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Plant Health

Work Package 5: Control

Task 5.3: OPM Control

Review of the impacts of Processionary (Thaumetopoea processionea) control methods on oak tree biodiversity

Rachel Down and Neil Audsley

31st March 2018

Table of contents

Abstract ……………………………………………………………………………………………………………………………………. 3

Chapter 1. Introduction ……………………………………………………………………………………………………………. 4

Chapter 2. Relative toxicities of Bacillus thuringiensis var. kurstaki, diflubenzuron and deltamethrin to and other invertebrates …………………………………………….. 7

Chapter 3. Invertebrate assemblages associated with oak trees ……………………………………………… 32

Chapter 4. Evaluation of methods for monitoring oak tree invertebrate biodiversity ……………… 66

Chapter 5. A review of similar studies: Short and long-term impacts of tree insecticides on invertebrate biodiversity and the wider environment ……………………………………… 72

Conclusions ………………………………………………………………………………………………………………………….…. 91

Recommendations ……………………………………………………………………………………………………………….…. 93

References ……………………………………………………………………………………………………………………………… 95

Appendix 1. List of Lepidoptera whose larvae feed on oak at some point between April and June ……………………………………………………………………………………………………………………………….. 106

Appendix 2. IUCN Red Data Book (RDB) categories and criteria ………………………………………….. 109

2 Impact of OPM control methods on oak tree biodiversity | March 2018 Abstract

Oak processionary moth Thaumetopoea processionea L. (Lepidoptera: Thaumetopoeidae) is an alien invasive that was first introduced to the UK circa 2005, and which poses a threat to native oak trees (through defoliation) and human health due to the tiny urticating hairs on the larval exoskeletons, which when shed can cause allergic reactions including skin rashes, eye and throat irritations, and breathing difficulties in susceptible individuals. As such there is a requirement that populations of this invasive pest are controlled.

At present there are several control methods approved for use in the UK including three insecticide sprays: the bacterial biopesticide Bacillus thuringiensis var. kurstaki (DiPel® DF), the growth- regulator diflubenzuron (Dimilin® Flo), and the synthetic pyrethroid deltamethrin (Bandu®), as well as physical nest removal.

There are two species of oak native to the British Isles, the pedunculate (common) oak L. and the sessile oak Q. petraea (Matt.) Liebl., and both support a vast array of species, providing an important food source for many species, and habitat for others. To date, over 2000 species of mosses, lichens, fungi, invertebrates, birds and mammals are known to be associated with oak, with over 800 invertebrate species associated with Q. petraea and over 1000 species of invertebrates associated with Q. robur. In particular, the invertebrate species could be directly at risk of non-target impacts from pesticides that may be applied to oak to control a pest species, therefore it is important to investigate and assess these potential non-target impacts. It is likely that there are in the region of 150-200 species of native Lepidoptera (potentially more) that feed on oak, and a significant proportion of these are present as larvae, feeding within the oak tree canopy, at the same time as oak processionary moth larvae, and therefore at risk of non-target impacts.

This review uses currently available literature to gather evidence on the potential impacts of the three insecticides currently approved to control oak processionary moth on invertebrate biodiversity within a tree environment, and to a lesser extent, implications for the wider environment by providing a detailed review on the mode of action, toxicology, specificity, mobility and persistence for each of the active ingredients. The review also seeks to collate detailed life-cycle and habitat information for the lepidopteran species associated with oak to establish which species are likely to be directly impacted upon by any insecticidal treatment.

3 Impact of OPM control methods on oak tree biodiversity | March 2018 Chapter 1. Introduction

The oak processionary moth Thaumetopoea processionea L. (Lepidoptera: Thaumetopoeidae) is a species that is native to central and southern and was first introduced into the London area of the UK as an invasive species circa 2005, almost certainly as eggs present on plants imported from continental Europe (CABI, 2018; Forestry Commission, 2018a). It has since spread to neighbouring counties (Surrey, Essex, Hertfordshire, Buckinghamshire, and west Berkshire) (Forestry Commission, 2018a).

The larvae of oak processionary moth feed on the foliage of oak and cause significant defoliation in Europe; this can leave a tree weakened and therefore vulnerable to other threats (Forest Research Advisory Note; Forestry Commission, 2018a). As such this invasive species poses a threat to the UK native oak species, the pedunculate (common) oak (Quercus robur L.) and the sessile oak ( (Matt.) Liebl.) as well as the introduced oak (Quercus cerris L.) (Forestry Commission 2018b). Oak processionary moth is most often found on urban trees, along forest edges and in amenity woodlands (Forestry Commission 2018b). It is reported that other species of tree such as hornbeam, , sweet chestnut, and can also be attacked by oak processionary moth, most notably when they are adjacent to severly defoliated oak trees (Forestry Commission 2018b). Oak processionary moth also poses a threat to human and health because the larvae produce tiny urticating hairs, which when shed can cause allergeric reactions, evident as skin rashes, eye irritations (such as conjunctivitis), sore throats (pharyngitis) and breathing difficulties (including asthma) in susceptible people that come into contact with them (Forestry Commission, 2018a,b). Contact with these tiny hairs is through direct contact with the caterpillars and with the nests, which contain huge numbers of shed hairs, but also occurs because these tiny hairs break off very easily and get dispersed in air currents (Forestry Commission, 2018a,b).

1.1 Biology

Eggs are laid in single layer rows, forming a plaque which is then covered with greyish scales, on twigs and small branches within the canopy from July to early September (Forestry Commission 2018b). Larvae hatch from the eggs the following April (sometimes earlier) and feed gregariously on the foliage from April to June, moving down the tree as they get larger (Forestry Commission, 2018a,b). When they are not feeding they rest in white, silken webbed communal nests found on the trunks and branches (only very rarely among the leaves), anywhere from ground level to high up in the tree (Forestry Commission, 2018a,b). Nests can vary enormously in size, from the width of a 50p coin to several feet (Forestry Commission, 2018a). When travelling between nest and feeding site, the lavae follow one another forming processions. Larvae typically complete their development, passing through six larval instars, in 9-12 weeks; they pupate within the nests and adult emerge one to two weeks later, sometimes up to four weeks later (Forestry Commission, 2018a,b).

4 Impact of OPM control methods on oak tree biodiversity | March 2018 1.2 Control

As an invasive pest species, and because of the threat it poses to both oak and human health, oak processionary moth is under surveillance, monitoring and control in the UK. There are currently three management zones for oak processionary moth: the Core Zone, the central part of the London outbreak area; the Control Zone, the buffer area; and the Protected Zone, which is free of the pest and where incursions must be eradicated by law (Forestry Commision, 2017). The government-funded oak processionary moth control programme (OPMCP) has been running since 2013; this currently enforces and/or carries out management of oak processionary moth in the Control and Protected Zones; management in the Core Zone is the responsibility of individual landowners (Forestry Commission, 2017). There are several possible methods for controlling oak processionary moth in the UK including nest removal and three currently approved insecticides sprays: the insect growth- regulator diflubenzuron (Dimilin® Flo) (MAPP 11056), the broad-spectrum synthetic pyrethroid deltamethrin (Bandu®) (MAPP 16153) and the bacterial biopesticide Bacillus thuringiensis var. kurstaki (DiPel® DF) (MAPP 17499). Bacillus thuringiensis var. kurstaki (Btk) is currently the preferred option as it is considered the most selective of the three insecticides (ADAS UK Ltd, 2015) and is the method normally used in the OPMCP, although sometimes Dimilin® Flo will be used (OPM Management, 2017). Both B. thuringiensis var. kurstaki and diflubenzuron are most effective against very young larvae, so timing of application is crucial (Forestry Commission, 2017). Whilst there were no plans to use deltamethrin within the OPMCP in the 2017-2018 season, its use may be considered in the Protected Zone as part of a robust response package (OPM Management, 2017) or by private landowners in the Core Zone; it may be the preferred insecticide against older larvae in circumstances when use of an insecticide is considered better than nest removal (Forestry Commission, 2017). Oak processionary moth management guidelines were provided within the TH0102 Project Final Report (ADAS UK Ltd, 2015). These include:

1. Spray programmes should begin as soon as egg hatch starts to occur; egg hatch can occur before oak leaf emergence, so spraying should start when at least some of the leaves have broken from bud.

2. Spray applications should be completed within 28 days.

3. Label recommendations for all three of the insecticides should be carefully followed.

4. Spray applicators should be calibrated in order to apply the correct rate.

5. Mist blowers provide better control than compression sprayers in terms of achieving a lower percentage of trees with nests and lower numbers of nests per tree.

6. Nest removal may be the only viable control option in some situations e.g. at some Sites of Special Scientific Interest.

Nest removal is also an option for control when larvae are too large for spraying with B. thuringiensis var. kurstaki or diflubenzuron to be effective (OPM Management, 2017).

5 Impact of OPM control methods on oak tree biodiversity | March 2018 In addition to the currently approved control methods, novel control methods are under investigation. For instance, the use of insect pathogenic nematodes, used in the Netherlands in some circumstances to control oak processionary moth, have given promising results in laboratory studies (ADAS UK Ltd, 2015).

1.3 Aims of this review

This literature review aims to address the following four questions to inform a costed research project design:

1. What are the relative toxicities of Bacillus thuringiensis var. kurstaki, diflubenzuron, and deltamethrin to Lepidoptera and other invertebrates?

2. Which invertebrate species are commonly associated with oak trees in urban parkland or similar habitats in southern England?

3. To evaluate the existing methods for monitoring oak tree invertebrate biodiversity.

4. How much can be inferred about the impact of OPM control methods on oak tree biodiversity from previous studies?

6 Impact of OPM control methods on oak tree biodiversity | March 2018 Chapter 2. Relative toxicities of Bacillus thuringiensis var. kurstaki, diflubenzuron and deltamethrin to Lepidoptera and other invertebrates

The aim of this chapter is to present an in-depth analysis of B. thuringiensis var. kurstaki, diflubenzuron and deltamethrin, specifically to compare:

 Toxicity – mode of action; chemical safety tests in terrestrial and aquatic environments;  Specificity – target and non-target organisms that are affected;  Solubility/mobility in the environment;  Stability/persistence in the environment;  Standard usage in the UK.

2.1 Bacillus thuringiensis Berliner subsp. kurstaki (formulated product DiPel® DF MAPP 17499)

2.1.1 Toxicity: Mode of action

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that occurs naturally in many species of , and within soil and leaf microbiota, across the world (Martin and Travers 1989; Reardon et al., 1994; Glare and O’Callaghan, 2000). Many subspecies (varieties) exist with different specificities towards different Orders of insect (Gill et al., 1992). This review focuses on the subspecies kurstaki (Btk), which has broad activity towards Lepidoptera (Krieg and Langenbruch, 1981; Glare and O’Callaghan, 2000), and is the active ingredient in the formulated product DiPel® DF. In addition to producing infective spores, B. thuringiensis also produces a protein crystal inclusion adjacent to the spore at the time of sporulation. The crystal inclusion contains inactive protoxins that are not toxic in themselves however, once they are ingested by an insect the protoxins are dissolved and digested in the insect gut, releasing smaller delta-endotoxin proteins (δ-endotoxins; insecticidal crystal proteins; ICPs), which are toxic (Reardon et al., 1994). Numerous δ-endotoxins exist, their primary structures varying according to the gene that encodes the protein. Toxins encoded by the cryI gene are toxic to Lepidoptera, and toxins encoded by the cryII gene are toxic to Lepidoptera and Diptera (Höfte and Whiteley, 1989; Gill et al., 1992; van Frankenhuyzen 2009). Specifically, the crystal inclusion of the B. thuringiensis var. kurstaki strain that is used in DiPel® DF, ABTS-351 (HD-1), contains three Cry1 (Cry1Aa, Cry1Ab, Cry1Ac) and two Cry2 (Cry2Aa and Cry2Ab) protoxins (Gill et al., 1992; Glare and O’Callaghan, 2000; Valent BioSciences, 2007; Konecka et al., 2014). Mommaerts et al. (2010) state that Btk (DiPel®) also contains Cry1Ia but this is the only reference to indicate this. The Valent BioSciences brochure indicates that the relative levels of the toxins within DiPel® are 15.3% Cry1Aa, 39.4% Cry1Ab, 23.1% Cry1Ac and 22.1% mixture of Cry2A/2Ab.

Whether or not an insect is susceptible to these δ-endotoxins is dependent on its ability to dissolve and digest the crystals within the gut environment (Aronson et al., 1991; Du et al., 1994; Reardon et al., 1994; Monnerat et al., 1999; van Frankenhuyzen, 2009). An alkaline midgut (pH > 8.0) and serine protease activity are required to dissolve and digest B. thuringiensis var. kurstaki crystal proteins (Dent, 1993; Lacey et al., 2015). Life stage and larval age also play an important part (Gilliland et al., 2002; van Frankenhuyzen 2009). Once released, the δ-endotoxin proteins bind to specific receptors on the membranes of the cells lining the midgut, penetrate through the cell membrane and form ion-

7 Impact of OPM control methods on oak tree biodiversity | March 2018 selective channels (Dent, 1993; Reardon et al., 1994). This disrupts the permeability of the cell membrane such that the cell absorbs water, swells and bursts (Gill et al., 1992; Bravo et al., 2007; Renzi et al., 2016). Ultimately, the gut becomes perforated, gut contents (including infective spores) leak into the haemolymph and the insect dies usually within 24-72 hours (in Mnif and Ghribi, 2015; DiPel® DF Product Label, 2016) through gut paralysis and cessation of feeding (Reardon et al., 1994). In less susceptible species death occurs as a result of septicaemia due to spore germination and bacteria multiplication (Reardon et al., 1994). Once the gut becomes perforated the insect becomes susceptible to non-specific infections by bacterial opportunists (Reardon et al., 1994). Despite the ability of B. thuringiensis var. kurstaki spores to germinate, multiply and re-sporulate once reaching the insect haemolymph, B. thuringiensis var. kurstaki does not naturally cause epizootics because when infected larvae die, their integument does not rupture (Reardon et al., 1994). Instead, the body usually drops to the ground, releasing spores and crystals into the soil as it decomposes rather than onto leaf surfaces that could subsequently be eaten by other susceptible individuals (Reardon et al., 1994). In some instances, larvae can recover from sublethal doses of B. thuringiensis through regeneration of the damaged cells in the midgut (laboratory study with spruce budworm; Fast and Regniere, 1984); on these occasions the larvae will stop feeding temporarily and development is slowed (Reardon et al., 1994).

Bacillus thuringiensis strains are classified by the industry ‘Insecticide Resistance Action Committee’ (IRAC) as Group 11 (microbial disruptors of insect midgut membranes).

2.1.2 Toxicity: Chemical safety tests

Commercial formulations of B. thuringiensis var. kurstaki contain both the spore and crystal (Reardon et al., 1994). Bacillus thuringiensis subsp. kurstaki strain ABTS-351 serotype 3a3b (strain HD-1; DiPel® ES Registration Report, 2014) is formulated as DiPel® DF (dry flowable) in the UK (MAPP 17499; DiPel® DF Safety Data Sheet, 2015) and as DiPel® ES (emulsifiable suspension) in most European countries (BPDB, 2018; DiPel® ES Registration report, 2014). As part of any registration process, chemical safety tests must be performed on a range of fauna. Acute toxicity data (the concentration (LC50) or dose (LD50) of the substance required (usually per body weight) to kill 50% of the test population) provided on the DiPel® DF Safety Data Sheet (2015) are tabulated in Table 1 and show no acute toxicity towards mammalian species in laboratory tests, as also confirmed by the Regulation (EU) No 528/2012 Assessment report for Bacillus thuringiensis subsp. kurstaki strain ABTS-351 serotype 3a3b (2016). Reardon et al. (1994) report no abnormal reactions in a variety of mammals when B. thuringiensis was injected, fed, inhaled or scratched onto.

Ecological toxicity details are also provided for a range of organisms in the DiPel® DF Safety Data Sheet (2015) and are tabulated in Table 2. No toxicity is observed towards birds. Moderate toxicity is recorded towards fish and aquatic invertebrates because 20% mortality was recorded for rainbow trout, and 100% mortality recorded for the invertebrate water flea Daphnia magna, although it remains unclear whether mortality was attributable to toxicity or high turbidity of suspended solids in the test media (Regulation (EU) No 528/2012 Assessment report, 2016). No adverse effects were observed on aquatic crustacea (copepods and shrimps; Regulation (EU) No 528/2012 Assessment report, 2016).

8 Impact of OPM control methods on oak tree biodiversity | March 2018 There has been some debate in the literature as to whether B. thuringiensis var. kurstaki is toxic to earthworms. In 1975, Benz and Altwegg (in Glare and O’Callaghan, 2000) demonstrated that the application of B. thuringiensis var. kurstaki over forests at a recommended field rate of 60 mg/m2 and 10x and 100x above this, and of Bactospeine® (Btk strain ABTS-351) applied at 30 mg/m2, did not result in a reduction in the number of Lumbricus terrestris (common earthworm). Smirnoff and Heimpel (1961) however, had previously reported very high doses (104-105 higher than normal levels) of Thuricide® (B. thuringiensis var. kurstaki) were fatal to L. terrestris; it has subsequently been suggested that this may have been due to high levels of β-exotoxins found in early products. More recently, laboratory work by Addison and Holmes (1996) showed that unformulated B. thuringiensis var. kurstaki, and aqueous DiPel® 8AF had no effect on the forest earthworm (Dendrobaena octaedra) at 1000x the expected environmental concentration (EEC). In contrast, a 1000x EEC of oil formulated DiPel® 8L (but not 100x EEC or lower) did reduce survival and have other adverse effects, but this was attributed to the oil rather than the product (Addison and Holmes, 1996). Glare and O’Callaghan (2000) considered B. thuringiensis to be safe for earthworms at field application rates, the exception being if excess exotoxin is present in the product. Bacillus thuringiensis var. kurstaki ABTS-351 (DiPel® DF) is not considered to be acutely toxic to earthworms in the Regulation (EU) No 528/2012 Assessment report (2016), which states an LC50 value > 1000 ppm/kg dry weight of soil was obtained in a 30 day long acute toxicity study with Eisenia fetida; this is in agreement with information provided in Table 2.

With regard to honey bees (Apis mellifera), Malone et al. (1999) tested two formulated B. thuringiensis var. kurstaki products (DiPel® 2X and Foray® 48B) on newly-emerged adult honey bees. When administered at a realistic dose (0.25% w/w in pollen) no adverse effects were observed however, reduced food consumption and reduced survival were observed when a very high dose (1% w/w) was tested. The authors therefore concluded that adult bees were unlikely to be harmed by the use of Btk- based biopesticides. Similarly, no adverse effects on the survival and foraging behaviour or reproductive effects were observed on bumblebee (Bombus terrestris) workers by Mommaerts et al. (2010) when they tested B. thuringiensis var. kurstaki (DiPel®) both dermally and orally, at a field recommended rate of 0.1%. Laboratory studies have indicated that B. thuringiensis var. kurstaki spores do not induce mortality in honey bees, but they do appear to differentially modulate some enzyme activities in honey bee, resulting in physiological modifications (Renzi et al., 2016). Neither do the Cry toxins produced by B. thuringiensis var. kurstaki (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab and Cry2Ac) affect survival of chronically exposed honey bees (Renzi et al., 2016), but Cry1Ab can reduce foraging activity in controlled flight-room conditions both during and after exposure (Ramirez-Romero et al., 2005). This response was not linked to repellency, and the bees still exhibited olfactory discriminating abilities, thus the authors suggested that the reduction in foraging might have been linked to affects on communication or learning abilities of individual bees. Subsequent work by these authors (Ramirez-Romero et al., 2008a) confirmed disturbed learning performances and demonstrated that honey bees took longer to feed on food containing the Cry1Ab toxin. Malone and Pham-Delègue (2001) reviewed the toxicity of some purified B. thuringiensis crystal toxins and Bt- transgene products on honey bees and bumblebees and concluded that Bt-transgene products were likely to be safe for both. Glare and O’Callaghan (2000) also concluded that Bt was safe for bees. The Regulation (EU) No 528/2012 Assessment report (2016) provides 48 hour LD50 values for acute oral and acute contact toxicity of Foray® 48B of > 542 µg per bee and > 555 µg per bee, respectively in

9 Impact of OPM control methods on oak tree biodiversity | March 2018 honey bees, and that Btk ABTS-351 technical material was not toxic to adult worker honey bees at a range of doses up to 4042 µg per bee over a 14 day oral toxicity study, the conclusion being that there is no adverse effect of B. thuringiensis var. kurstaki on bees. Similarly, DiPel® DF was not found to cause acute oral or contact toxicity in honey bees at all tested doses, with acute oral toxicity of > 222.41 µg per bee and acute contact toxicity of > 185 µg per bee for the formulated product (Table 2; DiPel® DF Safety Data Sheet, 2015).

2.1.3 Specificity

Bacillus thuringiensis var. kurstaki is well documented to have insecticidal activity towards more than 300 species of lepidopteran larvae (pest and non-pest species) both in the laboratory and in a wide range of field environments (Krieg and Langenbruch 1981; Navon 1993; Glare and O’Callaghan, 2000). Hence there is little doubt that non-target native species of Lepidoptera will be affected to some extent when B. thuringiensis var. kurstaki is used, for example, to control a pest species in a forest environment (Wagner et al., 1996; Glare and O’Callaghan, 2000).

A study reviewing the published data on toxicity of the B. thuringiensis toxins revealed that the lepidopteran-active toxins Cry1A and Cry2A were active towards more than 80% of the lepidopteran species tested; interestingly the and appeared to be the most susceptible whilst the had the highest proportion of unresponsive species (van Frankenhuyzen, 2009). This compares with laboratory bioassay data from Peacock et al. (1998) indicating that butterfly species appeared to be more susceptible to formulated B. thuringiensis var. kurstaki than moth species however, only four of the 42 species tested in this study were butterflies; these authors also concluded that the xylenine noctuids were insensitive. Field studies investigating the effects of B. thuringiensis var. kurstaki on non-target Lepidoptera in parkland and forest environments are documented in detail in Chapter 5.

The question remains, does B. thuringiensis var. kurstaki have any non-target effects on other species?

Studies indicate that B. thuringiensis is rarely directly harmful to spiders and beneficial invertebrates such as predators and parasitoids (Glare and O’Callaghan, 2000). DiPel® was rated as harmless to eight out of nine beneficial insects tested, making it the least harmful of 20 insecticides tested on beneficial by an IOBC/WPRS working group in 1983, and therefore recommended for use in integrated control programmes (in Glare and Callaghan, 2000). It has been classified as harmless to the ground beetles Pterostichus melanarius and Poecilus cupreus (Coleoptera), the staphylinid beetle Aleochara bilineata (Coleoptera), the seven-spotted ladybird Coccinella septempunctata (Coleoptera), the lacewing Chrysoperla carnea (Neuroptera), the predatory mite Typhlodromus pyri (Acari), the braconid wasp Aphidius rhopalosiphi (), the chalcid wasp Trichogramma cacoeciae (Hymenoptera), and the ichneumonid wasps Phygadeuon trichops and Coccygomimus turionellae (Hymenoptera) (DiPel® ES Registration report, 2014), all species commonly used in integrated pest management (IPM) schemes. The Regulation (EU) No 528/2012 Assessment report (2016) and the DiPel® DF product profile also indicates that Btk strain ABTS-351 is not likely to pose a risk to predatory mites and parasitoid wasps, and as such could be used in IPM.

