality 58% a ~ 50 * n 43.8 d 45 2012 40 35 31.8 2013 30 25 15.4 20 * 15 7.8 7.7 10 4.8

Percent EggPercent Mortality 5 2.2 2.3 0 Parasitism Chewing Sucking Unascribed Mortality Factor M a Parasitoid species composition in Maryland, 2012-2013 r -Trissolcus y brochymenae ll 2012 1.1% 2013 a -Trissolcus n euschisti d 0.4% Anastatus Anastatus -Trissolcus males males 0.2% 16.8% Anastatusmales males Anastatus reduvii 32.1%32.1% Anastatus Anastatus Anastatus -Telenomus 61.2% reduvii mirabilis reduvii podisi 0.04% 79.1% 0.8% 61.2% -Telenomus males Anastatus 0.04% pearsalli 3.9% -Ooencyrtus sp. 0.3% 5,638 parasitoids 9,719 parasitoids 24,124 eggs 32,073 eggs 1 : 2.05 M:F ratio 1 : 4.86 M:F ratio Summary

• Parasitism of sentinel and naturally laid eggs low throughout across years and locations (2006/2007 through 2015) • Survey of parasitism of non-BMSB naturally laid eggs suggests that native egg parasitoid species are present and active in the same habitats as BMSB • Failure to adapt? • Failure to recognize BMSB as a host? • BMSB defense mechanism to parasitism by native egg parasitoids? • Species composition of sentinel and naturally laid eggs: • dominated by Anastatus spp. (Eupelmidae) • highly variable between years • proportion of undeveloped eggs highly variable between year, partly due to state of egg mass at deployment (frozen vs. fresh) Summary – Maryland survey

• Egg mortality from all sources was approximately 58% • Mortality increased throughout the season

• Parasitism is the greatest cause of egg mortality (range 7-80% parasitism) • Female-biased sex ratio

• Anastatus reduvii was the most abundant parasitoid • BUT: Generalist across orders Logical next steps

• Continue sentinel surveys • Examine any adaptation by native natural enemies to BMSB

• Interaction between T. japonicus and local natural enemy communities and their impact on BMSB dynamics

• Long-term monitoring for successful native parasitoid populations (various locations) • laboratory selection • for augmentative biocontrol

• Examine what habitat factors influence various natural enemies • identification and conservation of those habitats/crops • Compare natural enemy complexes and impacts between different habitats Acknowledgements

• Funding source: USDA-NIFA SCRI Award # 2011-51181- 30937

• Brian Cutting, UD • Amanda Stout, UD • Hoelmer lab undergraduate students • Elijah Talamas, USDA-SEL • Matthew Buffington, USDA-SEL • VA Tech & NCSU undergraduate students assisting in the lab and field • Cerruti Hooks, UMD • Mike Raupp and lab, UMD • Galen Dively and lab, UMD • Shrewsbury lab, UMD • Raemelton Farm, Adamstown, MD • Ruppert Nursery, Laytonsville, MD • Many stakeholder cooperators, students, post-docs D e l Fate of sentinel BMSB eggs a w 12724 9538 17207 9780 14260 7599 1847 2512 2950 9784 N a 100% r e 90% 80% parasitized

70% Predation - Chew

60% Predation - Suck

50% Nymph - unhatched Nymph - hatched Egg Fate 40% missing 30%

20%

10%

0% 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015* DD

ee A. ll Fate of naturally laid BMSB eggs aa ww 756 849 2462 16928 5118 2014 1168 1395 567 N aa 100% rr ee 90% 80% parasitized 70% Predation - Chew 60%

50% Predation - Suck

Egg Fate 40% Nymph - unhatched

30% Nymph - hatched 20%

10%

0% 2007 2008 2009 2010 2011 2012 2013 2014 2015 Fate of Naturally Laid Eggs on Trees in Non-Managed NC Habitats 2014

100 Unemerged parasitoid 80 Parasitoid emerged Missing 60 Sucked

40 Chewed Percentage of total eggs oftotal Percentage Unhatched 20

Hatched 0 Catalpa Cherry Paulownia Tree of Walnut Yellowwood Heaven M a Parasitoid species composition in Maryland, 2012 r y l 2012 -Trissolcus a 2012: 5,638 brochymenae n parasitoids 1.1% d -Trissolcus euschisti 0.4% Anastatus males -Trissolcus males 32.1% 0.2% Anastatus reduvii Anastatus mirabilis 61.2% -Telenomus podisi 0.8% 0.04%

Anastatus pearsalli -Telenomus males 3.9% 0.04% -Ooencyrtus sp. Anastatus total: 98% 0.3% O r Parasitism and predation of eggs in landscape ornamentals e (Jana Lee) g o n

• 5 sites per host, with/out mesh cages at each site • 1 wk exposure • Monthly June – Aug/Sep • Frozen (!) sentinel eggs

Lee, Cave-USDA Corvallis Does Methyl salicylate enhance egg biocontrol through predation? (Jana Lee)

• Methyl salicylate (MeSA) is a plant volatile that attracts natural enemies • Sentinel egg masses next to MeSA had 4.8 X higher predation in Aug • Green lacewing adults and lady beetles were caught more in MeSA sites • Lacewing larva observed Commercial lure feeding on sentinel egg mass

Skillman, Lee -USDA Corvallis