10 Impact of OPM control methods on oak tree biodiversity | March 2018 With regard to (Hymenoptera), an early report cited in Porcar et al. (2008) suggested that field application of B. thuringiensis var. kurstaki resulted in mortality of sawfly larvae, but no further reports of sawfly mortality in the field exist, despite large-scale applications of B. thuringiensis var. kurstaki in the field to control forest pests (Porcar et al., 2008; see Chapter 5). Porcar et al. (2008) suggest that further studies, including laboratory assays, should be performed to confirm activity of B. thuringiensis strains against sawfly with a view to finding a strain to control pestiferous sawfly. Some strains do have hymenopteran activity for example, strain PS86Q3 (Porcar et al., 2008) which is listed as producing Cry5Ba1 endotoxin by Crickmore et al. (2016).

Glare and O’Callaghan (2000) report that a literature review by Surgeoner and Farkas (1989) concluded that there were no adverse affects of B. thuringiensis var. kurstaki in aquatic systems; Glare and O’Callaghan themselves report only minimal impacts on these communities in their review date 2000. Laboratory studies testing the effects of B. thuringiensis var. kurstaki on aquatic insects have failed to identify any sensitive species (in Glare and O’Callaghan, 2000). However, a single study within a forest stream in Canada by Kreutzweiser et al. (1994) demonstrated that whilst 10x the EEC of B. thuringiensis var. kurstaki did not significantly change taxonomic diversity it did result in short term alterations in the community structure, with stonefly (Leuctra tenuis) significantly reduced by 70%; Glare and O’Callaghan (2000) report that this may not have been due to B. thuringiensis var. kurstaki but rather some other unknown factor. There is some evidence in the literature to suggest that in laboratory studies a Cry1 toxin and B. thuringiensis var. kurstaki (DiPel® 2X DF) has molluscicidal activity against the fresh-water snail Biomphalaria alexandrina (Osman et al., 2011; Abd El-Ghany and Abd El-Ghany 2017).

Many years prior to the van Frankenhuyzen (2009, 2013) reviews on the toxicity of Bt endotoxins (see below), Glare and O’Callaghan (2000) produced a list of invertebrates that they found to be reported as susceptible in the laboratory and/or a field environment to the DiPel® DF (B. thuringiensis var. kurstaki) formulation produced by Abbott Laboratories. The publications used to compile this list all pre-date the year 2000 and formulations may well have advanced/improved since then; certainly, they no longer contain the exotoxins that were found in early formulations. The list includes cross-Order activity towards:

 four species of Coleoptera - Colorado potato beetle (Leptinotarsa decemlineata), 28-spotted potato ladybird (Henosepilachna (Epilachna) vigintioctopunctata); grain weevil (Sitophilus granarius) and red flour beetle (Tribolium castaneum);  one species of Dictyoptera - German cockroach (Blatella germanica);  six species of Diptera - vegetable (Liriomyza sativae), pea leaf miner (Liriomyza trifolii), okra stem fly (Melanagromyza hibisci), cabbage root fly (Delia radicum), the mosquitoes Culex molestus and Culex quinquefasciatus; note the Cry2 proteins were orginally classed as having both lepidopteran and dipteran activity;  eight species of - silverleaf whitefly (Bemisa tabaci), the aphids A. gossypii, B. brassicae, M. persicae, Indian cotton leafhopper (Amrasca biguttula), Assam black bug (Dimorphopterus gibbus), Lucerne bug (Adelphocoris lineolatus) and long-tailed mealybug (Pseudococcus longispinus);  four species of Hymenoptera - big-headed ant (Pheidole megacephala), sawfly Perga affinis affinis, lugens proxima, Pristiphora abietina and social wasps (Vespula sp.);

11 Impact of OPM control methods on oak tree biodiversity | March 2018  two species of Isoptera - termites Psammotermes hybostoma and Amitermes desertorum.

Cross-Class susceptibility was documented for four Acari species (ticks: Argas persicus, Hyalomma dromedarii, Boophilus annulatus (calcaratus) and Panonychus ulmi); two species of Nematoda (Meloidogyne javanica and Tylenchulus semipenetrans); and five species of Phthiraptera (lice: Eomenacanthus (Menacanthus) stramineus, Menopon gallinae, Bovicola (Damalinia) caprae, Linognathus stenopsis and Heterodoxus spiniger) (Glare and O’Callaghan, 2000).

Published data on the insecticidal activity of B. thuringiensis crystal proteins (bioassay data using spore-free preparations only) is incorporated into the B. thuringiensis toxin Specificity Database (http://www/glfc.cf.nrcan.gc.ca/bacillus) from which activity spectra for each toxin, if tested, can be obtained (van Frankenhuyzen, 2009, 2013). This database, along with data from the Non-target Effects of Bt Crops Database (http://delphi.nceas.ucsb.edu/btcrops; cited in van Frankenhuyzen, 2013), has been used by van Frankenhuyzen (2009, 2013) to compile reviews on the cross-order activity of B. thuringiensis toxins. Unfortunately, at the time of writing this review, links to both these databases appear to be broken so the databases were not accessible. Both the van Frankenhuyzen (2009, 2013) reviews conclude that an increasing number of toxin families have cross-Order activity, that the diversity of biological activity for the B. thuringiensis crystal proteins will be underestimated, and that more cross-Order activity is likely to be demonstrated as new data becomes available; this has certainly been the case between the author’s 2009 and 2013 reviews. Bacillus thuringiensis toxin activity is most commonly measured through mortality; however, it is thought that factors such as feeding inhibition and growth reduction are more sensitive indicators of sublethal effects (in van Frankenhuyzen 2009).

In the van Frankenhuyzen (2009) review, the author emphasises that cross-Order activity is not likely to threaten the environmental safety of Bt-based pest control products because when cross-Order activity occurs it tends to be considerably less toxic (orders of magnitude lower) to taxa outside of the primary specificity range (2009). This is the case for the reported toxicities of Cry1Aa, Cry1Ab and Cry2Aa towards Hemiptera (see following paragraphs). van Frankenhuyzen (2013) states that the lepidopteran toxins are generally active at a few µg/ml, and that they can be considered to have high toxicity when active in the 0.01 - 0.1 µg/ml range, medium toxicity when activity occurs in the 0.1 - 10 µg/ml range and low toxicity when active in the 10 - 1000 µg/ml range. With more data available by 2013 however, van Frankenhuyzen suggested that the current evidence for cross-reactivity should be taken into account when designing and approving pest control applications based on B. thuringiensis proteins. It is also important to understand that the absence of toxicity is impossible to prove because the list of potential target organisms is virtually endless (van Frankenhuyzen, 2013). The author suggests caution when interpreting positive cross-activity data because, whilst quantitative mortality data is provided, the evidence for cross-activity is not robust in the majority of cases (van Frankenhuyzen, 2013). They suggest that the only cross-activity discussed in the sections below that has been validated by independent studies is the toxicity of Cry1Ab towards aphids. Even if laboratory studies indicate a degree of toxicity, these need to be interpreted in terms of biological significance. In the case of Cry1A toxicities towards aphids (see following paragraphs), the laboratory tests indicated that toxicity was of a low level, and examples within the literature show no adverse effects on aphids in the field feeding on transgenic crops expressing the Cry1Ab protein (Ramirez-Romero et al., 2008b) and Cry1Ac protein (Zhang et al., 2008). Published toxicity data can be very difficult to

12 Impact of OPM control methods on oak tree biodiversity | March 2018 interpret because often it is simply not possible to relate the dosage information provided in these studies to field situations, susceptibility of an insect to a high dose in the laboratory does not necessarily translate into an obvious impact in the field, and sometimes the data relates to subspecies that are not used within the field (Addison, 1993; Glare and O’Callaghan, 2000). Whilst the following paragraphs concentrate on some of the Cry proteins known to be produced by B. thuringiensis var. kurstaki, it should be pointed out the van Frankenhuyzen (2013) paper discusses a number of other Cry proteins produced by different strains of B. thuringiensis. It was clear to van Frankenhuyzen after studying the cross-activity data available for as many of these proteins as was available, that whilst most of the reported cross-activities still required confirmation, a few had been validated, and indicated that these proteins are not as Order-specific as was originally believed. Glare and O’Callaghan (2000) argue that laboratory toxicity studies are unlikely to provide any further useful information, rather that ecosystem studies to investigate the sublethal and delayed impacts would fill a knowledge gap.

2.1.3.1 Specificity of the Cry1Ab and Cry1Ac toxins

Cross-Order activity for the Lepidoptera-active Cry1 family of toxins has been reported in laboratory assays, as has lack of cross-Order activity. van Frankenhuyzen (2013) report that the Cry1Ab toxin has been shown to have activity towards the dipteran yellow fever mosquito, Aedes aegypti (citing Haider et al. (1986)), but not the giant crane fly, Tipula abdominalis. However, Haider et al. (1986) actually report on the specificity of B. thuringiensis var. colmeri insecticidal δ-endotoxin, demonstrating structural homology between one of the var. colmeri polypeptides and one of the proteins present within the var. kurstaki P1 crystal (Cry1Aa, Cry1Ab or Cry1Ac). The authors state that whilst there is structural homology, the differences in amino acid sequence are enough to generate differences in the pattern of proteolysis and hence insect specificity between the toxins from the two B. thuringiensis subspecies (Haider et al., 1986).

Cry1Ab toxin also has reported activity (reduced survival) towards the coleopteran ladybird Cheilomenes sexmaculatus (Zig Zag Beetle) when larvae were directly fed on a sucrose solution containing the toxin; however, when larvae were fed on aphids reared on artificial diet containing the toxin, no adverse effects were observed on the ladybird larvae (Dhillon and Sharma 2009). Interestingly, reports of Cry1Ab toxicity towards the two-spot ladybird Adalia bipunctata are at odds with each other. Schmidt et al. (2009) report that solubilised and trypsin-activated Cry1Ab is toxic towards A. bipunctata under artificial laboratory feeding assays whilst other studies (Porcar et al., 2010; Álvarez-Alfageme et al., 2011) report no toxicity; Álvarez-Alfageme et al. (2011) go further and suggest that the apparent detrimental effects reported by Schmidt et al. (2009) were artefacts of poor study design and procedures. No activity towards other coleopteran species (Coleomegilla maculata, Propylea japonica, Oulema melanopus, Stethorus punctillum, Cryptolaemus montrouzieri and Atheta coriaria) is reported (in van Frankenhuyzen, 2013).

Toxicity of Cry1Ab towards hemipteran species has only been reported for the pea aphid Acyrthosiphon pisum albeit only at very high doses (500 µg/ml) and using a trypsin-digested toxin (Porcar et al., 2009), therefore not providing any information on the ability of the aphid to solubilise

13 Impact of OPM control methods on oak tree biodiversity | March 2018 and digest the protoxin crystal within the gut. Other species of aphids (Rhopalosiphum padi, Sitobion avenae feeding on Bt-maize; Ramirez-Romero et al., 2008b) and other Hemiptera (Nilaparvata lugens, Cyrtorhinus lividipennis, Orius majusculus, Orius insidiosus and Orius albidipennis) are reported as not susceptible (van Frankenhuyzen, 2013).

Conflicting literature also exists regarding toxicity of Cry1Ab towards the neuropteran C. carnea. Hilbeck et al. (1998; artificial diet studies using solubilized and trypsinized Cry1Ab protoxin, i.e. activated toxin), Hilbeck et al. (1999; feeding C. carnea larvae with lepidopteran larvae that had fed on Cry1Ab protoxin and toxin) and Dutton et al. (2002; C. carnea fed with aphids, mites and larvae of a lepidopteran species that had all fed on transgenic maize expressing Cry1Ab) all report toxic effects. In the case of the Dutton et al. (2002) study, only the C. carnea larvae that were fed on the (Bt-fed) lepidopteran larvae showed an increase in mortality and delayed development, whereas those that were fed on (Bt-fed) aphids and (Bt-fed) mites did not exhibit any adverse symptoms. In contrast, Pilcher et al. (1997; laboratory and field studies with Cry1Ab-expressing corn), Romeis et al. (2004; laboratory studies using artificial diet containing solubilized and trypsinized Cry1Ab protoxin, i.e. activated toxin) and Rodrigo-Simón et al. (2006; prey-mediated studies) all describe a lack of toxicity. Romeis et al. (2004) suggest that the earlier reported negative effects were due to the quality of the prey used in the experiments rather than a direct toxic effect. Rodrigo-Simón et al. (2006) found no indication that Cry1Ab (or Cry1Ac) was able to bind to the midgut epithelium of C. carnea in histopathological and biochemical studies.

Toxicity of Cry1Ab has been reported for one trichopteran species (Helicopsyche borealis; Rosi- Marshall et al., 2007) whilst Pycnopsyche scabripennis and Lepidostoma spp. were not killed by the toxin (in van Frankenhuyzen, 2013).

No cross-Order activity was observed towards two species of Thysanoptera (van Frankenhuyzen, 2013). Van Frankenhuyzen (2013) also reports a lack of cross-Class activity for Cry1Ab against Collembola (2 species), Amphipoda (1 species), Isopoda (2 species), Cladocera (1 species; D. magna), Acari (3 species), Araneae (two species), and no cross-Phylum activity (one Mollusc species and two species of earthworm Annelida).

Despite reports of cross-activity towards five non-lepidopteran orders of insect van Frankenhuyzen (2013) concludes that the only Cry1Ab cross-Order activity that has been substantiated is that towards the Hemiptera (A. pisum); this author concluded that (up to 2013) none of the other cross-activity reports for Cry1Ab have been unambiguously established.

Fewer instances of cross-Order toxicity of Cry1Ac have been reported. Activity of a protein found to be very similar to the Btk-73 Cry1Ac (one amino acid difference) has been observed towards the dipteran Glossina morsitans (Omolo et al., 1997), but Cry1Ac is reported as not active towards A. aegypti (in van Frankenhuyzen, 2013). The reported toxicity of the lepidopteran toxin Cry1Ac towards dipteran species falls within the toxicity range of dipteran-active toxins (van Frankenhuyzen, 2013).

Activity towards the hemipteran aphid A. pisum has been reported, but again only at a high concentration (500 µg/ml); however, these authors did use the protoxin and did demonstrate processing of the protoxin within the aphid gut (Li et al., 2011). In contrast, Cry1Ac is reported as

14 Impact of OPM control methods on oak tree biodiversity | March 2018 inactive towards the aphid species Myzus persicae, Brevicoryne brassicae and Aphis gossypii, and also towards four other hemipteran species (van Frankenhuyzen, 2013; Paula and Andow, 2016). Cry1Ac activity towards the aphid Macrosiphum euphorbiae is debated: van Frankenhuyzen (2009) hypothesise that the Walter and English (1995) study suggests a mixture of Cry1Aa and Cry1Ab is toxic towards M. euphorbiae rather than Cry1Ac toxin as reported by the authors of the study.

No Cry1Ac cross-Order activity has been reported towards Coleoptera (7 species), Hymenoptera (8 species including honey bee), Neuroptera (2 species including C. carnea), Blattodea (1 species), and no cross-Class activity is reported towards Collembola (2 species) and Acari (3 species) (in van Frankenhuyzen, 2013).

2.1.3.2 Specificity of the Cry2Aa and Cry2Ab toxins

The Cry2 family of toxins, originally classed as having activity towards both Lepidoptera and Diptera, appear to have more limited cross-Order activity than the Cry1 toxins although available data would suggest that the Cry2 toxins have been tested on fewer species. For example, Cry2A and Cry2Aa activity towards the hemipteran M. euphorbiae has been demonstrated but no activity has been recorded against a further three hemipteran species, including another aphid species (reported in van Frankenhuyzen 2009). Cry2Aa has a demonstrated lack of toxicity toward one neuropteran species however, there are conflicting reports regarding activity towards a second species (C. carnea; reported in van Frankenhuyzen, 2013). No cross-Order activity has been reported towards Coleoptera (5 species), Hymenoptera (4 species including the honey bee), Isoptera (1 species), Orthoptera (1 species), Blattodea (1 species), and no cross-Class activity has been reported towards Collembola (2 species) and Isopoda (1 species) (in van Frankenhuyzen, 2013).

No cross activity has been reported for Cry2Ab against Coleoptera, Hemiptera and Neuroptera (1 species each; in van Frankenhuyzen, 2013).

2.1.4 Solubility/mobility in the environment

The Safety Data Sheet for DiPel® DF (2015) states that the product is not classified as hazardous according to Regulation (EC) No. 1272/2008 [CLP], EU directive 67/548/EEC modified by Directive 2001/59/EC (results of experimental studies), Directives 1999/45/EC, 2001/60/EC and 2006/8/EC (based on concentration of active substance and ingredients) and Directive 2003/82/EC for pesticides (special risks and safety precautions). Nonetheless, precautionary statements are provided with regard to the environment (EUH 401) and general provisions state that waterways, drains and sewage systems should not be contaminated with the product or its container (SP 1, Directive 91/414/EEC). Bacillus thuringiensis does have the potential to enter waterways if applied by aerial spraying but would be relatively dilute. Potential drift from aerial spraying (by helicopter in mountainous areas of the USA) is reported to be at least 3 km downwind (Barry et al., 1993; Whaley et al., 1998) and lethal doses, resulting in mortality of non-target lepidopteran larvae, were observed in these drift areas (Whaley et al., 1998). Glare and O’Callaghan (2000) state that B. thuringiensis is rarely detected in

15 Impact of OPM control methods on oak tree biodiversity | March 2018 waterways for more than a few days post-application. The product is not considered to be a marine pollutant.

Solubility in water is low at 10 mg/L at a temperature of 20 °C (BPDB, 2018). The adsorption of the protoxins onto soil particles means that they are unlikely to leach into groundwater (Regulation (EU) No 528/2012 Assessment report, 2016).

No information is stated in the DiPel® DF Safety Data Sheet (2015) regarding mobility in the soil. However, the Regulation (EU) No 528/2012 Assessment report (2016) considers it to be non-mobile because both artificially and naturally irrigated conditions have shown a lack of mobility; a KOC value of 5000 is reported by BPDB (2018) – Koc measures affinity of the pesticide to sorb to organic carbon in soil, the higher the value the stronger the tendency to attach to the soil.

2.1.5 Stability/persistence in the environment

In terms of persistence and degradability, the DiPel® DF Safety Data Sheet (2015) states that B. thuringiensis var. kurstaki is naturally present in the environment. A number of abiotic factors are known to degrade B. thuringiensis var. kurstaki including UV light (causes a rapid loss of activity), with increasing humidity contributing further to this reduction, and high pH values (pH 9) also decreasing insecticidal activity (see below).

A nine-day monitoring period following spray application of B. thuringiensis var. kurstaki strain ABTS- 351 indicates a half-life of just 2.4 days in the atmosphere (Regulation (EU) No 528/2012 Assessment report, 2016).

DiPel® DF is biodegradable and dose not leave any detrimental residue on the crop when applied according to the recommendations for the use of DiPel® DF (Product Label, 2016). Residual toxicity is lost in numerous ways with degradation by sunlight, leaf temperatures, drying, washing off by rain, microbial degradation, leaf characteristics and chemistry, all capable of resulting in loss of activity (Kushner and Harvey, 1962; Pinnock et al., 1975; Leong et al., 1980; van Frankenhuyzen and Nystrom, 1989; Reardon et al., 1994). Wash-off by rain (van Frankenhuyzen and Nystrom, 1989) and UV (solar radiation) are the major sources of degradation (Morris, 1983; Pozsgay et al., 1987; Reardon et al., 1994); Ignoffo et al. (1977) estimated a half life of less than four hours when spores were exposed to an uninterrupted UV source indicative of the UV spectrum in natural sunlight. Formulated product (Foray® 48F) remains toxic on foliage for a short period of time when applied aerially: Sundaram et al. estimated a half-life in the field of 12-22 hours whilst Dubois estimated a half-life of 24-32 hours (in Reardon et al., 1994). Reardon et al. (1994) estimated that a 75 IU/cm2 deposition from a 90 Billion International Units (BIU)/ha application would still retain 50% of its activity after 4-6 days. Johnson et al. (1995) placed potted trees sprayed with B. thuringiensis var. kurstaki in exposed and below-canopy locations and demonstrated that the foliage remained toxic towards a (non-target) lepidopteran species for at least 30 days, with higher rates of mortality observed in the below-canopy locations. In contrast, Konecka et al. (2014) demonstrated that there was some persistence of B. thuringiensis var. kurstaki spores on hornbeam leaves up to six months after application but not after one year. Indeed, Scriber (2004) concludes that the reported persistence of toxic activity in forest ecosystems is highly

16 Impact of OPM control methods on oak tree biodiversity | March 2018 variable ranging from days to months and is likely to be linked to factors such as foliage, including its phytochemical composition, environmental conditions, and the relative susceptibilities of insect species.

Spores of B. thuringiensis var. kurstaki are can persist in soil for months and even years in ideal conditions (Van Cuyk et al., 2011; Konecka et al., 2014) and is thought to be linked to soil pH and microbial competition (West et al., 1985; Konecka et al., 2014). The DiPel® ES Registration report (2014) states a half life for the spores of over 100 days, comparing well with the 100-200 days reported in Regulation (EU) No 528/2012 Assessment report (2016). This is in contrast with the persistence of the endotoxins, which have half lives in soil that are measured in days (2.7-5.2 days) rather than months due to degradation by micro-organisms and adsorption onto soil particles (Glare and O’Callaghan, 2000; Konecka et al., 2014; Regulation (EU) No 528/2012 Assessment report, 2016). There appears to be some debate within the literature as to whether the spores are able to germinate in the soil environment (Regulation (EU) No 528/2012 Assessment report, 2016): some authors report no germination, proliferation or conjugation in soil under natural conditions (in Konecka et al., 2014) whilst others report that the spores can germinate and sporulate (1 million spores/g of soil have been recorded; Saleh et al., 1970; references cited in Reardon et al. (1994) and Konecka et al. (2014)). The product is considered to be non-persistent in the soil, with a typical soil degradation half-life (DT50 value) of 2.7 days (BPDB, 2018).

Although B. thuringiensis var. kurstaki is known to survive to some extent in water, viability is considered to be limited because conditions are far from optimal in aquatic environments, as such proliferation is not likely to occur (Regulation (EU) No 528/2012 Assessment report, 2016). Laboratory studies indicate that the half-life of the spores was highest in fresh water, being 50 days in lake water, whilst the half life of the Cry1Ab endotoxin is reported to be 4.4 days in surface water (Regulation (EU) No 528/2012 Assessment report, 2016).

The active ingredient of DiPel® DF is not considered to bioaccumulate because it is not pathogenic to non-target organisms, and it has not been observed to reproduce in non-target organisms; as such bioaccumulative and toxic (PBT), or very persistent and very bioaccumulative (vPvB) assessments were not required for registration (DiPel® DF Safety Data Sheet, 2015).

2.1.6 Standard usage in the UK

DiPel® DF is supplied as a water dispersal granule at a concentration of 540 g/kg (54% w/w) and a potency of 32,000 IU/mg. The product, which has standard approval for use as an insecticide by professionals, is marketed by Interfarm UK Limited and was first approved for use in the UK on 24/03/2016, with a current authorisation expiry date of 23/03/2020 (‘DiPel® DF’ formulations have been historically available for a number of years in the UK) (CRD Pesticides Register Database, 2018).

The product can be used on a number of crops against a range of lepidopteran larvae. On-label uses include amenity vegetation, aubergine (protected), broad bean (fresh; protected), broccoli/calabrese (outdoor), brussels sprout (outdoor), cabbage (outdoor), combining pea (outdoor), cucumber (protected), dwarf french bean (protected), edible podded pea (outdoor), globe artichoke (outdoor),

17 Impact of OPM control methods on oak tree biodiversity | March 2018 leek (outdoor), ornamental plant production, pepper and chilli (protected), raspberry, runner bean (protected), strawberry, tomato (protected) and vining pea (outdoor). A number of authorisations have been issued under EU 1107/2009 Article 51 ‘Extensions of Minor Use’ (EAMU) and as such, there is no assessment of efficacy for these authorisations. These EAMUs include forestry (oak processionary moth; Authorisation number 20160931), outdoor bulb onion, garlic and shallot (cutworm, noctuid spp; Authorisation number 20162623), outdoor red beet (cutworm, noctuid spp; Authorisation number 20162624), outdoor broad bean, dwarf French bean, runner bean (Silver Y moth, Autographa gamma; Authorisation number 20162625), a large number of outdoor and protected leafy vegetable and herb crops (caterpillars; see Authorisation Numbers 20162626 and 20162627 for full list of crops), protected and outdoor watercress (caterpillars; Authorisation number 20162628), outdoor leek (cutworm, noctuid spp; Authorisation number 20162629), outdoor sweetcorn (Silver Y moth caterpillars, Authorisation number 20162630), a number of root crops (cutworm, noctuid spp; Authorisation number 20162631), a number of protected and outdoor stone fruits, kiwi fruit, wine grapes, chestnut, hazelnut and walnut (Authorisation number 20162632), a number of outdoor or protected soft fruits grown on bushes (Winter Moth, Operophtera brumata; Authorisation number 20162633), protected and outdoor all edible crops (seed crop), protected and outdoor all non-edible crops (seed crop) (Authorisation number 20162634), and outdoor , cherry and (Authorisation number 20162635). The DiPel® DF product profile also indicates that it can be used to control Brown-tail Moth, Euproctis chrysorrhoea, in amenity situations.

Dosage rates are provided on the DiPel® DF Product Label (2016), along with information on appropriate water voluments, application timings, named target species. (These claims and recommendations for use are based on appropriately generated data and assessed to appropriate regulatory standards in accordance with EU (1107/2009). A recommended dose of 0.75 kg/ha is given for amenity vegetation; the maximum number of treatments allowed per year is eight. One to three applications should be made per caterpillar generation at intervals of 7 to 10 days until the end of the hatching period; application should commence as soon as the first larvae are seen, and preferably be done during an active feeding period. DiPel® DF should be applied to dry foliage and should not be applied if rain is expected within 12 hours of treatment; if heavy rain does occur then the application should be repeated. For optimum results, high light intensity conditions and temperatures outside the range of 10-20 °C should also be avoided during application (DiPel® DF Product Label, 2016).

Spray volumes of 1000-1500 litres (shrubs and trees) and 400-1000 litres (field crops, protected crops and amenity vegetation) are recommended, using the higher values when treating large trees and plants, or dense foliage. It is important to ensure that the spray particles are fine and homogeneous, and that the volume used completely covers the vegetation but does not run off (DiPel® DF Product Label, 2016). If applied by aerial spray, ultra-low volume microionisers should be used; if applied from the ground, mounted aerosolizing spray equipment or lances should be used (European Chemicals Agency, 2016). B. thuringiensis var. kurstaki should not be applied to wet foliage.

The DiPel® DF Product Label (2016) dosage recommendations are re-iterated in the following guidelines for using DiPel® DF to control oak processionary moth provided in the TH0102 Project Final Report (ADAS UK Ltd, 2015):

18 Impact of OPM control methods on oak tree biodiversity | March 2018  Use at a concentration of 0.75g/ha, calculated on the area beneath individual trees to be treated or the area of a group of trees to be treated;  A repeat application should be applied 7-10 days after the first application;  Apply to dry leaves and do not apply if rain is expected within six hours of treatment. If heavy rainfall does occur the treatment should be repeated;  Spray particles should be as fine and homogeneous as possible, using sufficient volume of water to ensure complete cover of the tree canopy.

These are in addition to the general guidelines provided in the TH0102 project final report (ADAS UK Ltd, 2015) outlined earlier in Section 1.2.

2.2 Diflubenzuron (formulated product Dimilin® Flo MAPP 11056)

2.2.1 Toxicity: Mode of action

Diflubenzuron ((1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea) is a benzoylphenylurea derivative, which acts as an insect growth regulator by interfering with chitin synthesis at the time of moulting thereby inhibiting the metamorphosis and moulting of juvenile stages of insects and crustaceans (Eisler, 1992; Beck et al., 2004; Maduenho and Martinez, 2008). However, reports suggest that diflubenzuron does not appear to affect other organisms that contain chitin e.g. fungi (Muzzarelli and Marks, 1986). Diflubenzuron acts by interfering in the final step of chitin formation, thus preventing the formation of chitin, a critical component of insect and crustacean exoskeletons (Marx, 1977; Eisler, 1992). In addition, Farlow (in Eisler, 1992) reports that cuticle chitinase (an enzyme that degrades chitin) and cuticle phenoloxidase (an enzyme involved in the tanning and hardening of the cuticle) activities are increased by diflubenzeron, resulting in a soft endocuticle and hardened exocuticle that can neither withstand the increase in pressure during ecdysis nor provide sufficient support during the moulting process. Ultimately, insects treated with diflubenzuron are unable to shed their exuviae (old exoskeleton), and death occurs from starvation or rupture of the new malformed cuticle (cited in Eisler, 1992). Dimilin® Flo, a formulated product containing diflubenzuron as the active ingredient, acts both via ingestion and contact (ADAS UK Ltd, 2015). It should be noted that due to its mode of action it is only effective at controlling immature stages of insects and is therefore most effective against very young lepidopteran larvae (Forestry Commission, 2017). There is some evidence that diflubenzuron may also act as an ovicide (in Butler et al., 1997) and prevent egg hatch (ADAS UK Ltd, 2015).

Diflubenzuron is classified by the industry ‘Insecticide Resistance Action Committee’ (IRAC) as Group 15 (Benzoylureas – inhibitors of chitin biosynthesis typo 0).

2.2.2 Toxicity: Chemical safety tests

Diflubenzuron is formulated as Dimilin® Flo in the UK (MAPP 11056).

Toxicological information indicates that diflubenzuron has relatively low toxicity towards mammals and no observed acute or short-term dietary toxicity towards birds (at the highest tested doses in bobwhite quail, Colinus virginianus) (Dimilin® Flo Safety Data Sheet, 2015; PPDB, 2018). LD50 values

19 Impact of OPM control methods on oak tree biodiversity | March 2018 for rats for both the product and active ingredient are provided and detailed in Table 1 to provide easy comparison [with each other and with DiPel® DF and Bandu®].

Ecological toxicity for the active ingredient (diflubenzuron) but not the product (Dimilin® Flo) itself are detailed in Table 2. The Dimilin® Flo Safety Data Sheet (2015) gives classifications according to Regulation (EC) NO. 1272/2008 [CLP] of H400 (Aquatic Acute 1; M-factor of 100) and H410 (Aquatic Chronic 1; M-factor of 1000), meaning that the product is very toxic to aquatic life with long lasting effects. As such, the product must not be discharged into the environment, or contaminate surface and ground water. The product authorisation for Dimilin® Flo (MAPP 11056) (CRD Pesticides Register Database, 2018) states a Local Environmental Risk Assessment for Pesticides (LERAP) Category of B, meaning the product is subject to 5 m buffer zones when applied with ground crop sprayers or 1 m buffer zones when applied with hand-held sprayers. A LERAP assessment must be applied when intending to reduce the size of the buffer zone used.

In the case of toxicity against fish species, LC50 values after 96 hours of exposure are given as > 0.13 mg/L for fish and > 0.2 mg/L for Cyprinodon sp. (pupfishes), therefore no toxicity was observed at the highest tested concentrations (Dimilin® Flo Safety Data Sheet, 2015; Table 2). An NOEC (no observed effect concentration) of 0.2 mg/L was given for Oncorhynchus mykiss (rainbow trout) after a prolonged 21 day exposure study at a nominal exposure concentration of 0.2 mg/L (Diflubenzuron Assessment Report, 2012; Dimilin® Flo Safety Data Sheet, 2015; Table 2). Previous reports observed no toxicity at up to 50 mg/L (LC50 > 50 mg/L; Fisher and Hall, 1992). 4-chloroaniline, one of the minor metabolites of diflubenzuron, is toxic to fish and has also been classified as mutagenic (Eisler, 1992). Budinsk (in Maduenho and Martinez, 2008) reports that when 4-chloroaniline is degraded via cytochrome P450, reactive oxygen species are created, which in turn can oxidise cell components, damaging the cytoskeleton and cell membrane, and ultimately leading to rupture of e.g. erythrocytes (red blood cells). Growing evidence suggests diflubenzuron can cause health disorders in fish such as haemolysis, hyperglycemic responses and hepatic alterations which could weaken normal liver function, as well as the activation of xenobiotic metabolic pathways and antioxidant defences at concentrations of 25 mg/L (neotropical fish, Prochilodus lineatus; Maduenho and Martinez, 2008).

Ecotoxicological data for other aquatic organisms, honey bees and earthworms were obtained from PPDB (2018) and are included in Table 2; extensive ecotoxicity data is also provided in Eisler (1992). Briefly, diflubenzuron is highly toxic to aquatic insects and also adversely impacts on aquatic arachnoids, molluscs and crustaceans. No toxicitiy was observed towards honey bees and earthworms at the highest tested doses. It is described as harmful to lacewings (C. carnea) but harmless to T. pyri (a predatory mite species); the Dimilin® Flo Product Label (2016) states that Dimilin® Flo has a negligible effect on many beneficial species, including parasitic wasps, predatory mites, midges, hoverflies, lacewings, ladybirds, bugs and spiders, and as such can be used with biological control agents as part of an integrated pest management programme.

2.2.3 Specificity

Diflubenzuron has the ability to disrupt chitin synthesis in all arthropod species: insects, arachnids, crustaceans and myriapods (Eisler, 1992), and as such it is capable of killing some other non-target

20 Impact of OPM control methods on oak tree biodiversity | March 2018 non-lepidopteran insects (ADAS UK Ltd, 2015) as well as non-target lepidopteran species. It is especially effective towards Lepidoptera, Coleoptera and Diptera but other insect species including cockroaches, ants and some beneficial insects are not so susceptible (Eisler, 1992). Marx (1977) hypothesized that this could be due to their feeding habits (such that they do not ingest much of the compound), differences in absorption of the chemical through the gut wall, and the ability to detoxify the compound. In 1983, the IOBC/WRPS-working group for “Pesticides and Beneficial Arthropods” concluded that Dimilin® Flo was harmless to most of the beneficial species tested (up to 13 beneficial arthropods were tested in their study overall) and the working group therefore recommended that it could be used in integrated control programmes (Hassan et al., 1983).

2.2.4 Solubility/mobility in the environment

Solubility of diflubenzuron in water is low (PPDB, 2018), and is reported to vary from 0.08-0.2 mg/L at 20 °C to 1.0 mg/L at 25 °C (Eisler, 1992; PPDB, 2018).

The Dimilin® Flo Safety Data Sheet (2015) does not offer any information on mobility of diflubenzuron in soil however, the Pesticides Properties Database reports that diflubenzuron is non-mobile in the soil (Kfoc = 4620; Kfoc measures affinity to sorb to organic carbon in soil) and has low leachability (PPDB, 2018). Eisler (1992) also reports that mobility and leachability of diflubenzuron in soil is low, such that residues are not usually detected past seven days, and that if the product is used correctly, it is unlikely to leach into ground water.

2.2.5 Stability/persistence in the environment

Eisler (1992) reports that diflubenzuron, when applied to foliage, remains adsorbed for several weeks, and absorption and translocation from the plant surfaces is low; values from a study by Bull and Ivie (1978) suggest < 5% photodegradation or metabolism in four weeks, < 7% absorption in 3-4 weeks and give losses of < 50% by volatisation and weathering in four weeks without rain. Most loss of product from plant surfaces is due to rain, wind abrasion or shedding of senescent leaves (Eisler, 1992). This compares with reports from Wimmer et al. (1993) stating that significant losses of diflubenzuron (ranging from 20% to 80%) occurred from the foliage during the first three weeks following application but further loss until leaf fall was negligible; 65% of their study trees retained more than 20% of diflubenzuron pesticide at leaf fall in October, this moves into the leaf litter layer at leaf fall.

Diflubenzuron seldom exists for extended periods of time in oil or water (Ivie et al., 1980; in Eisler 1992); however, the rate of degradation is dependent on pH. Ivie et al. (1980) suggest that the half life of diflubenzuron at a pH of 10 is less than three days, whilst degradation at pH 4 was undetected even after 56 days. Schaefer and Dupras (1976) also report that diflubenzuron is least stable when water temperature and pH are relatively high. Eisler (1992) similarly reports that diflubenzuron usually only persists in water for a few days with high pH, temperature, sediment and organic content all helping to degrade diflubenzuron more rapidly; more details on persistence in water, in relation to initial dose and other variables, can be found in that report.

21 Impact of OPM control methods on oak tree biodiversity | March 2018 Diflubenzuron is considered to be non-persistent in the soil (Schaefer and Dupras, 1977), a typical DT50 of three days is given (PPDB, 2018). Reports suggest that diflubenzuron is more persistent in post-harvest soils during the winter and spring months, especially if associated with plant material, but that higher temperatures in later months of the year promote a rapid decline (Bull and Ivie, 1978). For more details on persistence in the soil in relation to initial doses and other variables refer to Eisler (1992). A high abundance of soil micro-organisms, along with the application of formulations containing small particles (2-5 µm), helps to degrade diflubenzuron more rapidly (Eisler, 1992). However, diflubenzuron is described as not readily biodegradeable (Dimilin® Flo Safety Data Sheet, 2015) and is considered dangerous for the environment.

Dimilin Flo is not considered to be persistent, bioaccumulative and toxic (PBT), or very persistent and very bioaccumulative (vPvB) at levels of 0.1% and above; the BCF (bioconcentration factor; a measure of the extent of chemical sharing between an organism and its surrounding environment) for fish (bluegill sunfish) is quoted as 320 (Dimilin® Flo Safety Data Sheet, 2015). The PPDB (2018) record this value is at a threshold for concern, according to the general rule of thumb used by the US EPA whereby < 100 has a low potential for bioconcentration, 100-5000 is at a threshold for concern, and > 5000 has a high potential for bioconcentration. The Diflubenzuron Assessment Report (2012) noted that the BCF value of 320 was (1) not fully valid because of experimental short-comings but was considered indicative, and (2) required further assessment because it was > 100 for a non-biodegradable substance; the assessment report concluded that diflubenzuron has a low potential for bioconcentration.

2.2.6 Standard usage in the UK

In the UK Dimilin® Flo is marketed as an insecticide/acaricide and contains the active ingredient diflubenzuron (480g/L), supplied as a suspension concentrate that can be used aerially by professionals. It is marketed by Certis with Arysta LifeScience Registrations Great Britain Ltd being the authorisation holder (CRD Pesticides Register Database, 2018). The product has had provisional approval for use in the UK since 2002, with a current authorisation expiry date of 31/12/2021; it is not approved for use in the aquatic environment. However, Dimilin Flo (MAPP numbers 08769 and 11056) has a withdrawal of use on all edible crops, issued on 7th September 2017 under the Plant Protection Products Regulation (EC) No 1107/2009 (Amendment numbers 1717 of 2017 and 1718 of 2017, respectively; CRD Pesticides Register Database, 2018). Uses to be withdrawn include apple, blackcurrant, broccoli/calabrese, Brussels sprout, cabbage, cauliflower, mushroom, pear and plum. From 31st December 2017 new stock can only be introduced into the supply chain if it complies with Regulation 2017/855 and must have all references to edible crops removed from the label from that date. Disposal, storage and use of existing stocks on edible crops may continue until 8th September 2018. Use may continue on non-edible crops as specified on the product authorisation and label; this includes amenity vegetation, forest, forest (aerial), hedgerow, livestock housing, manure heap, ornamental plant production and refuse tip (Dimilin® Flo Product Label, 2016). Diflubenzuron was approved for use in the EU on 01/01/2009 but approval expires on 31/12/2018 (EU Pesticides database, 2016).

22 Impact of OPM control methods on oak tree biodiversity | March 2018 Dimilin® Flo is considered most active on early instar larvae and therefore should be sprayed from egg lay to early instar stage (Dimilin® Flo Product Label, 2016). Whilst the label does not specifically mention the use of Dimilin Flo to control oak processionary moth, it does suggest control of other pest species within forestry. In these circumstances, the recommended rate of use, for good foliage cover using a ground application, is given as 150 ml/ha. When aerially sprayed for control of forest lepidopteran larvae, it should be applied at 150 ml/ha in a tank mix with a suitable adjuvant in a volume of 1-10 litres/ha; controlled droplet application/rotary atomiser equipment should be used (Dimilin® Flo Product Label, 2016).

In addition to the general guidelines provided in Section 1.2, the following guidelines for using Dimilin® Flo to control oak processionary moth have been provided within the TH0102 Final Project Report (ADAS UK Ltd, 2015):

 Apply to dry leaves; however, it is rain-fast one hour after application;  Apply in a high volume of water (400 litres/ha and above);  Apply at the recommended rate for the control of caterpillars in amenity trees i.e. 13 ml/100 litres;  The Dimilin® Flo Product Label (2016) recommends a repeat application after 3-4 weeks. This is likely to be too late for oak processionary moth control because most remaining caterpillars will be too old and therefore less susceptible.

2.3 Deltamethrin (formulated product Bandu® MAPP 16153)

2.3.1 Toxicity: Mode of action

Deltamethrin, ((S)-cyano(3-phenoxyphenyl)methyl (1R,3R)-3-(2,2-dibromoethenyl)-2,2- dimethylcyclopropane-1-carboxylate), is a broad-spectrum synthetic pyrethroid insecticide with non- systemic contact and stomach action (PPDB, 2018). It modulates sodium channels (PPDB, 2018), interfering with the normal production and conduction of nerve signals; essentially it delays the closing of the sodium channel activation gate in nerve membranes (NPIC, 2011). Deltamethrin is a Type II pyrethroid; Type II pyrethroids exert long lasting inhibition of the sodium channel activation gate, leading to prolonged permeability of the nerve cell to sodium (NPIC, 2011). This results in a series of repetitive nerve signals in sensory organs, nerves and muscles (NPIC, 2011), thus it is able to exert widespread and potent actions on the nervous system of the insect, effectively paralysing the nervous system and resulting in death, at very low concentrations (Miller and Adams, 1982; Toxipedia, 2014). Type II pyrethroids may also be able to affect other ion channels in the nervous system besides sodium channels (NPIC, 2011). Deltamethrin is a fast-acting insecticide and is considered the most powerful and toxic of the pyrethroid pesticides (Toxipedia, 2014). Deltamethrin can be used against older larval stages of oak processionary moth (Forestry Commission, 2017).

Deltamethrin is classified by the industry ‘Insecticide Resistance Action Committee’ (IRAC) as Group 3A (pyrethroids, pyrethrins – sodium channel modulators).

23 Impact of OPM control methods on oak tree biodiversity | March 2018 2.3.2 Toxicity: Chemical safety tests

Deltamethrin is highly toxic to mammals (Acute Toxicity 3: H331, H301 for deltamethrin; Acute Toxicity 4: H302, H332 and Aspiration Toxicity 1: H304 for the formulated product Bandu®1) and aquatic organisms (Acute Toxicity 1: H400) with long lasting effects (Chronic Toxicity 1: H410), acting as a neurotoxin, and also known to have weak oestrogenic activity and be an IARC Group 3 carcinogen in humans (Bandu® Product Label, 2014; Bandu® Safety Data Sheet, 2015; PPDB, 2018); toxicity and ecotoxicity data are provided in Tables 1 and 2, respectively. Deltamethrin however, is relatively non- toxic to birds with a low threshold for concern; no acute or short-term dietary toxicity was observed for bobwhite quail at the highest tested doses (PPDB, 2018).

Deltamethrin is toxic towards honey bees but relatively non-toxic towards earthworms (PPDB, 2018; Table 2). With regard to ecotoxicity testing against other invertebrates, a variety of results are reported in the PPDB (2018). For example, a high threshold for concern is advised for aquatic crustaceans (Americamysis bahia; Acute 96 h LC50 = 0.0000017 mg/l), and a moderate threshold for concern reported for sediment dwelling organisms (Chironomus riparius; Chronic 28-day NOEC for static water, 0.01 mg/l); mesocosm study data gives a NOEAEC (no observed ecologically adverse effect concentration) value of 0.0032 mg/l for the aquatic community. Deltamethrin is reported as being toxic to larvae of the seven-spot ladybird larvae (100% mortality using a dose of 13.5 g/ha) but does not adversely affect the ability of the adult parasitoid wasp Trichogramma cacoeciae to parasitise eggs (100% parasitism).

Bandu® is included in the Local Environment Risk Assessment for Pesticides (LERAP) scheme. Applications using a horizontal boom sprayer or broadcast air assisted sprayer must have a LERAP conducted or else the statutory buffer zone must be maintained.

2.3.3 Specificity

Deltamethrin is a broad-spectrum insecticide (National Pesticide Information Center, 2011) and as such is used to control a range environmental, agricultural and horticultural species of insects and other invertebrates from the Orders Diptera, Hemiptera, Lepidoptera, Coleoptera, Blattodea, Hymenoptera, Thysanoptera, Siphonaptera, Thysanura, Araneae and Acari (Toxipedia, 2014; CRD Pesticides Register Database, 2018; see section 2.3.6). As such, deltamethrin has the potential to adversely affect a large number of non-target species from a wide range of insect Orders. Susceptibility of an insect is dependent on its physiological structure, but also on environmental conditions (e.g. flies are more susceptible at dawn) (Toxipedia, 2014)

2.3.4 Solubility/mobility in the environment

1 Hazard statements: H301=Toxic if swallowed, H302=Harmful if swallowed, H304=May be fatal if swallowed and enters airways, H332=Harmful if inhaled, H400=Very toxic to aquatic life, H410=Very toxic to aquatic life with long lasting effects (Bandu® Safety Data Sheet, 2015).

24 Impact of OPM control methods on oak tree biodiversity | March 2018 Deltamethrin has low aqueous solubility. It is also non-mobile in the soil, with a Koc of 10240000 (Bandu® Safety Data Sheet, 2015; PPDB, 2018) and as such has a low potential to leach into ground water (GUS leaching potential index -4.26). It is therefore considered not mobile in the environment because of its strong adsorption to particles (Toxipedia, 2014).

2.3.5 Stability/persistence in the environment

Bandu® is not rapidly biodegradeable (Bandu® Safety Data Sheet, 2015); however, deltamethrin is not considered persistent, bioaccumulative and toxic (PBT) or very persistent and very bioaccumulative (vPvB) (Bandu® Safety Data Sheet, 2015). In the soil, a typical DT50 value is 13 days, or 21 days in the field, with a range of 18-35 days given in EU dossier laboratory studies (PPDB, 2018). The PPDB (2018) states that it is stable in water, with DT50 in the water phase of 17 days (slow) and DT50 in water sediment of 65 days (moderately fast). Due to its low solubility, deltamethrin is stable in the physical environment even when exposed to air and sunlight; it does not degrade at 40 °C, even after two years (Toxipedia, 2014). Deltamethrin is considered dangerous to the environment (R50/532) (Bandu® Safety Data Sheet, 2015).

2.3.6 Standard usage in the UK

Bandu® (MAPP no. 16153; marketed by Headland Agrochemicals Limited), containing 25 g/l of the active ingredient deltamethrin in the form of an emulsifiable concentrate has standard approval for use by professionals as an insecticide to control aphids, caterpillars and a range of other pests in a number of crops. On-label uses include amenity vegetation (outdoor), apple, spring and winter barley, broad bean, brussels sprout, cabbage, cauliflower, combining and vining pea, protected cucumber, field bean, outdoor lettuce, spring and winter mustard, spring and winter oats, spring and winter oilseed rape, outdoor and protected ornamental plant production, pear, protected pepper, raspberry, sugar beet, swede, protected tomato, turnip, and spring and winter wheat (CRD Pesticide Register Database, 2018; Bandu® Product Label, 2014). Bandu® is not currently authorised for aerial or aquatic use (CRD Pesticide Register Database, 2018). The authorised holder for the product is Bayer CropScience Ltd; authorisation was first given in 2013 and expires on 30/04/2020 (CRD Pesticides Register Database, 2018). Extensions of authorisation for minor use (EAMU) include protected and outdoor forest nursery (Authorisation No. 20131856); the control of Liriomyza trifolii and Frankliniella occidentalis in protected aubergine (Authorisation No. 20132523), protected courgette, summer squash and gherkin (Authorisation No. 20132524) and protected ornamental plant production (Authorisation No. 20141102); the control of Drosophila suzukii and F. occidentalis in strawberry (Authorisation No. 20132527); the control of aphids, cereal flea beetle, gout fly, saddle gall midge and yellow cereal fly in Durum wheat, rye and Triticale (Authorisation No. 20141100); the control of leaf miners in protected spring onion (Authorisation No. 20141101); the control of cutworms and leaf moths in outdoor leek (Authorisation No. 20141103); the control of aphids, caterpillars and flea beetle

2 Risk phrases: R50/53=Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.

25 Impact of OPM control methods on oak tree biodiversity | March 2018 in outdoor cress, endive, lamb’s lettuce, land cress and outdoor leaves and shoots (Authorisation No. 20141104); the control of carrot fly and cutworms in outdoor carrot, horseradish, mallow and parsnip (Authorisation No. 20141105); the control of bramble shoot moth and raspberry beetle on outdoor blackberry (Authorisation No. 20141106); and the control of aphids on seed crop grassland (Authorisation No. 20141107). Dosage rates and volumes differ depending on the crop and are detailed on the Bandu® Product label (2014); latest time of application is seven days before harvest. Deltamethrin is also approved for the control of non-agricultural pest species such as cockroaches, ants, fleas, silverfish, bedbugs, mosquitoes, house flies, beetles, spiders and bird mites within commercial, industrial and public buildings, public health situations and stored produce (PPDB, 2018).

It should be noted that an amendment notice (2222 of 2016) was issued on the 5th September 2016 stating that the authorisation for deltamethrin (Bandu®; M16153) ends on 31st October 2018, and that by 30th April 2019 sale and distribution of existing stocks is prohibited by any persons, stating a final date of 30th April 2020 for the disposal, storage and use of existing stocks (CRD Pesticides Register Database, 2018).3

In addition to the general guidelines provided in Section 1.2, the following guidelines were provided in the TH0102 Final Project Report (ADAS UK Ltd, 2015) for using Bandu® to control all stages of oak processionary moth:

 Deltamethrin should only be applied in a high volume of water;  Deltamethrin should be used at the recommended rate for amenity vegetation (70 ml/ 100 litres of water).

3 Since this report was submitted on 31st March 2018, the deltamethrin (Bandu®) licence has been extended until 30th April 2021. More information can be found in amendment notice 20180737 released on 18th April 2018: https://secure.pesticides.gov.uk/pestreg/getfullproduct.asp?productid=28304&pageno=1&origin=prodsearch.

26 Impact of OPM control methods on oak tree biodiversity | March 2018

Diflubenzuron Dimilin® Flo Bandu® Bacillus thuringiensis (deltamethrin) subsp. kurstaki, 540 g/kg water dispersible granule Oral LD50 > 4640 mg/kg LD50 5000 LD50 416 mg/kg LD50 > 5050 mg/kg mg/kg (OECD 401) Dermal LD50 > 2000 mg/kg LD50 2000 LD50 > 2000 mg/kg LD50 > 2020 mg/kg mg/kg (OECD 402) Inhalation LC50 > 2.49 mg/L/4h LC50 > 5 mg/L LC50 2.69 mg/L/4h LC50 > 5.15 mg/L/4h (irritating to (nose only) (OECD 425) respiratory system)

Table 1. LC50 and LD50 toxicity values for Dimilin® Flo, Bandu® and DiPel® DF and/or their corresponding active ingredients as listed on the Safety Data Sheets for the products. Information is provided for rats except for the dermal toxicity value for Bacillus thuringiensis subsp. kurstaki, which is provided for rabbit. The Bandu® Safety Data Sheet (2014) states that the toxicological data refer to a similar formulation. Where, LC50 is the concentration of the product at which there is mortality in 50% of the test population during the observation period; LD50 is the dose at which there is mortality in 50% of the test population during the observation period.

27 Impact of OPM control methods on oak tree biodiversity | March 2018

Diflubenzuron Bacillus thuringiensis subsp. Bacillus Bandu® kurstaki, technical grade thuringiensis subsp. kurstaki, 540 g/kg water dispersable granule Fish 96 h LC50 > 0.13 Fish (Lepomis > 2.87 x 109 cfu/L test media (> mg/L macrochirus) 143.5 mg a.s./L) (FIFRA 154-19) LC50 Infectivity/pathogenicity 32 days Rainbow trout 96 h NOEC 0.2 Rainbow trout > 2.87 x 109 cfu/L test media (> Rainbow trout 96 h LC50 (Oncorhynchus mg/L (Oncorhynchus mykiss) 143.5 mg as/L) (FIFRA 154-19) (Oncorhynchus 0.00925 mg/L mykiss) LC50 Infectivity/pathogenicity 32 mykiss) days Pupfish (Cyprinodon 96 h LC50 > 0.2 sp.) mg/L Water flea (Daphnia 48 h EC50 0.0026 Water flea (Daphnia 21 day EC50 (adult Water flea (Daphnia 48 h EC50 magna) mg a.s/L magna) mortality/immobility) 14 mg/L magna) 0.00011 mg/L NOEC < 5 mg/L (FIFRA 154-20) Freshwater green 120 h EC50 > 0.3 Freshwater green algae Acute toxicity 72 Freshwater green 96 h EC50 60.5 algae mg/L (Pseudokirchneriella h algae mg/L (Pseudokirchneriella subcapitata = EC50 50.84 mg/L (Pseudokirchneriella subcapitata = Selenastrum (OECD 201) subcapitata = Selenastrum capricornatum) NOEC 10 mg/L Selenastrum capricornatum) capricornatum) Honey bee (Apis Acute contact 48 h Honey bee (Apis Oral toxicity 14 day LD50 > 4042 Acute oral Honey bee (Apis Acute oral mellifera) LD50 > 30 µg/bee mellifera) µg/bee (FIFRA 154A-24) toxicity 48 h mellifera) toxicity 48 h Acute oral 48 h LD50 > 222.41 LD50 0.079 LD50 > 25 µg/bee µg/bee (OECD µg/bee 213)

28 Impact of OPM control methods on oak tree biodiversity | March 2018 Acute contact Acute contact toxicity 48 h toxicity 48 h LD50 > 185.0 LD50 0.0015 µg/bee (OECD µg/bee 214) Earthworm (Eisenia Acute 14 day LC50 Earthworm (Eisenia Toxicity 30 day LC50 > 1000 Earthworms Acute 14 day fetida) > 500 mg/kg fetida) mg/kg soil (no effect) (OECD LC50 > 1290 207) mg/kg soil Bird, bobwhite quail Acute LD50 > 5000 Bird, bobwhite quail Toxicity 5 day NOEC > 2857 Bird, bobwhite quail Acute LD50 > (Colinus virginianus) mg/kg (Colinus virginianus) mg/kg b.w./d (FIFRA 154A-16) (Colinus virginianus) 2250 mg/kg Short term dietary Short term LC50/LD50 > 1206 dietary mg/kg LC50/LD50 > bodyweight/day 5620 ppm Bird (Mallard duck) Toxicity 5 day NOEC > 2857 mg/kg b.w./d (FIFRA 154A-16) Table 2. Ecological toxicity values for Dimilin® Flo, Bandu® and DiPel® DF and/or their corresponding active ingredients as listed on the Safety Data Sheets for the products and on the Pesticides Properties DataBase (PPDB, 2018). NB. No data is available for the product Dimilin® Flo, only for its active ingredient. The Bandu® Safety Data Sheet (2014) states that the ecotoxicological data refer to a similar formulation. Where, LC50 is the concentration of the product at which there is mortality in 50% of the test population during the observation period; LD50 is the dose at which there is mortality in 50% of the test population during the observation period; EC50 is the concentration that gives half-maximal effect; NOEC is the concentration at which there is no observed effect.

29 Impact of OPM control methods on oak tree biodiversity | March 2018 Summary

1. Bacillus thuringiensis var. kurstaki and diflubenzuron are not toxic towards mammals; deltamethrin is highly toxic towards mammals (including humans).

2. Bacillus thuringiensis var. kurstaki, diflubenzuron and deltamethrin are all relatively non-toxic to birds.

3. Deltamethrin and diflubenzuron are both considered highly toxic to aquatic life; B. thuringiensis var. kurstaki is considered moderately toxic towards aquatic life.

4. Bacillus thuringiensis var. kurstaki, diflubenzuron and deltamethrin are considered relatively non-toxic towards earthworms.

5. Deltamethrin is highly toxic towards honey bees; B. thuringiensis var. kurstaki and diflubenzuron have no observed toxicity towards honey bees.

6. Bacillus thuringiensis var. kurstaki, diflubenzuron and deltamethrin all have low solubility in water and are all considered non-mobile in the soil.

7. The persistence of B. thuringiensis var. kurstaki on leaves is variable (days to months have been reported in the literature). Persistence of diflubenzuron is thought to be 3-4 weeks although reports are variable; a proportion is reported as still present on the leaves at leaf fall. Deltamethrin is stable in the physical environment.

8. Bacillus thuringiensis var. kurstaki spores can persist in the soil for many months (half-life 100- 200 days; NB. B. thuringiensis naturally occurs in the soil environment) whilst the δ-endotoxins are not thought to persist for long in the soil (half-life measured in days); B. thuringiensis var. kurstaki is therefore considered non-persistent. Diflubenzuron is described as non-persistent in the soil. Deltamethrin has a reported persistence in the soil of 13-35 days.

9. Bacillus thuringiensis var. kurstaki and deltamethrin are both described as not bioaccumulative. Diflubenzuron is also described as not bioaccumulative although the PPDB (2018) suggests that the potential for bioconcentration in fish is at a threshold to cause concern.

10. DiPel® DF (Bacillus thuringiensis var. kurstaki) has an extension of minor use authorisation in forestry specifically for the control of oak processionary moth (Authorisation number 20160931) until 23/03/2020.

11. Dimilin® Flo (diflubenzuron) was provisionally approved for use in the UK until 31/12/2021. Use of Dimilin® Flo has been withdrawn on edible crops although it appears that use on non- edible crops may continue as specified on the product authorisation and label. EU approval for the use of diflubenzuron expires on 31/12/2018 (EU Pesticides database, 2016).

12. An amendment notice (2222 of 2016) was issued on the 5th September 2016 stating that the authorisation for deltamethrin (Bandu®; M16153) ends on 31st October 2018, and that by 30th April 2019 sale and distribution of existing stocks is prohibited by any persons, stating a final

30 Impact of OPM control methods on oak tree biodiversity | March 2018 date of 30th April 2020 for the disposal, storage and use of existing stocks. [Note that since this report was submitted on 31st March 2018, the deltamethrin (Bandu®) licence has been extended until 30th April 2021 (amendment notice 20180737)].

13. Bacillus thuringiensis var. kurstaki and diflubenzuron are most effective towards early instar oak processionary moth larvae whereas deltamethrin will effectively kill larvae of all stages.

14. Lepidopteran species within the families Pyralidae and Tortricidae may be the most susceptible to the B. thuringiensis var. kurstaki lepidopteran-active toxins (Cry1A and Cry2A) whilst the family Noctuidae may have the highest proportion of unresponsive species (reviewed by van Frankenhuyzen, 2009).

15. Bacillus thuringiensis var. kurstaki was originally considered to be specific to Lepidoptera. The literature suggests that this may not be the case. Data presented within Glare and O’Callaghan (2000) suggest that some species of Coleoptera, Dictyoptera, Diptera, Hemiptera, Hymenoptera and Isoptera are susceptible to the product DiPel® (as well as some species of ticks, lice and nematode). In addition, cross-Order activity is reported for three of the δ- endotoxins known to be produced by B. thuringiensis var. kurstaki strain ABTS-351 (Cry1Ab, Cry1Ac and Cry2Aa) across a total of five insect Orders.

16. Data on cross-Order/cross-Class activity of the B. thuringiensis δ-endotoxins must be interpreted with caution for a variety of reasons, such as the dose used, how the product/toxin was delivered, and the biological relevance of any toxic response observed in laboratory studies. van Frankenhuyzen (2013) suggest that the only reported cross-Order activity for Cry1Ab, Cry1Ac and Cry2Aa to be validated is that observed towards aphids. However, it is also important to point out that not all species of aphid appear to be susceptible.

17. Diflubenzuron is not specific to lepidopteran species. It has the potential to disrupt chitin synthesis in the immature stages of any arthropod but not all species are susceptible. Many beneficial species of insect for example are not thought to be affected by use of diflubenzuron. Species within the families Lepidoptera, Coleoptera and Diptera are particularly susceptible to diflubenzuron.

18. Deltamethrin is a broad-spectrum pesticide used to control a wide range of insect pests across several different Orders. It can therefore be expected to adversely impact on a wide variety of non-target insect species.

19. Glare and O’Callaghan (2000) ask the question “Are studies underestimating the impact of Bacillus thuringiensis?” because reported studies most often assess larval mortality rather than adult emergence and sublethal effects.

31 Impact of OPM control methods on oak tree biodiversity | March 2018 Chapter 3. Invertebrate assemblages associated with oak trees

The aim of this chapter is to provide details of the Lepidoptera and other invertebrate species commonly associated with oak trees in parkland, open woodland and woodland edge habitats in southern England. Comparisons between different oak species, habitat types (parkland/open woodland/dense woodland), geographical regions, rarity, food source and typical life cycles are detailed where known. This will enable an assessment of how vulnerable they may be to measures put in place to control oak processionary moth.

There are two native species of oak in Britain, the pedunculate (common or English) oak Q. robur, and the sessile oak Q. petraea, and in addition there are the introduced species such as Turkey oak (Quercus cerris L.), northern red oak () and the evergreen holm oak (Quercus ilex L.).

Oak trees in the UK support a vast array of species: they provide an important food source for many insect species (discussed in more detail later in this chapter) and some mammals and birds, and a habitat for other species including mosses, lichens, birds and bats (SEFARI, 2018). Mammals (such as mice, squirrels, badgers and deer) and birds (such as jays) are known to feed on the acorns (The Woodland Trust, 2018). In addition, the many insect species present on oak, particularly lepidopteran larvae present in the spring time provide an important source of food for many species of birds (The Woodland Trust, 2018). Several species of bats feed on insects present in the canopy, and also roost under areas of loose bark or in old woodpecker holes (The Woodland Trust, 2018). Holes and crevices within the bark also provide valuable nesting sites for bird species such as the pied flycatcher and marsh tit (The Woodland Trust, 2018). Once fallen, the leaves form a rich leaf mould on the ground and support invertebrate species such as the stag beetle and several species of fungi (such as milkcap) (The Woodland Trust, 2018). Information has been gathered on over 2000 species of mosses, lichens, fungi, invertebrates, birds and mammals associated with oak trees (SEFARI, 2018) and is soon due to be released as a downloadable database. A decline in oak tree health (which can sometimes be related to severe defoliation) poses significant risks to those species that are reliant upon them, and any control options applied to control pestiferous insects on oak have the potential to impact on the vast array of oak-associated invertebrates, which in turn has the potential to impact upon the species higher up in the food chain (such as birds and bats) that are reliant upon them for food.

There are a number of reasons why the diversity on oak is so rich (Sobczyk, 2014):

 Oak provides a habitat for a very long time because of its longevity;  Oak has a high leaf regeneration potential;  The death process in oak is slower than for other tree species, resulting in the provision of a variety of structures;  All parts of oak provide a primary food source, including the lichens and fungi present on the bark;  Oak trees provide habitat for spiders;  The insects feeding upon oak can themselves provide food for predators and hosts for parasitoids.

The two native oak species have the highest number of associated insect species of any of the native trees in Britain. Southwood (1961) reports a total of 284 species of insects associated with deciduous

32 Impact of OPM control methods on oak tree biodiversity | March 2018 Quercus (Q. robur and Q. petraea) in Britain. Of these species, 106 are macrolepidoptera, 81 are microlepidoptera, 50 belong to the Coleoptera and 47 belong to the hemipteran sub-families of Heteroptera (37 species) and Auchenorrhyncha (10 species). Twenty three years later, a revised and expanded list was published indicating a total of 423 species: 106 species of macrolepidoptera, 83 species of microlepidoptera, 67 species of Coleoptera, seven species of Diptera, 81 species of Hemiptera, 70 species of Hymenoptera, seven species of Thysanoptera and two species of Acarina (Kennedy and Southwood, 1984; Table 3).

More recently, Southwood et al. (2004, 2005) report on a detailed study of the invertebrate fauna associated with the two native and two introduced species of oak, over five years, in woods near Oxford, UK. The authors used the knockdown technique and branch sampling to assess the invertebrate fauna. Their 2005 publication (Southwood et al., 2005) reports on the composition of the arthropod fauna within the canopies. No details of individual invertebrate species are given; however, the native oak species had nearly double the number of species (excluding Nematocera) (801 species on Q. petraea, 1149 species on Q. robur) compared with the two non-native oak species (543 species on Q. cerris and 431 species on Q. ilex). These numbers are considerably higher than those reported by Southwood (1961, 1984). The proportions of each invertebrate Order that Southwood et al. (2005) reported (based on the ordinal counts) for Q. robur and Q. petraea are provided in Table 3.

Seasonal patterns occur with species richness peaking in the summer and early autumn, reflecting high populations of spiders (Southwood et al., 2005). The chewing insects peak in May, followed by the sap-sucking insects, then the leaf miners and finally the gall formers (Southwood et al., 2004). Phytophages, predators and parasitoids are much higher in abundance and species richness on the native oak trees compared with the non-native oak trees (Southwood et al., 2004, 2005). The two native species have very similar species compositions, species numbers, richness and biomass in terms of overall numbers, and ordinal composition, and the guild composition in terms of numbers, species richness and biomass were closely correlated. The proportion of species classified by guild on the native oak species are as follows (Southwood et al., 2005):

Phytophages – 30.9% (Q. petraea), 33.3% (Q. robur)

Predators – 21.6% (Q. petraea), 21.6% (Q. robur)

Parasitoids – 32.8% (Q. petraea), 29.4% (Q. robur)

Scavengers – 6.4% (Q. petraea), 6.5% (Q. robur)

Epiphytes – 8.3% (Q. petraea), 9.2% (Q. robur)

Both the epiphyte fauna and tourist species have no direct trophic link to the trees.

33 Impact of OPM control methods on oak tree biodiversity | March 2018 Taxon Numbers of species Taxon Proportion of ordinal reported on native numbers reported by Quercus by Kennedy and Southwood et al. (2005) Southwood (1984) Q. petraea Q. robur Acarina 2 Aranae 3.4 2.6 Coleoptera 67 Coleoptera 6.9 6.3 Dermaptera 0.1 0.0 Diptera - 7 Diptera 12.0 10.0 Cecidomyiidae Heteroptera 38 Heteroptera 6.7 6.9 Hemiptera - 21 Hemiptera 7.8 5.9 Auchenorrhyncha Hemiptera - 1 Psylloidea Hemiptera - 15 Aphidoidea Hemiptera - 1 Aleyrodoidea Hemiptera - 5 Coccoidea Hymenoptera - 17 Hymenoptera - 0.3 0.2 Symphyta Symphyta/Aculeata Hymenoptera - 53 Hymenoptera - 14.9 12.9 Cynipoidea Parasitica Isopoda 0.1 0.0 Macrolepidoptera 106 Lepidoptera 11.5 11.2 Microlepidoptera 83 Neuroptera 0.3 0.5 Opiliones 0.3 0.6 Orthoptera 0.5 0.3 34.5 41.2 Thysanoptera 7 Thysanoptera 0.7 1.3 Total 423 100% 100% Table 3. The numbers of species (Kennedy and Southwood, 1984) and the proportion of ordinal counts (Southwood et al., 2005) representing the invertebrate taxa found on native oak.

Table 4 provides extensive details of the lepidopteran species in the UK whose larvae feed on oak. These details include life-cycle, larval food plants, preferred habitats, and their abundance and geographical distribution within the British Isles along with any other pertinent details. As such the oak-feeding Lepidoptera are not discussed any further within the text, and the reader is referred to Table 4 for further information.

The reported numbers of Coleoptera associated with oak are variable, for instance Stork et al. (2001) report 144 species found on Q. robur whereas Stork and Hammond (2013) indicate 150 coleopteran species. As expected, some coleopteran species have a preference for the canopy region whilst others are more abundantly found near the trunk; some move between the ground, trunk and canopy during the year (Stork and Hammond, 2013). Overall, coleopoteran abundance and species diversity is highest in late June. However, similar levels are observed throughout the year for the oak specialist

34 Impact of OPM control methods on oak tree biodiversity | March 2018 beetles whereas the generalist beetles peak in July; predatory and herbivorous species peak in May to June whereas the fungivores and scavengers peak July to August (Stork and Hammond, 2013). Sobczyk (2014) reports that 90% of oak-associated coleopteran species prefer free-standing trees with good light.

However, it is not just the invertebrate fauna in the oak trees themselves that is at risk when an insecticide is applied to control an insect pest within the tree. The fauna associated with the plants growing along ridges and clearings within woodlands, and in the grassland habitat that surrounds the trees in open parklands, are also at risk from chemical control methods used to control oak processionary moth. In particular, the moths and butterflies within these habitats are potentially at risk of non-target impacts if B. thuringiensis var. kurstaki (and indeed any other insecticide) is applied to control oak processionary moth. Lepidoptera species are already in significant decline in areas of the UK, including the southern half of the country where oak processionary moth is present. With this in mind, Parsons and Hill (2016) identified the lepidopteran species in these habitats most at risk from B. thuringiensis var. kurstaki if applied to oak to control oak processionary moth in the Wisley (Surrey) area. Parsons and Hill (2016) report the following findings:

1. A minimum of 395 species (23 species of butterfly, 267 species of larger moths and 105 species of micro-moths) are associated with woodlands in the Wisley (Surrey) area. Larvae of all 23 species of butterfly would be feeding during April to June, although only one, the Purple Hairstreak (Neozephyrus quercus), feeds on oak. 2. Ninety one (91) species of larger moths that feed on oak as larvae were recorded in the Wisley area, 77 of which feed during the April to June period. Thirty five (35) of these 77 species are external feeders (that is to say their feeding habit is unlikely to afford them any protection from an application of B. thuringiensis var. kurstaki (or any other insecticide)). 3. Two hundred and sixty seven (267) species of larger moths were identified as woodland species, with 253 of them feeding during the April to June period, and 13 of these species feeding internally or under bark. 4. Forty two (42) species of micro-moths feed on oak as larvae, with 31 of these feeding during the April to June period. In addition, three species feed on the lichens and mosses of tree trunks and their larvae would be active for at least part of the April to June period. 5. One hundred and five (105) species of micro-moths were considered woodland species. Ninety four (94) of these species feed as larvae during the April to June period, 20 of which have internal feeding habits. 6. In total, 395 lepidopteran species in the Wisley area were identified as woodland species: 134 are associated with oak, of which 109 will feed during April to June. 7. Three hundred and thirty seven (337) of the 395 woodland species of Lepidoptera were considered potentially at risk from spraying in a woodland environment during April to June (assuming that internal feeders are not affected by spraying). 8. The number of species at risk would increase considerably if other habitats were considered. 9. In addition to the three Section 41 species that feed on oak (see Table 4), a further three Section 41 species occur in woodland in the Wisley area (Barred Tooth-striped Trichopteryx polycommata, Sloe Carpet Aleucis distincta (both associated with scrubby habitats) and the

35 Impact of OPM control methods on oak tree biodiversity | March 2018 Forester Adscita statices (associated with open and grassland habitats). All three of these species feed as larvae during April to June. 10. Four Nationally scarce species are associated with oak (see Table 4) but only one feeds between April and June (Great Oak Beauty Hypomecis roboraria; Table 4). A further 22 species of larger moths considered to be scarce or threatened are woodland species whose larvae feed during April to June. 11. Two Nationally scarce micro-moths are associated with oak (Carpetolechia decorella and Povolnya leucapennella; Table 4) and feed during the April to June period. A further four Nationally scarce or threatened species were identified as associated with woodland and feeding between April and June (Perittia obscurepunctella, zieglerella, Paratalanta hyalinalis and Hellinsia lienigiana). 12. Nine species of butterfly are associated with woodland habitat in the Wisley area and feed during April to June. Seven of these species are Section 41 species: Dingy Skipper (Erynnis tages), Brown Hairstreak (Thecla betulae), White Admiral (Limentis camilla), and Grayling (Hipparchia semele) are classed as vulnerable; Wood White (Leptidea sinapsis) and White- letter Hairstreak (Satyrium w-album) classed as endangered; and Small Blue (Cupido minimus), Purple Emperor (Apatura iris) and the Wall (Lasiommata megera) classified as near- threatened. The Grizzled Skipper (Pyrgus malvae; Section 41 vulnerable) is found in open woodland and the Silver-studded Blue (Plebejus argus (Section 41 vulnerable) is a heathland species feeding between April and June.

The reader is referred to Parsons and Hill (2016) for more details.

Summary

1. Over 2000 species (mosses, lichens, fungi, invertebrates, birds and mammals) are associated with oak. 2. The two native speices of oak (Q. robur and Q. petraea) have the highest number of associated insect species of any of the native trees, estimated as 284 (in 1961), 423 (in 1984), and 1149 for Q. robur and 801 for Q. petraea (in 2005). 3. Details of the lepidopteran species that are directly associated with Q. robur and Q. petraea are provided in Table 4. One hundred and seventy (170) species were identified, 98 of which feed at some point between April and June. 4. In total, Parsons and Hill (2016) identified 395 woodland lepidopteran species in the Wisley (Surrey) area: 134 which are associated with oak, and of these 109 will feed during April to June.

36 Impact of OPM control methods on oak tree biodiversity | March 2018 Family/Sub- Species name Vernacular National Foodplant Assoc- Habitat Geographical Life cycle Notes References family name status iated with location which Oak species? Agonoxenidae Dystebenna Oak Cosmet Nationally Oak Quercus Open mature Occurs only in parts Moths fly late June to Larvae feed in Robinson et al., stephensi Scarce A woodland, of south and south- September the bark 2010; Kimber, parkland east England, with 2018; Sussex occasional records Moth Group; further north and Hants Moths west into the Midlands Arctiidae Callimorpha Scarlet Tiger Local Larvae feed on Quercus Damp areas Southern and south- Larvae feed August to Overwinters as Robinson et al., dominula Moth various such as fens, west England, south May. larvae 2010; Kimber, herbaceous marshes, Wales and some Moths fly in May and 2018; Butterfly plants especially riverbanks, areas in north-west June during the Conservation comfrey rocky cliffs England daytime near the sea Bucculatrix ulmella Oak Bent- Common Oak and Quercus England, Wales and Leaf mines July and The mine is a Robinson et al., wing hornbeam Scotland September to short and 2010; Kimber, October. Adults fly contorted 2018; Norfolk late April to mid-June gallery close to Moths; Sussex and July to August. the midrib of Moth Group Double-brooded the leaf Quercus Case-bearer bipennella Coleophora Scarce pRDB3 Oak, hornbeam Quercus Woodland South-east England, Larvae present in Leaf miner and Robinson et al., currucipennella Wood Case- and sallow containing Cheshire, June case-bearer 2010; Pitkin et bearer oak and Denbighshire, North al., 2018; hornbeam Somerset, Shropshire, Norfolk Moths Stafford Coleophora Tipped Oak Common Deciduous and Quercus Oak Distributed widely Larvae feed Case-bearer. Robinson et al., flavipennella Case-bearer evergreen oak woodland, but locally September to Larvae diapause 2010; Kimber, prefers throughout most of November and May. over winter 2018; Norfolk margins of Britain Adults fly July to Moths; Suffolk rides and August. Single- Moth Group isolated oak brooded trees

37 Impact of OPM control methods on oak tree biodiversity | March 2018 Coleophora Forest Case- Local Oak Quercus Oak Mainly in south-east Larvae feed Leaf miner and Robinson et al., ibipennella bearer woodland, England September to case-bearer. 2010; Kimber, along rides October and April to Larvae diapause 2018; Norfolk and on edges May. Adults fly late- over winter Moths; Suffolk of oak July to August. Single Moth Group woodland brooded. Coleophora Common Common Oak, possibly Quercus Oak England, Wales and Larvae feed Case-bearer. Robinson et al., lutipennella Oak Case- also sweet woodland, southern Scotland September to Larvae diapause 2010; Kimber, bearer chestnut preferring November and May. over winter 2018; Norfolk margins of Adults fly July to Moths; Suffolk rides and August. Single Moth Group isolated oak brooded trees Coleophora White Oak Local Oak Quercus Oak woodland South-east England, Larvae feed Case-bearer. Robinson et al., kuehnella Case-bearer and scrub, ranging northwards September to Larvae diapause 2010; Kimber, preferring to the Midlands and October and May. over winter 2018; Norfolk margins and Derbyshire Adults fly late-July to Moths; Suffolk rides with August. Single- Moth Group plenty of brooded sunshine Coleophora lutarea Stitchwort Nationally Larvae feeds on Q. robur, Open Southern England and Larvae feed August- Pupates in the Robinson et al., Case-bearer Scarce B greater Q. woodland, Wales September. Moths fly bark of a tree. 2010; Kimber, stitchwort, petraea shady in May. Single- Larvae diapause 2018; Norfolk consuming the situations brooded over winter Moths; Suffolk seeds Moth Group Cossidae Cossus cossus Goat Moth Section 41; Various Quercus Open Locally widespread in Larvae feed at some Larvae burrow Parsons and Nationally broadleaved woodland and the south, scarcer time during April to into trunks of Hill, 2016; Scarce B trees including scrub, further north in June. Adults fly in various Kimber, 2018; oak, sallows, orchards, Britain June/July deciduous trees ukmoths.org.uk; , ash, gardens and and feed on the Norfolk Moths; poplars, birch, parks wood. Larvae Suffolk Moth apple often take up to Group five years before pupation due to long digestion period for wood

38 Impact of OPM control methods on oak tree biodiversity | March 2018 Cymatophorina Oak Local Pedunculate Q. robur Mature oak Throughout England Larvae April – June. British C. diluta Robinson et al., diluta Lutestring and sessile oak and Q. woodland Adults fly August and are a separate 2010; Kimber, petraea September. Single- subspecies, 2018; Norfolk brooded hartwiegi Moths; Suffolk Moth Group; Hants Moths Ochropacha Common Common Birch, alder, Quercus Heathland, Throughout most of Larvae August to Robinson et al., duplaris Lutestring hazel and oak open Britain October. Adults fly 2010; Kimber, woodland and June to August. 2018; Norfolk scrub where Single-brooded Moths; Suffolk birch grows Moth Group Polyploca ridens Frosted Local Pedunculate, Q. robur, Mature oak Southern half of Larvae May to July. Robinson et al., Green sessile and Q. woods England and Wales, Adults fly April and 2010; Kimber, Turkey Oak petraea becomes scarcer May. Single-brooded 2018; Norfolk and Q. further north Moths; Suffolk cerris Moth Group Watsonalla binaria Oak Hook- Common Pedunculate, Q. robur, Oak woodland Southern half of Larvae June to Robinson et al., tip sessile and Q. and parkland, Britain September. Adults fly 2010; Kimber, Turkey Oak petraea scrub oak on May to June and 2018; Norfolk and Q. heathland, again in August. Moths; Suffolk cerris fens, Double-brooded Moth Group grassland Eriocrania Common Common Oak Quercus Woodland Throughout Britain Mines during May. Larvae feed Robinson et al., subpurpurella except very north of Adults fly April to internally 2010; Kimber, Scotland May. Single-brooded (miner) 2018; Norfolk Moths; Suffolk Moth Group Winter Oak Nationally Deciduous oak Woodland Widespread in Larvae feed April to Larvae feed Parsons and decorella Groundling Scarce B or dogwood and scrub mainland Britain June. Pupated in July. inside folded Hill, 2016; () (also gardens Adults emerge in July leaf Kimber, 2018; in Surrey). and overwinter until Freed and Found April/May. Single- Reeve, 2014; associated brooded Norfolk Moths; with isolated Suffolk Moth trees Group Psoricoptera Humped Local Deciduous oak Quercus Mature Southern England and Larvae feed May to Larvae feed in Robinson et al., gibbosella Groundling woodland south Wales June. rolled leaf 2010; Kimber, 2018; Norfolk

39 Impact of OPM control methods on oak tree biodiversity | March 2018 Adults fly July to Moths; Suffolk October. Single- Moth Group brooded Stenolechia Black- Local Oak Quercus Woodland Widely distributed in Larvae feed June. Larvae feed on Robinson et al., gemmella dotted England and Wales Adults fly July to young shoots; 2010; Kimber, Groundling September. Single- adults rest in 2018; Norfolk brooded. bark crevices Moths; Suffolk Moth Group luculella Crescent Common Deciduous oak Quercus Oak woods Occurs in most of Larvae feed during Larvae feed Robinson et al., Groundling England and Wales September; adults fly between spun 2010; Kimber, but not in Scotland in May and June. leaves and 2018; Single-brooded overwinter as Norfolk Moths; pupa on the Suffolk Moth ground Group Pseudotelphusa Tawny Nationally Deciduous oak Quercus Depends on Oak-feeding race in Larvae feed August to Two distinct Robinson et al., paripunctella Groundling Scarce B and bog myrtle the race (see southern Britain, Bog- September. Adults fly races: one 2010; Norfolk notes) myrtle-feeding race in May to June. Single- feeding on oak Moths; Suffolk northern England and brooded in woodland Moth Group; Scotland edges, heaths Hants Moths and hedgerows; the second race feeding on bog myrtle in moorland, fens, bogs, damp heathland and mosses Geometridae Spring Common Mainly on Q. robur Mature oak England and Wales, Larvae feed April to Females are Robinson et al., leucophaearia Usher pedunculate and Q. woodland, local in Scotland June. Adults emerge wingless and 2010; Kimber, and sessile oak petraea heathland February and March. climb up tree 2018; Norfolk with scrub Single-brooded trunks; males Moths; Suffolk oak are weak fliers Moth Group Agriopis marginaria Dotted Common Pedunculate Q. robur Woodland, Throughout British Larvae feed April to Freed and Border and sessile oak, and Q. gardens, Isles June. Adults fly late- Reeve, 2014; hawthorn, petraea heathland and January to early-May. Norfolk Moths, blackthorn, scrub Single-brooded Suffolk Moth birch, hazel, Group; Hants , , Moths sycamore,

40 Impact of OPM control methods on oak tree biodiversity | March 2018 apple, plum and heather Alsophila aescularia March Moth Common Oak, hawthorn, Quercus Woodland but Throughout British Larvae feed May to Females are Freed and hazel, crab can occur in Isles June. Adults fly late- wingless Reeve, 2014; apple and most habitats February to April. Norfolk Moths, blackthorn Single-brooded Suffolk Moth Group; Hants Moths Apocheima Small Local Mainly Q. robur Mature oak Southern England and Larvae feed April to Females are Robinson et al., hispidaria Brindled pedunculate and Q. woodland Wales, can be found June. Adults emerge wingless and 2010; Kimber, Beauty and sessile oak, petraea locally as far north as in February and may be found 2018; Norfolk occasionally Yorkshire March. Single- on the trunks of Moths; Suffolk hazel and elm brooded trees Moth Group Biston strataria Oak Beauty Common Deciduous trees Quercus Oak Throughout England Larvae feed May to Freed & Reeve and shrubs woodland, and southern July. Adults fly mid- (2014); Norfolk including oak, heathland Scotland February to April. Moths, Suffolk hazel, aspen, with scrub, Single-brooded Moth Group; alder, elm and gardens Hants Moths sallow miata Autumn Local Rowan, sallow, Quercus Woodland Throughout Britain Larvae feed June to Robinson et al., Green birch, alder and and August. Adults 2010; Kimber, Carpet lime, wild scrubland, emerge in the 2018; Norfolk and other hedgerows, autumn and Moths; Suffolk broadleaved gardens in overwinter. Flies Moth Group trees upland areas September and October and less commonly in March and April. Single- brooded Chloroclysta Red-green Common Various Quercus Mainly Much of Britain Larvae feed June to Robinson et al., siterata Carpet deciduous trees woodland, August. Adults 2010; Kimber, especially oak scrub and emerge in the 2018; Norfolk and rowan gardens autumn and Moths; Suffolk overwinter. Flies Moth Group September and October and early spring. Single- brooded

41 Impact of OPM control methods on oak tree biodiversity | March 2018 Comibaena Blotched Local Pedunculate Q. robur Established Southern half of Larvae feed April- Rare double- Robinson et al., bajularia Emerald and sessile oak and Q. woodland England and parts of May. Adults fly June brood with 2010; Kimber, petraea with mature Wales and July larvae feeding 2018; Suffolk , scrub in September Moth Group; Norfolk Moths Cyclophora porata False Mocha Nationally Pedunculate Q. robur Scrubby oak Distributed in Larvae feed July and Robinson et al., Scarce B and sessile oak and Q. woodland southern half of October. Adults fly 2010; Kimber, petraea England and Wales; May to June and 2018; Suffolk local and uncommon again August to Moth Group; September. Double- Norfolk Moths brooded Cyclophora Maiden’s Local Pedunculate, Q. robur, Oak South of England, Larvae feed July and Robinson et al., punctaria Blush sessile and Q. woodland, scarcer further north October. Adults fly 2010; Kimber, Turkey oak petraea heathland, into Scotland May-June and again 2018; Norfolk and Q. scrub in August. Double- Moths; Suffolk cerris brooded Moth Group Cyclophora Blair’s Scarce Evergreen oak Q. ilex Most records have Larvae feed in Continental Robinson et al., puppillaria Mocha Immigrant (Q. ilex), been on the south October species 2010; Kimber, strawberry-tree, coast (a few inland 2018; Norfolk rock and further north) Moths; Suffolk Moth Group Deileptenia ribeata Satin Beauty Common Coniferous Quercus Deciduous Mainly in southern Larvae feed August Robinson et al., trees including and half of England and through to May. 2010; Kimber, Scots pine, coniferous Wales; few scattered Adults fly June to 2018; Norfolk Norway spruce, woodland colonies northwards August. Single- Moths; Suffolk , Douglas to Scotland brooded Moth Group and yew erosaria September Common Oak, birch, lime, Quercus Woodland, England and Wales, Larvae May to July. Robinson et al., Thorn beech parkland and scarcer in north of Adults fly July to 2010; Kimber, gardens England and Scotland October. Single- 2018; Norfolk brooded Moths; Suffolk Moth Group Ennomos quercaria Clouded Oak Quercus Doubtfully recorded Adults fly June to July Mainland Robinson et al., August in Britain Europe species 2010; Kimber, Thorn 2018 Erannis defoliaria Mottled Common Various Quercus Woodland, Most of Britain, Larvae feed April to Females are Robinson et al., Umber broadleaved suburban especially in the June. Males fly wingless; 2010; Kimber, trees and areas, scrub, south October to 2018; Glare and shrubs hedgerows, O’Callaghan,

42 Impact of OPM control methods on oak tree biodiversity | March 2018 gardens and December. Single- susceptible to 2000; Norfolk heathland brooded. DiPel® Moths; Suffolk Moth Group Brindled Common Pedunculate Q. robur Oak Much of Britain, Larvae feed May to Robinson et al., abbreviata Pug and sessile oak and Q. woodland, except far north of June. Adults fly April 2010; Kimber, and hawthorn petraea heathland, Scotland to May. Single- 2018 oak scrub brooded Eupithecia Oak-tree Common Pedunculate Q. robur Deciduous Widespread in Larvae feed June to Robinson et al., dodoneata Pug oak (leaves) and woodland, England and Wales August. Adults fly 2010; Kimber, flowers of suburban May to June. Single- 2018; Norfolk hawthorn habitats, brooded Moths; Suffolk hedgerows Moth Group Eupithecia irriguata Marbled Nationally Q. robur, Quercus Mature oak Mainly in southern Larvae feed June to Robinson et al., Pug Scarce B possibly Q. woodland, England, notably in July. Adults fly late- 2010; Kimber, petraea hedgerows the New Forest April to early-June. 2018; Norfolk with mature Single-brooded Moths; Suffolk oak Moth Group Hemithea aestivaria Common Common Hawthorn, Quercus Open Throughout much of Larvae feed August to Larvae diapause Freed and Emerald blackthorn, woodland, lowland England and September and April. over winter Reeve, 2014; hazel, oak, hedgerows, Wales Adults fly late-June to Norfolk Moths, willow, birch, scrub, mid-August. Single- Suffolk Moth sallow and field grassland, brooded Group; Hants heathland and Moths gardens Hypomecis Pale Oak Common Pedunculate Q. robur Broadleaved South of England and Larvae feed July to Robinson et al., punctinalis Beauty oak, birch and woodland, Wales August. Adults fly 2010; Kimber, other trees scrub and May to July. Single- 2018; Norfolk heathland brooded Moths; Suffolk Moth Group Hypomecis Great Oak Nationally Pendunculate Q. robur Ancient and South of England and Larvae feed August to Larvae diapause Robinson et al., roboraria Beauty Scarce B oak mature oak Wales, ranging October and April to over winter 2010; Kimber, woodland northwards locally to May. Adults fly June 2018; Parsons the Midlands to July. Single- and Hill, 2016; brooded Suffolk Moth Group; Hants Moths lactearia Little Common Range of trees Quercus Open Throughout England Larvae feed Robinson et al., Emerald including oak, woodland, and Wales September-October. 2010; Kimber, mature 2018; Norfolk

43 Impact of OPM control methods on oak tree biodiversity | March 2018 birch and hedgerows Adults fly May-June. Moths; Suffolk hawthorn and scrub Single-brooded Moth Group Parectropis Brindled Local Pedunculate Q. robur Broadleaved Occurs in the south of Larvae feed July to Robinson et al., extersaria White-spot oak, birch, woodland, England and Wales September. Adults fly 2010; Kimber, (similaria) hazel, hawthorn hedgerows, May to June. Single- 2018; Norfolk heathland brooded Moths; Suffolk Moth Group Plagodis dolabraria Scorched Local Range of Quercus Broadleaved Widely distributed in Larvae July to Robinson et al., Wing deciduous trees woodland, England and Wales, September. Adults fly 2010; Kimber, including oak, heathland and less common in May to June. Single- 2018; Norfolk birch, sallow, scrub Scotland brooded Moths; Suffolk beech and Moth Group sweet chestnut Operophtera Winter Common Broadleaved Quercus Any, Throughout much of Larvae feed April to Can be a serious Freed and brumata Moth trees and wherever British Isles June. Adults fly mid- pest, especially Reeve, 2014); shrubs including trees and October to February. in orchards Norfolk Moths, alder, birch, shrubs grow. Single-brooded Suffolk Moth field maple, Very common Group; Hants elm, hawthorn, in woodland, Moths hazel, parks and blackthorn, oak, gardens sallow, wild cherry, ash and Selenia lunularia Lunar Thorn Local Range of Quercus Open Distributed over Larvae feed July to Immigrant Robinson et al., deciduous trees woodland and much of Britain September. Adults fly species 2010; Kimber, including oak, scrub May to June. Single- 2018; Norfolk birch, ash, brooded Moths; Suffolk blackthorn, Moth Group dog-rose Selenia tetralunaria Purple Common Range of Quercus Woodland, Southern England and Larvae feed June to Double- Robinson et al., Thorn deciduous trees heathland, Wales, scarcer September. Adults fly brooded except 2010; Kimber, including hazel, scrub and northwards into April to May and in northern 2018; Norfolk birch, oak, alder fens southern Scotland again in July to extreme of its Moths; Suffolk and hawthorn August range (where it Moth Group flies in May) Acrocercops Brown Oak Local Oak, including Quercus Open Mainly in southern Larvae feed June. Larvae make Robinson et al., brongniardella Slender evergreen oak (includes woodland, England, occasional Adults fly at the end mines in the 2010; Kimber, Q. ilex) upper 2018; Norfolk

44 Impact of OPM control methods on oak tree biodiversity | March 2018 hedgerows, sporadic populations of July. Single- epidermis. Moths; Suffolk scrub further north brooded Adults can also Moth Group overwinter and fly April to June Yellow- Common Oak Quercus Oak woodland Throughout much of Larvae feed July to Larvae form Robinson et al., alchimiella triangle British Isles October. Adults fly mines then 2010; Kimber, Slender May to July. Single- subsequently 2018; Norfolk brooded feed in leaf Moths; Suffolk folds. Moth Group; Identification Hants Moths requires examination of genitalia Caloptilia robustella New Oak Common Oak, beech, Quercus Woodland, Throughout Britain Larvae feed May to Larvae are leaf Robinson et al., Slender sweet chestnut heathland and July and September miners. 2010; Kimber, scrub to October. Adult Identification 2018; Norfolk moths fly mid-April to requires Moths; Suffolk June and mid-July to examination of Moth Group; early-September. genitalia Hants Moths Double-brooded Phyllonorycter Scarce pRDB2 Oak Quercus Old East Kent, Larvae are leaf Robinson et al., distentella midget established Gloucestershire, miners 2010; Pitkin et oak Herefordshire, al., 2018; Hants woodlands Monmouthshire Moths; Davis 2012 Phyllonorycter White Oak Common Oak Quercus Deciduous Throughout much of Larvae feed June to Leaf miner Robinson et al., harrisella Midget woodland, Britain July and September 2010; Kimber, scrub, to October. Adults fly 2018; Norfolk hedgerows May to June and Moths; Suffolk again late-July to Moth Group; August. Double- Hants Moths brooded Phyllonorycter Pale Oak Common Oak Quercus Oak woodland Throughout England, Larvae feed July and Leaf miner Robinson et al., heegeriella Midget and areas Wales and Scotland September to 2010; Kimber, with scattered except in far north October. Adults fly 2018; Norfolk oak May and August. Moths; Suffolk Double-brooded Moth Group; Hants Moths

45 Impact of OPM control methods on oak tree biodiversity | March 2018 Phyllonorycter Scarce Oak pRDB3 Deciduous oak Quercus Woodland Breeding in Norfolk Larvae feed in July Leaf miner in Robinson et al., kuhlweiniella Midget where fairly common and September to the higher 2010; Kimber, (hortella) but very localised. November. Adults fly branches 2018; Sussex (saportella) Found in Bedfordshire in May and again in Moth Group; in 2014. late July to August. Norfolk Moths; Double-brooded Suffolk Moth Group; Hants Moths Phyllonorycter Fiery Oak Local Deciduous oak Quercus Woodland Throughout much of Larvae feed July and Leaf miner. Robinson et al., lautella Midget and areas British Isles September to Larvae show a 2010; Kimber, with scattered October. Adults fly preference for 2018; Norfolk oaks May and again in young trees and Moths; Suffolk August. Double- saplings Moth Group; brooded Hants Moths Phyllonorycter Garden Common Evergreen and Quercus, Woodland, Throughout much of Adults fly April- Leaf miner Robinson et al., messaniella Midget deciduous oak, including scrub, the British Isles, more November. More or 2010; Kimber, beech, sweet Q. ilex hedgerows so in the south less continuously 2018; Norfolk chestnut, and fens brooded, with larvae Moths; Suffolk hornbeam, throughout the year Moth Group; birch, plum, Hants Moths apple Phyllonorycter Western Nationally Oak Quercus Ancient oak Distributed mainly in Larvae mine during Leaf miner Robinson et al., muelleriella Midget Scarce B woodland the south Midlands July and again 2010; Kimber, and Welsh borders, September to 2018; Hants into northern England October. Adults fly Moths; Davis, and parts of Scotland May and again in 2012 August. Double- brooded Phyllonorycter Common Common Deciduous oak Quercus Oak woodland Throughout most of Larvae feed July and Leaf miner Robinson et al., quercifoliella Oak Midget and areas the British Isles September to 2010; Kimber, with scattered except the Northern October. Adults fly 2018; Norfolk oaks Isles April to May and Moths; Suffolk again in August to Moth Group; September. Double- Hants Moths brooded Phyllonorycter Gold-bent Nationally Deciduous oak Quercus Mature oak Distributed Adults fly in June. Leaf miner Robinson et al., roboris Midget Scarce B woodland throughout England Single-brooded 2010; Kimber, and eastern Wales 2018; Norfolk Moths; Suffolk

46 Impact of OPM control methods on oak tree biodiversity | March 2018 Moth Group; Hants Moths Povolnya Sulphur Nationally Oak, also Quercus, Oak woodland Distribute throughout Larvae feed at some Leaf miner, Parsons and leucapennella Slender scarce B evergreen oak including much of Britain, less point during April to then creates Hill, 2016; Q. ilex common in the south June. Adults fly July to fold at edge of Kimber, 2018 and east October. Single- leaf, later rolling brooded the leaf tip sericiella Oak Satin Local Oak Quercus Woodland, Throughout much of Adults fly mid-April to Twig and leaf Robinson et al., Lift heathland, Britain May. Single-brooded miner. 2010; Kimber, hedgerows, Descends to the 2018; Norfolk scrub, where ground to Moths; Suffolk foodplant pupate Moth Group; occurs Hants Moths Hepialus humuli Ghost Moth Common Grasses and Quercus Rough Throughout much of Larvae appear to take Larvae feed Robinson et al., small meadows, Britain more than a year to underground on 2010; Kimber, herbaceous grasslands, complete roots 2018; Norfolk plants e.g. fens, development. Adults Moths; Suffolk stinging nettle woodland fly June to early- Moth Group; rides, August Hants Moths heathland and gardens Lasiocampidae Malacosoma The Lackey Common Hawthorn, Quercus Woodland, Mainly in the Larvae feed April to Larvae feed Glare and neustria blackthorn and gardens, southern half of June. Adults fly June gregariously O’Callaghan, other scrub and Britain to August. Single- within a silk 2000; Robinson broadleaved heathland brooded web; et al., 2010; trees and susceptible to Kimber, 2018; shrubs (locally DiPel® Norfolk Moths; on oak) Suffolk Moth Group; Hants Moths Limacodidae Apoda limacodes The Festoon Nationally Oak (possibly Quercus Mature Restricted to Larvae feed July to Larvae diapause Robinson et al., Scarce B only (possibly broadleaved southern counties, October. Adults fly over winter 2010; Kimber, pedunculate only Q. woodland, mainly from Dorset to June to July. Single- 2018; Norfolk oak) and beech robur) hedges and Kent brooded Moths; Suffolk heathland Moth Group; with mature Hants Moths oaks

47 Impact of OPM control methods on oak tree biodiversity | March 2018 Lycaenidae Neozephyrus Purple Common Oak Quercus Primarily oak Throughout southern Larvae feed mid- Robinson et al., quercus (Quercusia/ Hairstreak woodland but England and Wales March to mid-June. 2010; Parsons Favonius quercus) anywhere Adults fly late-June to and Hill, 2016; that oak August. Single- UK Butterflies; occurs brooded Hants Moths including lanes, parks and other urban areas Lymantriidae Lymantria monacha Black Local Pedunculate Q. robur Mature Mainly throughout Larvae feed April to Susceptible to Glare and Arches and sessile oak, and Q. woodland, southern England and June. DiPel® O’Callaghan, sometimes on petraea heathland and Wales Adults fly July to 2000; Robinson other deciduous scrub early-September. et al., 2010; and coniferous Single-brooded Kimber, 2018; tress Norfolk Moths; Suffolk Moth Group; Hants Moths Orgyia antiqua The Common Broadleaved Quercus Open Throughout British Larvae feed May to Freed and Vapourer trees and woodland, Isles July. Adults fly July to Reeve, 2014; shrubs including heathland, October. Single- Norfolk Moths, birch, hazel, moorland, brooded Suffolk Moth sallow, hedgerows, Group; Hants hawthorn, fens, scrub, Moths blackthorn, elm, urban gardens lime and oak and parks Orgyia recens Scarce RDB3 Range of Q. robur Heathland, Confined to a few Larvae feed August to Larvae diapause Robinson et al., Vapourer deciduous trees and Q. damp locations in the north- September and April over winter. 2010; Kimber, such as petraea woodland, east of England to May. Partial second 2018; Norfolk pedunculate fens and bogs Adults fly June to July. brood flies Moths; Suffolk and sessile oak, Single-brooded August to Moth Group; hawthorn, October. Hants Moths; sallow, birch, Females are Butterfly blackthorn, wingless COnservation alder buckthorn, hazel and also rose, bramble, heather,

48 Impact of OPM control methods on oak tree biodiversity | March 2018 common sorrel, rosebay willowherb and meadowsweet White- Common Oak Quercus Wherever Distributed Larvae mine during Leaf miner Robinson et al., albifasciella banded foodplant throughout England August to October. 2010; Kimber, Pigmy occurs, and Wales and into Adults fly in June. 2018; Norfolk woodland, southern half of Single-brooded Moths; Suffolk gardens, Scotland. Moth Group; orchard and Hants Moths parks Ectoedemia Oak-bark Unknown Bark of oak Quercus Woodland Occurs in south-east Adults fly June to Larvae mine the Robinson et al., atrifrontella Pigmy agg. and southern England September bark, preferring 2010; Kimber, /longicaudella younger 2018; Hants branches. Moths Requires examination of genitalia for accurate identification Ectoedemia heringi White-spot Local Oak Quercus Woodland, South-east England Larvae leaf mine Mines can be Robinson et al., Pigmy orchards, and parts of Wales October to located in fallen 2010; Kimber, gardens and December. leaves. Requires 2018; Norfolk parks Adults fly in July examination of Moths; Suffolk genitalia for Moth Group; accurate Hants Moths identification Ectoedemia Five-spot Local Oak Quercus, Oak woodland Mostly in south-east Larvae leaf mine Mines can be Robinson et al., quinquella Pigmy especially England September to located in fallen 2010; Kimber, Q. robur November. leaves 2018; Norfolk Adults fly June to July. Moths; Suffolk Single-brooded Moth Group; Hants Moths Ectoedemia Spotted Common Pedunculate Q. robur Wherever the South-east England, Larvae leaf mine Leaf miner Robinson et al., subbimaculella Black Pigmy and sessile oak and Q. foodplant up to Yorkshire October to 2010; Kimber, petraea occurs, November. Adults fly 2018; Norfolk woodland, June to July. Single- Moths; Suffolk gardens and brooded Moth Group; parklands Hants Moths

49 Impact of OPM control methods on oak tree biodiversity | March 2018 Stigmella Black- Common Pedunculate, Q. robur, Wherever the Throughout British Larvae feed June to Leaf miner Robinson et al., atricapitella headed sessile and Q. foodplant Isles July and September 2010; Pitkin et Pigmy Turkey oak petraea occurs to November. Adults al., 2018; and Q. fly May to June and Norfolk Moths; cerris August to September. Suffolk Moth Double-brooded Group; Hants Moths Stigmella Base- Common Oak Quercus Wherever the South-eastern Larvae present June Leaf miner Robinson et al., basiguttella spotted foodplant England, up to to July and 2010; Pitkin et Pigmy occurs, Yorkshire September to al., 2018; woodland, October. Double- Norfolk Moths; gardens, brooded Suffolk Moth orchards and Group; Hants parks Moths Stigmella roborella Common Common Pedunculate Quercus Woodland, Widespread across Larvae mine June to Leaf miner. Robinson et al., Oak Pigmy oak and other gardens and Britain July and October to Identification 2010; Kimber, species of parkland November. requires 2018; Pitkin et deciduous oak Adults fly May to June examination of al., 2018; and August to genitalia Norfolk Moths; September. Double- Suffolk Moth brooded Group; Hants Moths Stigmella Red-headed Common Oak Quercus Wherever the Widespread across Larvae feed June and Leaf miner. Robinson et al., ruficapitella Pigmy foodplant Britain August to September. Eggs laid on the 2010; Kimber, occurs Adults fly May to June upper leaf 2018; Pitkin et and again July to surface al., 2018; August. Double- Norfolk Moths; brooded Suffolk Moth Group; Hants Moths Stigmella samiatella Chestnut Local Oak and sweet Quercus Wherever the Widespread in Larvae mine June to Leaf miner. Robinson et al., Pigmy chestnut foodplant England July and again Bivoltine 2010; Kimber, occurs September to 2018; Pitkin et October. Adults fly al., 2018; May to June and Norfolk Moths; again July to August. Suffolk Moth Double-brooded Group; Hants Moths

50 Impact of OPM control methods on oak tree biodiversity | March 2018 Stigmella Holm-oak Local Evergreen oak Q. ilex Mainly coastal Mainly in southern Larvae mine October Leaf miner. Robinson et al., suberivora Pigmy wherever England and Wales to March and again Eggs are laid on 2010; Kimber, foodplant is June to July. Adults fly upper leaf 2018; Pitkin et found, May and September. surface. al., 2018; gardens, Double-brooded Bivoltine Norfolk Moths; orchard and Suffolk Moth parks Group; Hants Moths Stigmella svenssoni Orange- Local Oak Quercus Woodland Widespread across Larvae mine July to Leaf miner. Robinson et al., headed Britain August. Identification 2010; Kimber, Pigmy Adults fly May to requires 2018; Pitkin et June. Single-brooded examination of al., 2018; NBN genitalia Atlas; Norfolk Moths; Suffolk Moth Group; Hants Moths Noctuidae auricoma Scarce Extinct as a Range of food Q. robur Formerly resident in Larvae feed June and Robinson et al., Dagger breeding plants including and Q. south-east of September. Adults fly 2010; Kimber, species; pedunculate petraea England. May to early-June 2018; Norfolk occasional and sessile oak and again mid-July to Moths; Suffolk immigrant and bilberry August. Double- Moth Group; brooded Hants Moths Acronicta leporina The Miller Local Mainly birch Quercus Broadleaved Across much of Larvae feed July to Robinson et al., and alder but woodland, Britain but more September. 2010; Kimber, on other heathland, common in the south Adults fly late-May to 2018; Norfolk deciduous trees moorland, mid-August. Single- Moths; Suffolk as well, fens, scrub, brooded Moth Group; including oak urban areas Hants Moths Amphipyra berbera Svensson’s Common Pedunculate Q. robur Woodland, Throughout much of Larvae feed April to Robinson et al., Copper oak, lime, scrub, England, Wales and May. Adults fly mid- 2010; Kimber, Underwing hornbeam, gardens, southern Scotland, July to September. 2018; Norfolk willow hedgerows more frequent in the Single-brooded Moths; Suffolk and parks north Moth Group; Hants Moths Amphipyra Copper Common Range of trees Quercus Woodland, Throughout much of Larvae feed April to Robinson et al., pyramidea Underwing and shrubs, scrub, fens, England, Wales and May. 2010; Kimber, mainly oak, gardens, southern Scotland, Adults fly mid-July to 2018; Norfolk hawthorn, parks, fairly common over mid-October. Single- Moths; Suffolk blackthorn, crab brooded

51 Impact of OPM control methods on oak tree biodiversity | March 2018 apple, hazel, heathland and southern half of Moth Group; honeysuckle hedgerows Britain Hants Moths Brachionycha The Common Range of Q. robur Broadleaved Distributed Larvae feed May to Robinson et al., (Asteroscopus) Sprawler deciduous woodland, throughout England June. Adults fly late- 2010; Kimber, sphinx trees, gardens and October to 2018; Norfolk pedunculate areas with November. Single- Moths; Suffolk oak, hazel, field scattered brooded Moth Group; maple, trees Hants Moths hawthorn, blackthorn, elm, lime, willow The Immigrant Evergreen oak Q. ilex Southern England Adults fly May to July Sporadic Robinson et al., Alchemist and again in migrant to the 2010; Kimber, September. Limited British Isles. 2018; Hants evidence of breeding. Moths Double-brooded Catocala promissa Light RDB Pedunculate Q. robur, Mature oak New Forest and south Larvae feed April to Robinson et al., Crimson oak, possibly possibly woodland Wiltshire May. Adults fly mid- 2010; Kimber, Underwing sessile oak Q. July to early- 2018; Norfolk petraea September. Single- Moths; Suffolk brooded Moth Group; Hants Moths Catocala sponsa Dark RDB3 Pedunculate Q. robur Large mature Breeds only in the Larvae feed April to Robinson et al., Crimson and sessile oak and Q. oak woods New Forest and June. Adults fly late- 2010; Kimber, Underwing petraea Hampshire, otherwise July to early- 2018; Norfolk occasionally recorded September. Single- Moths; Suffolk as a migrant brooded Moth Group; Hants Moths White- Various Quercus Broadleaved Scattered localities Larvae feed May to Larvae have not Robinson et al., leucographa marked herbaceous and woodland, throughout England June. Adults fly March definitely been 2010; Kimber, woody plants mature and Wales to April. Single- found in Britain 2018; Suffolk hedgerows brooded Moth Group; and scrub Hants Moths Conistra vaccinii The Common Oak, elm, Quercus Broadleaved Throughout the Larvae feed May to Freed and Chestnut blackthorn, woodland, British Isles June. Adults fly mid- Reeve, 2014; hawthorn, scrub, September to mid- Norfolk Moths; downy birch heathland, May. Larvae diapause Suffolk Moth and herbaceous hedgerows during July. Single- Group; Hants and gardens brooded Moths

52 Impact of OPM control methods on oak tree biodiversity | March 2018 plants such as dock Cosmia trapezina The Dun-bar Common Various Quercus Woodland, Throughout the Larvae feed April to Larvae eat the Freed and broadleaved heathland, British Isles May. Adults fly mid- larvae of other Reeve, 2014; trees and scrub, fens, June to mid- moth species Norfolk Moths; shrubs including grassland with September. Single- especially Suffolk Moth hawthorn, elm, scrub brooded Winter Moth Group; Hants sallow, larvae Moths blackthorn, oak, hazel, field maple Griposia (Dichonia) Merveille Common Pedunculate Q. robur, Broadleaved Widespread but Larvae feed March to Larvae feed on Robinson et al., aprilina du jour oak, and possibly woodland, thinly scattered over June. Adults fly mid- the buds and 2010; Kimber, possibly sessile Q. parkland, most of Britain September to early- flowers, and 2018; Norfolk and Turkey oak petraea hedgerows November. Single- later on the Moths; Suffolk and Q. and gardens brooded leaves Moth Group; cerris Hants Moths Dicycla oo Heart Moth Section 41; Pedunculate Q. robur Woodland; Very local species Larvae feed April to Larvae feed Robinson et al., Rare; RDB3 oak parkland and around the home June. Adults fly late- during the night 2010; Parsons suburban counties June to mid-July. on young and Hill. 2016; habitats Single-brooded leaves, hide Kimber, 2018; (farmland in during the day Suffolk Moth Surrey) in spun Group; Sussex together leaves Moth Group Dryobotodes Brindled Common Pedunculate Q. robur Broadleaved Throughout the Larvae feed March to Larvae feed Robinson et al., eremita Green and sessile oak and Q. woodland, British Isles June. Adults fly initially on buds 2010; Kimber, petraea parkland, August to September. and later on 2018; Norfolk scrub and Single-brooded leaves Moths; Suffolk gardens Moth Group; Hants Moths Herminia grisealis Small Fan- Common Range of Q. robur Broadleaved Throughout most of Larvae feed August to Larvae often Robinson et al., foot deciduous trees woodland, British Isles October. Adults fly feed on 2010; Kimber, including scrub, fens, late-May to mid- withered and 2018; Norfolk pedunculate heathland and August. Single- fallen leaves Moths; Suffolk oak, birch, gardens brooded Moth Group; hazel, Hants Moths hawthorn, sallow, alder, bird cherry,

53 Impact of OPM control methods on oak tree biodiversity | March 2018 bramble, Traveller’s joy croceago Orange RDB1 Pedunculate Q. robur Oak Not been seen in Adults fly October- Adults Robinson et al., Upperwing and sessile oak and Q. woodland, Britain for a number November and again overwinter 2010; Kimber, petraea hedgerows of years March-May. Single- 2018; Hants and areas brooded Moths; Sussex with scattered Moth Group trees Lithophane Grey Common Pedunculate Q. robur, Broadleaved Southern parts of Larvae feed May to Larvae are Robinson et al., ornitopus Shoulder- oak, possibly possibly woodland and England and Wales July. Adults fly mid- cannibalistic. 2010; Kimber, knot sessile and Q. parkland September to early Adults 2018; Norfolk Turkey oak petraea May. Single-brooded overwinter Moths; Suffolk and Q. Moth Group; cerris Hants Moths Lithophane Tawny Local Mainly ash Q. robur Open Southern parts of Larvae feed May to Adults Robinson et al., semibrunnea Pinion broadleaved England and Wales, June, larval diapause overwinter 2010; Kimber, woodland, occasionally reaching in July. Adults fly 2018; Norfolk parks, northern counties October to May. Moths; Suffolk gardens and Single-brooded Moth Group; marshy places Hants Moths Minucia lunaris Luna Rare Pedunculate Q. robur Woodland South-eastern Larvae feed July to Robinson et al., Double- immigrant oak England. Temporarily August. Adults fly 2010; Kimber, stripe established in Kent mid-May to June. 2018; Norfolk and East Sussex Single-brooded Moths; Suffolk (1940s/1950s) Moth Group; Hants Moths Moma alpium Scarce RDB Pedunculate Q. robur Mature Very south and south- Larvae feed July to Early instar Robinson et al., Merveille oak and and deciduous east of England September. Adults fly larvae are 2010; Kimber, du Jour probably sessile probably (oak) early-June to mid- gregarious 2018; Norfolk oak Q. woodland July. Single-brooded feeders Moths; Suffolk petraea Moth Group Orthosia cerasi Common Common Oak, sallow, Quercus Lowland Widespread Larvae feed May to Robinson et al., Quaker hazel, birch, elm habitats throughout Britain, June. Adults fly mid- 2010; Kimber, and hawthorn including except in the far February to May. 2018; Norfolk woodland and north where it is Single-brooded Moths; Suffolk gardens scarcer Moth Group Orthosia cruda Small Common Oak, Willow, Quercus Deciduous Throughout British Larvae feed May to Robinson et al., Quaker birch, sallow, woodland, Isles June. Adults fly mid- 2010; Kimber, 2018; Norfolk

54 Impact of OPM control methods on oak tree biodiversity | March 2018 hazel, field wooded areas February to mid-May. Moths; Suffolk maple and heathland Single-brooded Moth Group; Hants Moths Orthosia gothica Hebrew Common Many Quercus Any habitat Throughout the Larvae feed May to Freed and Character deciduous trees British Isles June. Adults fly mid- Reeve, 2014; and shrubs February to mid-June. Norfolk Moths; including oak, Single-brooded. Suffolk Moth birch, Group; Hants hawthorn, Moths sallow, lime and bilberry. Also, herbaceous plants such as stinging nettle and meadowsweet Orthosia incerta Clouded Common Broadleaved Quercus Broadleaved Throughout the Larvae feed May to Robinson et al., Drab trees and woodland, British Isles June. Adults fly late- 2010; Kimber, shrubs, scrub, February to May. 2018; Norfolk including birch, heathland, Single-brooded Moths; Suffolk sallow, hazel, gardens Moth Group; hawthorn, elm, Hants Moths lime and oak Orthosia miniosa Blossom Local, Pedunculate Q. robur Mature oak Southern England and Larvae feed May to Young larvae Robinson et al., Underwing immigrant and sessile oak, and Q. woodland and Wales, less frequent June. Adults fly March feed 2010; Kimber, and later on low petraea mature in north-west England to April. Single- gregariously 2018; Norfolk herbaceous hedgerows brooded within a silken Moths; Suffolk plants web. Separate Moth Group; as they get Hants Moths older and then may also feed on low plants Anorthoa (Orthosia) Twin- Common Broadleaved Q. robur Broadleaved Throughout England Larvae feed May to Robinson et al., munda spotted trees including and Q. woodland and and Wales June. Adults fly March 2010; Norfolk Quaker pedunculate petraea scrub to early-May. Single- Moths; Suffolk and sessile oak, brooded Moth Group; birch, sallow, Hants Moths aspen, field maple, ash,

55 Impact of OPM control methods on oak tree biodiversity | March 2018 honeysuckle and hop Paracolax tristalis Clay Fan- Nationally Pedunculate Q. robur Broadleaved South-eastern Larvae feed April to Larvae diapause Robinson et al., foot Scarce A oak, probably woodland England June. Adults fly late- over winter. 2010; Suffolk other June to early-August. Larvae reported Moth Group; broadleaved Single-brooded to prefer moist Hants Moths trees and fallen oak shrubs leaves, completing growth on herbaceous plants Pechipogo strigilata Common Section 41; Pedunculate Q. robur Open ancient Parts of southern Larvae feed July to Larvae feed on Robinson et al., Fan-foot Nationally oak woodland England September. Adults fly withered leaves 2010; Parsons Scarce A late-May to early- and Hill, 2016; July. Single-brooded Kimber, 2018); Norfolk Moths; Suffolk Moth Group; Hants Moths Trisateles Olive RDB3 Beech, oak and Quercus Mature In the east Larvae feed August to Larvae feed on Robinson et al., emortualis Crescent hornbeam woodland October. Adults fly withered leaves 2010; Sussex mid-June to early- Moth Group; August. Single- Suffolk Moth brooded Group bicolorana Scarce Local Pedunculate Q. robur, Broadleaved Throughout much of Larvae feed Freed and Silver-lines oak and possibly woodland, England and Wales September and again Reeve, 2014; probably sessile Q. areas with April to May. Adults Norfolk Moths; oak, silver and petraea scrub oak fly June to early Suffolk Moth downy birch August. Larvae Group; Hants diapause October Moths through to March. Single-brooded Pseudoips Green Common Broadleaved Quercus Woodland, Widespread Larvae feed August to Robinson et al., prasinana Silver-lines trees including heathland, September. Adults fly 2010; Norfolk oak, birch, scrub, May to July. Single- Moths; Suffolk beech, hazel, grassland and brooded Moth Group sweet chestnut, gardens aspen and elm

56 Impact of OPM control methods on oak tree biodiversity | March 2018 Meganola strigula Small Black Nationally Pedunculate Q. robur Mature oak Local in southern Larvae feed Larvae diapause Robinson et al., Arches Scarce A oak woodlands England September and May. over winter 2010; Norfolk Adults fly late June to Moths; Suffolk July. Single-brooded Moth Group Nola confusalis Least Black Local Various Quercus Woodland, Widespread Larvae feed July to Robinson et al., Arches broadleaved parks, and throughout Britain August. Adults May to 2010; Norfolk trees including other well- June. Single-brooded Moths; Suffolk lime, oak, wooded Moth Group; downy birch, areas, Hants Moths blackthorn, heathland, buckthorn scrub and gardens Nycteola revayana Oak Local Pedunculate Q. robur, Broadleaved Widespread Larvae feed June to Formerly known Robinson et al., Nycteoline oak, possibly possibly woodland, throughout British July. Adults fly late- as Large 2010; Norfolk sessile oak Q. areas with Isles March to early- Marbled Tortrix Moths; Suffolk petraea scrub oak and August and mid- Moth Group; gardens August to mid- Hants Moths October. Double- brooded Notodontidae Drymonia Marbled Local Pedunculate Q. robur Mature oak Much of England, Larvae feed July to Robinson et al., dodonaea Brown oak and sessile and Q. woodland, Wales and parts of September. Adults fly 2010; Norfolk oak petraea heathland, Scotland May to early-August. Moths; Suffolk grassland with Single-brooded Moth Group; scrub oaks Hants Moths Drymonia ruficornis Lunar Common Pedunculate Q. robur Woodland, Throughout the Larvae feed July to Susceptible to Glare and Marbled oak and sessile and Q. heathland British Isles August. Adults fly DiPel® O’Callaghan, Brown oak petraea with scrub April to May. Single- 2000; Robinson oaks, brooded et al., 2010; grassland Norfolk Moths; Suffolk Moth Group; Hants Moths Peridea anceps Great Local Pedunculate Q. robur Mature oak Throughout England Larvae feed June to Robinson et al., Prominent oak and sessile and Q. woodland, and Wales August. Adults fly 2010; Norfolk oak petraea broadleaved April to mid-June. Moths; Suffolk woodland, Single-brooded Moth Group; heathland Hants Moths with scrub oaks

57 Impact of OPM control methods on oak tree biodiversity | March 2018 Phalera bucephala Buff-tip Common Broadleaved Quercus Heathland, Widespread Larvae feed July to Robinson et al., trees including open throughout British September. Adults fly 2010; Norfolk birch, sallow, woodland, Isles May to early-August. Moths; Suffolk oak and hazel scrub, Single-brooded Moth Group; hedgerows Hants Moths and gardens Phalera raya Q. serrata Robinson et al., 2010 Stauropus fagi Lobster Common Birch, alder, Quercus Mature Throughout much of Larvae feed July to Robinson et al., Moth oak, beech woodland southern England August. Adults fly 2010; Norfolk May to mid-July. Moths; Suffolk Single-brooded Moth Group; Hants Moths quercana Long- Common Various Quercus Mature oak Throughout the Larvae feed April to Larvae diapause Robinson et al., horned Flat- deciduous trees and beech British Isles June and September over winter 2010; Freed and body woodland, to October. Adults fly Reeve, 2014; heathland, July to August. Single- Norfolk Moths; scrub, brooded. Suffolk Moth hedgerows Group; Hants and suburban Moths gardens fagella March Tubic Common Oak, beech, Quercus Deciduous Throughout much of Larvae feed June to Larvae feed Freed and hornbeam, (oak or birch) the British Isles, not in October. Adults fly between leaves Reeve, 2014; birch, willow, woodland, the Scottish Highland March to May. Single- spun together Norfolk Moths; blackthorn, scrub, or islands of Scotland brooded with silk Suffolk Moth aspen, hazel hedgerows Group; Hants Moths Diurnea lipsiella (D. November Local Oak, especially Quercus, Oak woodland Throughout much of Larvae feed June to Robinson et al., phryganella) Tubic sessile oak, especially British Isles but not in July. Adults fly 2010; Norfolk bilberry, aspen, Q. the Highlands and October to early- Moths; Suffolk small-leaved petraea Islands of Scotland November. Single- Moth Group; lime brooded Hants Moths Incurvariidae Incurvaria Common Common Bilberry, Q. Heathland, Throughout much of Larvae August to Larvae mine Robinson et al., oehlmanniella Leaf-cutter cloudberry petraea/Q moorland, British Isles April. Adults fly June bilberry, living 2010; Norfolk dogwood, .robur damp to July. Single- in moveable Moths; Suffolk blackthorn woodland brooded case, Moth Group; subsequently Hants Moths feeds on fallen leaves

58 Impact of OPM control methods on oak tree biodiversity | March 2018 Psychidae Sterrhopterix fusca Endanger- Grasses, oak, Quercus Parts of England and Larvae feed in Kimber, 2018; ed hawthorn, birch Wales movable case, Hants Moths; and heather overwintering NHK twice Pyralidae Broad- Local Oak Quercus Woodland, Throughout most of Larvae feed Larvae feed in Robinson et al., consociella barred heathland, British Isles but not in September to spun leaves. 2010; Norfolk Knot-horn scrub and northern Scotland October and May to Larvae diapause Moths; Suffolk gardens June. Adults fly late- over winter Moth Group; June to mid-August. Hants Moths Single-brooded Acrobasis Warted Common Oak Quercus Woodland Throughout England Larvae feed May to Larvae feed in Robinson et al., repandana Knot-horn and scrub June. Adults fly mid- spun leaves 2010; Norfolk (Conobathra June to mid-August Moths; Suffolk repandana) Moth Group; Hants Moths Acrobasis sodalella Oak Quercus Adults fly June to July Robinson et al., 2010; Wikipedia Acrobasis tumidana Oak Quercus Adults fly July to Larvae feed in Robinson et al., early-September spun leaves. 2010. Kimber, Probable 2018 immigrant. Larvae rarely, if ever seen in Britain Cryptoblabes Double- Local Various Quercus Oak England and Wales Larvae feed August to Robinson et al., bistriga striped deciduous trees woodland, October. Adults fly 2010; Norfolk Knot-horn including oak heathland, May to June and mid- Moths; Suffolk and alder grassland and July to August. Moth Group; fens Double-brooded Hants Moths similella Nationally Pedunculate Q. robur Oak Home counties of Larvae feed July to Larvae feed in Robinson et al., Scarce B oak woodland, south-east England August. Adults fly spun leaves 2010; Kimber, parkland and June to July. Single- 2018; Hants some gardens brooded Moths; Suffolk (areas with Moth Group mature oak trees) Nephopterix Q. Robinson et al., tumidella petraea/Q 2010 .robur

59 Impact of OPM control methods on oak tree biodiversity | March 2018 Phycita roborella Dotted Oak Common Oak, pear and Quercus Woodland, Throughout much of Larvae feed Larvae feed in Robinson et al., Knot-horn crab apple heathland, England September to spun leaves 2010; Freed and gardens, fens October and April to Reeve, 2014; and grassland May. Adults fly late Norfolk Moths, June to mid-August. Suffolk Moth Single-brooded Group; Hants Moths Sesiidae Synanthedon Yellow- Nationally Pedunculate Q. robur Open Central southern Larvae feed August to Larvae feed Robinson et al., vespiformis legged Scarce B oak, possibly woodland, England, northwards May. Adults fly May internally in the 2010; Parsons Clearwing other oaks, parks and to Yorkshire to mid-August. Single- bark and Hill, 2016; sweet chestnut, hedgrows, brooded Norfolk Moths, birch and elm fens and Suffolk Moth Marshes Group; Hants Moths Sphingidae Mimas tiliae Lime Hawk- Common Limes, , Quercus Broadleaved Throughout much of Larvae feed July to Robinson et al., moth and woodland and southern England, August. Adults fly 2010; Norfolk alder suburban north to Yorkshire May to early-July. Moths, Suffolk habitats, Single-brooded Moth Group; gardens, Hants Moths heathland Tischeriidae Tischeria dodonaea Small Carl Local Oak, sweet Quercus Deciduous Throughout much of Larvae feed Leaf miner. Robinson et al., chestnut woodland Britain, north to Lake September to Larvae diapause 2010; Norfolk District October over winter Moths, Suffolk Moth Group; Hants Moths Tischeria Oak Carl Common Oak, sweet Quercus Woodland, Throughout the Larvae feed Leaf miner. Robinson et al., ekebladella chestnut where British Isles September to Larvae diapause 2010; Norfolk foodplant October over winter Moths, Suffolk occurs Moth Group; Hants Moths Tortricidae ferrugana Rusty Oak Common Oak, sallow Quercus Woodland, Throughout much of Larvae feed May to Larvae live Robinson et al., Button heathland the British Isles June and August. between spun 2010; Norfolk Adults fly mid- leaves. Need to Moths, Suffolk October to mid-April identify by Moth Group; and July to August. examination of Hants Moths Double-brooded genitalia Acleris literana Lichen Local Oak Quercus Woodland Throughout much of Larvae feed May to Larvae live Robinson et al., Button Britain June. Adults fly May between spun 2010; Norfolk leaves Moths, Suffolk

60 Impact of OPM control methods on oak tree biodiversity | March 2018 to August. Single- Moth Group; brooded Hants Moths Yellow Oak Common Oak, hornbeam, Quercus Woodland, Throughout British Larvae feed May. Larvae live in Robinson et al., loeflingiana Button maple parkland, Isles Adults fly June to July. spun/rolled leaf 2010; Norfolk scrub and Single-brooded. Moths, Suffolk gardens Moth Group; Hants Moths Red Roller Common Oak and beech Quercus Deciduous Throughout British Larvae feed July to Larvae live Robinson et al., mitterbacheriana woodland, Isles September. Adults fly within spun or 2010; Norfolk heathland mid-May to June. rolled leaf. Moths, Suffolk with scrub Single-brooded Larvae diapause Moth Group; over winter Hants Moths crataegana Brown Oak Local Oak, elm, ash, Quercus Woodland Throughout much of Larvae feed April to Larvae live Robinson et al., Tortrix sallow, lime, rides, forests England and Wales May. Adults fly mid- within spun or 2010; Norfolk willow and orchards June to July. Single- rolled leaf. Moths, Suffolk brooded Moth Group; Hants Moths Archips xylosteana Variegated Common Variety of Quercus Woodland, Throughout British Larvae feed April to Larvae live Freed and Golden deciduous trees scrub and Isles June. Adults fly mid- within spun or Reeve, 2014; Tortrix including oak, gardens June to July. Single- rolled leaf. Norfolk Moths, elm, lime, hazel, brooded Suffolk Moth field maple. Group; Hants Sycamore and Moths ash splendana Marbled Common Oak, sweet Quercus Woodland, Throughout Britain, Larvae feed August to Larvae feed on Robinson et al., Piercer chestnut, heathland more frequent in October. Adults fly the acorn, and 2010; Norfolk walnut with oak south-east mid-July to fruit of sweet Moths, Suffolk scrub, fens September. Single- chestnut Moth Group; and gardens brooded Hants Moths grotiana Brown- Common Oak, hawthorn Quercus Deciduous Throughout British Larvae feed August to Larvae diapause Robinson et al., barred and brambles woodland, Isles October and in May over winter 2010; Norfolk Tortrix heathland, Moths, Suffolk sand dunes Moth Group; and fens Hants Moths Apple Nationally Mostly on apple Quercus Woodland Southern Britain Adults fly July to Larvae feed Robinson et al., porphyrana Marble Scarce A August within a spun or 2010; Kimber, rolled leaf 2018; Hants Moths

61 Impact of OPM control methods on oak tree biodiversity | March 2018 Eudemis Diamond- Common Oak Quercus Oak woodland England and Wales Larvae feed in June. Larvae live in a Robinson et al., profundana back Marble and scrub Adults fly July to mid- spun or rolled 2010; Norfolk September. Single- leaf Moths, Suffolk brooded Moth Group; Hants Moths Common Common Various trees Quercus Open Throughout much of Larvae feed Larvae live in a Robinson et al., dealbana Cloaked and shrubs woodland, British Isles September and April spun or rolled 2010; Norfolk Shoot including hazel, hedgerows, to June. Adults fly leaf Moths, Suffolk hawthorn, scrub, fens late-June to August. Moth Group; poplar, sallow and heathland Single-brooded Hants Moths; Freed and Reeve, 2014 Lobesia reliquana Oak Marble Local Oak, birch and Quercus Open Throughout British Larvae feed July to Robinson et al., blackthorn woodland, Isles August. Adults fly 2010; Norfolk hedgerows, mid-May to June. Moths, Suffolk parks, Single-brooded Moth Group; orchards and Hants Moths gardens Pammene Blotched Local Oak Quercus Woodland Mainly south and east Larvae feed Larvae feed on Robinson et al., albuginana Piercer and heathland England September to the inside of 2010; Norfolk with scrub October. Adults fly galls made by Moths, Suffolk oaks June. Single-brooded other insects Moth Group; Hants Moths Pammene argyrana Black- Local Oak Quercus Oak Throughout British Larvae feed June to Larvae feed as Freed and bordered woodland, Isles September. Adults fly an inquiline in Reeve, 2014; Piercer heathland mid-April to May. young galls Norfolk Moths, with scrub Single-brooded Suffolk Moth oaks Group; Hants Moths Pammene fasciana Acorn Common Oak and sweet Quercus Woodland, Throughout much of Larvae feed August to Larvae diapause Robinson et al., Piercer chestnut scrub, England and Wales, October. Adults fly over winter. 2010; Norfolk parkland, especially in the June to mid-August. Larvae feed in Moths, Suffolk heathland, south Single-brooded the acorn Moth Group fens and gardens Pammene germana Black Local Possibly on wild Quercus Open Throughout much of Adults fly May to Larvae feed on Robinson et al., Piercer plum, oak, woodland and England but recently June. Single-brooded shoots 2010; Norfolk hawthorn and hedgerows seldom found north Moths, Suffolk blackthorn of the Midlands

62 Impact of OPM control methods on oak tree biodiversity | March 2018 Moth Group; Hants Moths Pammene Early oak Nationally Oak-apple galls Quercus Woodland Throughout much of Larvae feed June to Larvae are Robinson et al., giganteana (P. Piercer Scarce B and heaths Britain except August. Adults fly inquiline in oak 2010; Norfolk inquilina) northern Scotland April to early-May. galls Moths, Suffolk Single-brooded Moth Group; Hants Moths Pammene Drab Oak Nationally Deciduous oak Quercus Oak woods, Throughout much of Larvae feed June to Larvae feed Robinson et al., splendidulana Piercer Scarce B leaves mixed British Isles. Very July. Adults fly April to between two 2010; Freed and woodland and local in Surrey and early-June. Single- leaves flatly Reeve, 2014; commons Suffolk brooded spun together. Norfolk Moths, where oak is Pupates under Suffolk Moth plentiful bark, in rotten Group; Hants wood, old oak- Moths apple galls, sponge galls Pandemis cerasana Barred Common Broadleaved Quercus Woodland, Throughout British Larvae feed April to Larvae diapause Freed and Fruit-tree trees and fens, gardens, Isles May. Adults fly mid- over winter. Reeve, 2014; Tortrix shrubs including heathland and June to early August. Larvae live Norfolk Moths; maple, alder, scub Single-brooded within spun or Suffolk Moth hazel, oak, rolled leaf Group; Hants willow, bilberry, Moths lime, birch, elm, yellow loosestrife and rowan Pandemis corylana Chequered Common Broadleaved Quercus Woodland, Throughout British Larvae feed May to Larvae live Freed and Fruit-tree trees including heathland and Isles June. Adults fly late- within spun or Reeve, 2014; Tortrix hazel, ash, scrub July to mid- rolled leaf Norfolk Moths; cherries, September. Single- Suffolk Moth blackthorn, brooded Group; Hants bramble and Moths oak Ptycholoma Brindled Common Variety of trees Quercus Wooded Throughout much of Larvae feed August to Larvae live in a Robinson et al., lecheana Tortrix and shrubs areas, scrub British Isles September and April spun or rolled 2010; Norfolk and orchards to May. Adults fly leaf. Larvae Moths, Suffolk late-May to June. diapause over Moth Group; Single-brooded winter Hants Moths

63 Impact of OPM control methods on oak tree biodiversity | March 2018 Strophedra nitidana Little Oak Local Oak Quercus Oak woodland Throughout much of Larvae feed July to Larvae live in Robinson et al., Piercer England and Wales September. Adults fly between leaves 2010; Norfolk May to June. Single- spun together Moths, Suffolk brooded Moth Group; Hants Moths Syndemis Dark-barred Common Bramble, birch, Quercus Open Throughout the Larvae feed July to Larvae live in a Freed and musculana Tortrix oak, Japanese woodland, British Isles September. Adults fly spun or rolled Reeve, 2014; larch, shore heathland, mid-April to mid- leaf. Larvae Norfolk Moths, pine, spruce hedgerows, June. Single-brooded diapause over Suffolk Moth high winter Group; Hants moorland and Moths scrub Tortricodes Winter Common Oak, hornbeam, Quercus Open, Throughout Britain Larvae feed May to Larvae live in Robinson et al., alternella Shade birch, hazel, deciduous June. Adults fly mid- between leaves 2010; Norfolk hawthorn, woodland, January to March. spun together Moths, Suffolk blackthorn and especially oak Single-brooded Moth Group; lime woodland Hants Moths Tortrix viridana Green Oak Common Oak and other Quercus Oak woods, Throughout much of Larvae feed May to Susceptible to Glare and Tortrix deciduous trees open British Isles June. Adults fly June DiPel® O’Callaghan, woodland and to mid-July. Single- 2000; Robinson gardens brooded et al., 2010; Norfolk Moths, Suffolk Moth Group; Hants Moths Zeiraphera isertana Cock’s-head Common Oak Quercus Oak Throughout the Larvae feed May to Larvae live Robinson et al., Bell woodland, British Isles June. Adults fly late- within a spun or 2010; Norfolk heathland, May to August. rolled leaf Moths, Suffolk scrub and Single-brooded Moth Group; other areas Hants Moths where oak occurs Yponomeutidae Oak-bark Local Oak, horse Quercus Open Throughout Britain Larvae feed March to Leaf miner. Robinson et al., glaucinella Argent chestnut woodland April. Adults fly June Identification 2010; Norfolk to July. Single- requires Moths, Suffolk brooded dissection of Moth Group; genitalia Hants Moths

64 Impact of OPM control methods on oak tree biodiversity | March 2018 Ypsolopha alpella Barred Local Oak Quercus Oak woodland Throughout much of Larvae feed May to Larvae feed Robinson et al., Smudge and scrub southern England and June. Adults fly beneath a silken 2010; Norfolk southern Wales, August to September. web Moths, Suffolk north to Yorkshire Single-brooded Moth Group; Hants Moths Ypsolopha lucella Plain Nationally Oak Quercus Oak woodland South of England, Larvae feed May to Larvae feed Robinson et al., Smudge Scarce A northwards to June. Adults fly July to beneath a silken 2010; Norfolk Staffordshire and The August. Single- web. Males are Moths, Suffolk Wash brooded rarely seen. Moth Group; Thought to be Hants Moths largely partheno- genetic Ypsolopha White- Common Oak, hazel, Quercus Woodland, Throughout much of Larvae feed May to Robinson et al., parenthesella shouldered hawthorn, scrub, British Isles except June. Adults fly July to 2010; Norfolk Smudge hornbeam, heathland and the Outer Hebrides mid-October. Single- Moths, Suffolk birch fens and Shetland brooded Moth Group; Hants Moths Ypsolopha sylvella Wood Local Oak Quercus Woodland Throughout much of Larvae feed June. Larvae feed Robinson et al., Smudge and scrub southern England and Adults fly mid-July to beneath a silken 2010; Norfolk Wales early-October. Single- web. Moths, Suffolk brooded Moth Group; Hants Moths Ypsolopha ustella Variable Common Oak Quercus Woodland, Throughout the Larvae feed May to Larvae feed Robinson et al., Smudge heathland, British Isles June. Adults fly beneath a silken 2010; Norfolk grassland, August to April. web. Moths, Suffolk fens and Single-brooded Moth Group; scrub Hants Moths Table 4. Details of the lepidopteran species associated with oak trees in the British Isles. The list of species associated with oak was obtained from the Natural History Museum website using the “HOSTS – a database of the world’s lepidopteran hostplants” search function (Robinson et al., 2010) and Freed and Reeve (2014). A summary list of species with larvae that feed at some point between April and June can be found in Appendix 1. IUCN Red Data Book (RDB) categories can be found in Appendix 2.

65 Impact of OPM control methods on oak tree biodiversity | March 2018 Chapter 4. Evaluation of methods for monitoring oak tree invertebrate biodiversity

The aims of this chapter are to evaluate the methods for monitoring oak tree invertebrate biodiversity at different layers, specifically

 The canopy layer – sticky traps, destructive foliage sampling, beating and leaf miner sampling;  The ground layer – pit-fall traps and water traps.

And to assess the methods and ease of identification of potential target groups, including Lepidoptera.

The most common forms of data required to assess the impact of a pesticide on terrestrial invertebrates are quantifications of population levels, relative abundance and species composition in a treated area, which can be statistically compared with this same data collected from untreated areas (Tingle, 2002). This type of assessment needs considerable resources and time if it is to be done thoroughly and it is imperative to avoid collecting data that is impossible to analyse, and therefore interpret.

Tingle (2002) recommends a number of requirements that should be incorporated into any study designed to assess the impacts on non-target invertebrates, as follows:

1. A replicated experimental design should be used, and replication should be high enough for statistical analyses to be able to demonstrate effects that are over and above natural variation (variance between plots can be high, and this becomes problematic with low replicate numbers). 2. At least one month of pre-treatment data should be collected but ideally this phase should span a year. 3. The study design should take into account seasonal abundance patterns (a common trait in terrestrial invertebrates (Tingle, 2002; Grootaert, 2010) such that misleading comparisons for a given taxa are not made between different months. 4. The study should ideally be long-term (3-5 years) or repeated short-term (e.g. 2-3 months each year for 3-5 years) due to high year to year variability seen with terrestrial invertebrate species (Tingle, 2002; Grootaert, 2010). 5. Diurnal rhythms should be accounted for. Many invertebrates are strongly influenced by time of day and weather (Ausden, 1996). 6. Control (untreated) plots should always be included, and plots should be matched between untreated and treated. 7. Plot sizes should be appropriate for the scale of pesticide application and the activity of the invertebrate groups of interest. 8. Treated and untreated plots should be sufficiently far apart so as to avoid contamination of the control plots with pesticide and movement of species between the treated and untreated plots. 9. Detailed environmental data should be collected for each plot. 10. The study design should be in proportion to the resources available.

66 Impact of OPM control methods on oak tree biodiversity | March 2018 An array of techniques is required to sample invertebrate species according to their ecological, behavioural and habitat diversities. Generally, a number of sampling methods should be chosen dependent on the target taxa of interest (Tingle, 2002; Grootaert, 2010). The types of trap used will be dependent on the habitat to be sampled and the target invertebrates of interest, which will in turn be dependent on the pesticide under investigation (Tingle, 2002).

4.1 Canopy Layer sampling

4.1.1 Sticky traps

Sticky traps are a form of passive collecting and are basically coloured sheets, which can be made from a variety of materials, and covered with a thin layer of weatherproof glue such as Tanglefoot® Tangle- Trap® insect trap coating. They are versatile in that they can come in a range of sizes and colours and can be deployed in hard to reach places such as the canopy (Grootaert, 2010). The colour of the trap should be chosen in accordance with the species of interest for trapping. Bright yellow is used to collect a broad array of low flying insects, especially Hymenoptera and predacious fly species; white is a good colour for catching Syrphidae and Dolichopodidae dipteran species, although arboreal dolichopodids are best caught with blue, and red should be used for soil-dwelling dolichopodids (Grootaert, 2010). Other dipteran species, whose larvae are associated with plant material are also attracted to blue traps (Grootaert, 2010). If the aim of the trapping is to assess overall species diversity, then Grootaert (2010) recommends yellow and white traps.

The main disadvantage of sticky traps is that it is hard to remove the insects without damaging them, so this could be problematic if identification of small insect species is required; however, chemicals (such as kerosene) can be used to dissolve the glue (Grootaert, 2010). Sticky traps also require a frequent sampling regime to ensure that the insects caught are identified before they deteriorate and become unidentifiable (L. Collins pers. comm). Sticky traps are considered to be a useful supplementary technique for catching ants, long-legged flies, xylobiont beetles, and species of Collembola, Thysanura and Blattodea; interestingly, Grootaert (2010) does not include this technique for sampling hymenopteran parasitoids, instead recommending malaise traps as the most suitable methods for collecting these insects.

4.1.2 Destructive foliage sampling

Destructive foliage sampling is a direct search method of sampling, involving the removal of foliage from the tree, and as such is probably the most useful method for surveying the invertebrates on the canopy foliage. This method will also need to be used if leafminer larvae are to be assessed. This method of sampling has been used in all the studies reviewed in Chapter 5. Upon removal, the sample is sealed into a bag to contain the fauna. Invertebrates are removed from the foliage by tapping it into a box, or something similar, and then the foliage (and bag) are searched for any remaining invertebrates that failed to dislodge. Some active insects may be disturbed during the process and fly off, and some may escape while samples are sorted, meaning that the numbers of some mobile

67 Impact of OPM control methods on oak tree biodiversity | March 2018 species may be underestimated. Samples can be standardised for example by counting the number of leaves per sample or by weight of the foliage (Ausden, 1996).

4.1.3 Beating

Beating involves tapping the canopy branches to dislodge invertebrates and catching them as they fall in a beating tray held underneath. Insects are then collected from the tray using a pooter. Sampling can be standardised by beating for a set number of times. Whilst very quick and easy to do, and often resulting in a large catch, it does have disadvantages. Primarily, many species will fly away and so the technique is biased towards collecting insect species that are easily dislodged but do not take to the wing when disturbed (Ausden, 1996). It can be a good method for catching bugs, adult lacewing and beetles (adults and larvae). Lepidopteran larvae are not easily dislodged from foliage (Ausden, 1996), so whilst some larvae will be caught using this technique, it is unlikely that representative samples will be obtained.

4.1.4 Leaf miner sampling

Leaf miner sampling requires the collection of foliage. There are four insect orders that contain species with larvae that mine: Lepidoptera, Diptera, Hymenoptera, and Coleoptera. In total, 45 species of British miners are recorded on Quercus spp: one Hymenoptera (sawfly), four Coleoptera (beetles) and 40 Lepidoptera (Pitkin et al., 2018). The leaf mines can be categorised as gallery (corridor), blotch, and blister however, some mines can be a combination of these categories (Dunlop, 2016). Frass (dung) patterns are an important aide in leaf miner identification: a single line is indicative of lepidopteran miners, multiple lines are indicative of dipteran larvae whilst a distributed frass pattern is indicative of sawfly miners (Dunlop, 2016). Shape of the larvae is also another consideration in identifying leafmining larvae: dipteran miners have larvae that are bullet shaped whereas larvae that are ‘round- shouldered’, and with or without pro-legs, are likely to be sawfly larvae. Pitkin et al. (2018) provide an online key including descriptions and photographs for the purpose of identifying leafminer species. In addition to Pitkin et al. (2018), two other useful online resources are available for the identification of leaf miners: the British Leaf miner website (http://www.leafmines.co.uk/index.htm) provides a downloadable key and Ellis (2017) provides an online key for the leafminers and plant galls of Europe.

4.2 Ground layer sampling

4.2.1 Pitfall traps

Pitfall traps, which actually measure ‘activity abundance’ are good for providing data on the presence/absence and relative abundance of a broad range of invertebrate species active at ground level (Tingle, 2002). They are most suitable for sampling Collembola, Thysanura, Carabid beetles, and apterous/brachypterous Diptera, and can be used as a supplementary sampling technique for ants, phorid flies, Blattodea and some orthopteran family species, but are not considered an appropriate

68 Impact of OPM control methods on oak tree biodiversity | March 2018 technique for catching Lepidoptera (Grootaert, 2010). They can be widely used in variety of habitats, including woodland/forest, orchards/plantations and grassland but do have limitations (Tingle, 2002). Some species are known to avoid pitfall traps or be able to escape easily. Trap catches are also influenced by a whole host of factors, which need to be standardised as much as possible, these include surrounding vegetation, ground-surface irregularities, trampling around the trap, species selectivity, whether or not the trap is covered, climatic conditions, the shape, form and diameter of the trap along with the material from which it is made, and the preserving agent used (Tingle, 2002). Some of these can be minimised/standardised:

 Tingle (2002) recommend that at least 30 traps per treatment area should be used but does not relate this number to size of the area; traps should be arranged in a line or grid with 2 m between each trap (Ausden, 1996; Tingle, 2002);  The same size and type of container should be used for the duration of the study (preferably glass or plastic, 6 cm diameter and not less than 12 cm deep); Traps with a larger diameter will collect a greater number of invertebrates (Ausden, 1996; Tingle, 2002);  The same preservative should be used throughout the study (picric acid ideally, or formalin as an alternative; Tingle, 2002). Ausden (1996) recommends ethylene glycol if traps are to be checked once a month;  The trap should be place in a sleeve that is permanently set in the ground to minimise disturbance when emptying and resetting the traps (Tingle, 2002).

Pitfall traps have the advantage that they are easily deployed and can be emptied on a daily, weekly or monthly sampling regime if the correct preservative for the time interval required is used. Only one person is required to empty and reset them although two would be more preferable. In terms of the number of personnel needed to sort and identify the catches, this will be dependent on the number of traps, quantities of invertebrates trapped and the level of identification to be achieved; however, Tingle (2002) suggests that three to five staff would be ideal.

4.2.2 Water/Pan traps

Water traps are used to collect a variety of flying insects, predominantly aphids, flies, hymenopteran species and some coleopteran and lepidopteran species, and measure activity abundance (Tingle, 2002). They consist of a coloured dish containing water with detergent added to break the surface tension (Tingle, 2002; Grootaert, 2010). As with sticky traps different colours can be used according to the target insect; yellow is particularly good for catching flies, aphids, chalcidoid wasps and some beetles (Tingle, 2002). Ideally, they should be set at a standard height above the vegetation (Tingle, 2002), Grootaert (2010) suggests at a height of 60 cm to collect arboreal species. They can also be deployed at ground level however, if used at ground level then sinking the trap into the soil such that the rim of the trap is at surface level greatly improves the catch both in terms of diversity and abundancy (Grootaert, 2010).

Whilst they are easy to deploy, the catch is dependant on a number of factors such as type of vegetation, climatic conditions (temperature, wind speed, rainfall, season) as well as the design and siting of the trap itself (Tingle, 2002). They also need to be checked, and catches removed, at frequent

69 Impact of OPM control methods on oak tree biodiversity | March 2018 intervals; Tingle (2002) suggests daily with two to three staff to sort and identify the catch. They can be checked less frequently if a preservative is used. Grootaert (2010) suggests that if a 5% preserving solution is used, they can be left for seven days, and for two weeks using a 10% preservative. However, the amount of rainfall will influence the effectiveness of the preserving agent; a large amount of rainfall will dilute the preserving solution. Traps are also at risk of filling up when it rains. This can be overcome to some extent by drilling a series of small holes around the perimeter of the surface to allow the water to drain and prevent the insects on the top of the water from floating out of the trap (Grootaert, 2010). Presumably this will only be effective if the holes do not get clogged up, and the surrounding ground does not become saturated.

Other methods of monitoring ground level invertebrates include the use of quadrats (in Tingle, 2002), direct counts (Ausden 1996) and tethered litter bags (Tingle, 2002). Tussocks of grass and other vegetation can be collected by slicing off the tussock at root level and then shaking and cutting it open over a tray to remove the species that feed/hibernate within this habitat (Ausden, 1996). Suction sampling can also be used to sample invertebrates in low vegetation (less than 15 cm in height) for example, using a D-vac. However, they are known to under-estimate large invertebrates (> 3mm in length), those that tend to be firmly attached to their substrate (e.g. Lepidoptera), and species that live low down in taller vegetation; some species will also take evasive action when they sense the disturbance (Ausden, 1996).

4.3 Invertebrate identification methods

Accurate identification of lepidopteran larvae is reported to be notoriously difficult (Freed and Reeve, 2014). The authors report that photography and rearing larvae to final instar/adulthood both proved to be invaluable for identification; rearing was relatively straightforward except for larvae with exacting ecological requirements, which did not survive. Other authors (e.g. Wagner et al., 1996) also report that rearing larvae to adulthood was required for identification of some species. In some instances, for both mining and non-mining species, dissection of the adult genitalia is required to enable identification to species level. Freed and Reeve (2014) report that dead larvae, especially those that were shrivelled, long-dead, or had died as a result of ingesting Btk were not usually identifiable beyond family level.

Identification of leaf mining species is discussed above in Section 4.1.4.

Summary

1. Destructive foliage sampling is the most useful sampling method for assessing invertebrates on canopy foliage but is likely to underestimate mobile species with a tendency to fly off when disturbed. 2. Pitfall traps and ground level water traps are suitable for collecting a wide range of active species at ground level but are not considered suitable for catching lepidopteran species. 3. Identification of lepidopteran species is difficult. It is useful to photograph specimens, and some larvae will need to be reared to adulthood for identification to species level. In some

70 Impact of OPM control methods on oak tree biodiversity | March 2018 instances, adult genitalia will need to be dissected if accurate identification to species level is required.

71 Impact of OPM control methods on oak tree biodiversity | March 2018 Chapter 5. A review of similar studies: Short and long-term impacts of tree insecticides on invertebrate biodiversity and the wider environment

The aim of this chapter is to review studies that have investigated:

 Short and long-term impacts of tree insecticides (B. thuringiensis var. kurstaki, diflubenzuron and deltamethrin) on invertebrate biodiversity and the wider environment.

Direct (observable) or indirect (secondary) effects, which can be both positive and negative, on non- target organisms may occur as a result of insecticide treatment (Strazanac and Butler, 2005). Indirect effects occur as a result of the direct impacts on the foliage feeding larvae, and may be positive at the same trophic level if competition from susceptible foliage feeders is reduced, and at higher trophic levels if susceptible larvae become weakened and more vulnerable to predation and parasitism. However, immediate and sustained indirect negative effectives could occur when foliage feeders are killed by the treatment resulting in both a void in the food chain and a lack of larvae available for parasitism (Strazanac and Butler, 2005).

5.1 Studies with B. thuringiensis var. kurstaki

Bacillus thuringiensis var. kurstaki is well documented to have insecticidal activity towards more than 300 species of lepidopteran larvae (pest and non-pest species) both in the laboratory and a wide range of environments (Krieg and Langenbruch, 1981; Navon, 1993; Peacock et al., 1998; Glare and O’Callaghan, 2000). Hence there is little doubt that non-target native species of Lepidoptera will be affected to some extent when B. thuringiensis var. kurstaki is used, for example, to control a pest species in a forest environment (Wagner et al., 1996; Glare and O’Callaghan, 2000). Glare and O’Callaghan (2000) concluded from their review that even though non-target impacts of B. thuringiensis var. kurstaki have been observed for some non-target Lepidoptera in ecosystem studies, the impacts were not sufficient to endanger any of the species that had been assessed; however, these authors commented that studies assessing larval mortality, rather than adult emergence or sublethal effects, may underestimate impacts. The extent of non-target impact in the field is dependent on a number of factors including the dose rates used, method of application, inherent variability in the susceptibility of different insect populations, differences in insect feeding behaviours and environmental factors (Glare and O’Callaghan, 2000).

Preliminary studies in the USA estimated that 92-98% of 223 species of Lepidoptera (from 22 families) present in the National Parks of Washington DC would be vulnerable to Bt-sprays, based on their host- sharing, larval habitat and phenological overlap with gypsy moth (in Scriber et al., 2004).

In 2014, Sobczyk produced a large document (written in German) detailing the history, biology, hazards and control of oak processionary moth in Germany. Chapter 8 discusses the impact of the control options on non-target Lepidoptera. Of the 366 lepidopteran species found on oak in Germany, 288 of them have larvae at the same development stage as oak processionary moth larvae during April to June, when control methods would be applied. Sobczyk (2014) suggests that 74 of these 288 species would not be affected, or only minimally affected, by surface applied insecticides because of their biology e.g. they develop in wood, under bark or in leaf mines; leaving 214 species that could be

72 Impact of OPM control methods on oak tree biodiversity | March 2018 directly affected. This hypothesised protection due to biology and feeding habits does not always prove to be the case in other pest/control situations (reported in Sobczyk, 2014), and certainly does not appear to be the case for the leaf-rolling species (Tortricidae) when subjected to spray applications of B. thuringiensis var. kurstaki (Strazanac, 2005a; Freed and Reeve, 2014; Sobczyk,2014), which are therefore now thought of as directly affected. Sobczyk (2014) presents a graphic of the assumed impact of B. thuringiensis var. kurstaki (DiPel® ES) on the 366 species of Lepidoptera associated with oak in Germany. The graphic predicts that species of Cossidae, Sesiidae, Limacodidae, Oecophoridae, Chimabachidae, Yponomeutidae, Gracillariidae, Bucculatricidae, Tineidae, Tischeriidae, Incurvariidae, Heliozelidae and Nepticulidae would not be affected, whilst all species of Arctiidae, Lymantriinae, Drepanidae, Lycaenidae, Sphingidae, Saturniidae, Coleophoridae, Ypsolophidae and Eriocraniidae species, and the majority of Noctuidae, Geometridae, Lasiocampidae, Tortricidae, Gelechiidae and Psychidae species would be affected. However, this is a prediction, and therefore may not be truly representative. For example, some authors suggest that the Noctuidae are less susceptible to B. thuringiensis var. kurstaki than might be expected (van Frankenhuyzen, 2009), although significant adverse impacts on noctuids have certainly been observed in the field in some studies (see below; e.g. Freed and Reeve, 2014). On the basis of this prediction (that 214 of 366 species of oak tree Lepidoptera are likely to be directly affected by applications of B. thuringiensis var. kurstaki), Sobczyk (2014) conclude that both area-wide applications and repeat applications on the same site should not be done, and also suggest that control measures taken at the edges of oak tree habitats, and within forests with openings, are likely to impact on an even greater number of very diverse species inhabiting the grasslands and hedgerows in these areas. Sobczyk (2014) also concludes that these predictions signal a risk of extinction of rare species with isolated populations.

The most relevant study investigating the non-target impacts of spraying with B. thuringiensis var. kurstaki to control oak processionary moth is that of Freed and Reeve (2014). Their study within Richmond Park (UK) specifically investigated the effects of spraying with B. thuringiensis var. kurstaki to control oak processionary moth on non-target species of Lepidoptera, particularly leaf-chewing larvae (although leaf miners and all other invertebrates were recorded) on Q. robur. This study utilised three groups (A, B, C) of 12 trees matched in terms of age, size and aspect; group A were sprayed three times within one week during mid-May (14th-22nd) 2013, group B had been sprayed once in May of the previous year (8th May 2012) and had not been sprayed since, and group C – the control group – had never been sprayed. A sampling protocol was devised to establish whether a spring application of B. thuringiensis var. kurstaki could affect later lepidopteran communities. Sampling was performed at three timepoints in the year: late May/early June (seven days after the final spray application for Group A), late June, and late September. On each sampling occasion, three branch tips (measuring approximately 70 cm) were cut from each tree, from different parts of the outer canopy, from the browse-line to a maximum height of 6.2 m. Sampling was always performed in the early morning to take account of possible diurnal activity rhythms of the larvae (Freed and Reeve, 2014). Branches were sorted as soon as possible after collection: lepidopteran and non-lepidopteran larvae (by-catch) were removed by bashing the cut branches into a box followed by foliar examination for any larvae that were not dislodged by bashing (Freed and Reeve, 2014). Leaf mines were counted and identified to and species level where possible. Cannibalistic larvae and other large noctuids were removed immediately and separated. All lepidopteran larvae and pupae were kept in labelled plastic boxes for photographing and rearing through to the adult stage, if required, to aid identification. Freed and

73 Impact of OPM control methods on oak tree biodiversity | March 2018 Reeve (2014) report that photography and rearing larvae to final instar/adulthood both proved to be invaluable for identification; rearing was relatively straightforward except for larvae with exacting ecological requirements, which did not survive. Live lepidopteran larvae and pupae were grouped together for total counts. Dead larvae, especially those that were shrivelled, long-dead, or had died as a result of ingesting B. thuringiensis var. kurstaki were not usually identifiable beyond family level and were recorded separately (Freed and Reeve, 2014). By-catches were preserved in 80% ethanol for analysis at a later date. A number of lepidopteran larvae were found to be parasitised and when possible the emerging parasitoid wasps were included in the by-catch data. Data were log10 transformed before analysing by one-way parametric Analysis of Variance (one-way ANOVA) for independent samples (two-tailed) followed by Tukey post-hoc analysis to establish inter-group differences (Freed and Reeve, 2014). When the transformed data failed homogeneity of variance checks, untransformed data were analysed using the non-parametric Kruskal-Wallis test.

Freed and Reeve (2014) recorded a total of 89 species of Lepidoptera in the study (Table 5), the majority of which belonged to the Tortricidae family. These larvae, and those from the Pyralidae family, were often found within leaf rolls, folds and spinnings, densely woven spinnings within oak infloresences, in the axils of twigs and branches, under lichen and amongst other debris (Freed and Reeve, 2014). Sixty one of the recorded species were oak leaf-chewers for all or part of their lives, with the remaining species identified as leaf miners (22 species), lichen-moss feeders (2 species), xylophagous (1 species) and those using other food sources (3 species) (Freed and Reeve, 2014).

Data from the three sampling time points indicated differences in the leaf-chewing lepidopteran abundance and biodiversity between the three groups of trees. At all three sampling times the numbers of live larvae and numbers of identifiable species, were significantly lower on trees that had been sprayed with B. thuringiensis var. kurstaki in 2013 compared with the untreated trees (Freed and Reeve, 2014). This was particularly apparent for the spring-feeding species (e.g. Carcina quercana, angustiorana, Gypsonoma dealbana, Phycita roborella) contrasting with numbers of some exclusively autumn-feeding species (e.g. Teleiodes luculella) which were unchanged. Numbers of live larvae and identifiable species were also significantly lower on trees sprayed in 2013 compared with those sprayed the previous year during the first and third sampling points but not at the second sampling time point (Freed and Reeve, 2014). When compared with the untreated group, trees sprayed in 2012 but not during the sampling year supported significantly fewer numbers of living larvae at the first and second sampling points but not the third, and the number of different species observed was significantly less at the first sampling point but not at the two later sampling points (Freed and Reeve, 2014). These authors conclude that the consistent ranking both in terms of numbers of larvae and biodiversity on the untreated trees > trees sprayed the previous year > trees sprayed during the survey year indicate a clear negative effect on the lepidopteran community as a result of spraying with B. thuringiensis var. kurstaki (Freed and Reeve, 2014). This significant negative effect was not only observed immediately after spraying but also four months and one year on from spraying; however, the data suggests that although the overall numbers of larvae are still lower, the biodiversity (i.e. the number of species) starts to recover one year on from spraying (Freed and Reeve, 2014).

The authors go on to discuss the biodiversity data in more detail. In summary, 56 species were associated with the untreated trees compared with 45 species for trees treated in 2012 and 32 species

74 Impact of OPM control methods on oak tree biodiversity | March 2018 for trees treated in 2013 (the sampling year; group A); thirteen species were found only in the untreated trees whilst five were only found in group B and two were only found in group A. The spring defoliating totricids, Tortrix viridana and Zeiraphera isertana, and some geometrids (e.g. Spring Usher Agriopis leucophaearia) and noctuids showed a significant decline in numbers in both treatment groups, suggesting that spraying with B. thuringiensis var. kurstaki the previous year had significantly reduced numbers, and populations had not recovered much by the following year. Freed and Reeve (2014) suggest that the species most affected are spring defoliators and weak fliers. Larvae of the spring defoliators will be present on the leaves at the time of spraying for oak processionary moth, and as such have the potential to ingest B. thuringiensis var. kurstaki, resulting in death. This also means that fewer numbers will survive to adulthood meaning fewer eggs will be laid for the next generation (Freed and Reeve (2014). Recolonisation of sprayed sites will be slower for species that are weak fliers, have wingless (apterous) females or females with much reduced wings (brachypterous), as is the case for many geometrid species (Freed and Reeve, 2014).

Freed and Reeve (2014) also report some unexpected results. For instance (although numbers were too low for statistical analysis), more Purple Hairstreak (Neozephyrus quercus) larvae were found in the 2012 treated trees during the first sampling than on trees in either of the other groups. The authors suggest that, whilst not explaining the low count at the untreated site, this result could reflect a combination of warm weather conditions during the 2012 summer favouring dispersal and recolonisation, and less competition from other oak folivores at site B. Other species showing noticeably higher counts at site B include the oecophorid, Carcina quercana (thought to be able to monopolise a niche created by spraying during the previous year) and the gelechiid, Teleiodes luculella (avoids direct effects of B. thuringiensis var. kurstaki because of its autumn-feeding habits) (Freed and Reeve, 2014).

Table 5 also lists the 22 species of leaf miner found in the Freed and Reeve (2014) study. For the purposes of analysis, count and identification, Freed and Reeve (2014) grouped leaf mine data into assemblages because related species often have very similar mines. The assemblages were:

 Ectoedemia albifasciella group;  Stigmella ruficapitella group;  Caloptilia alchimiella/robustella;  Phyllonorycter quercifoliella group;  Coleophora lutipennella/flavipennella;  Eriocrania subpurpurella/Orchestes spp.

Overall, the authors found few significant differences in the abundance of leaf mines and the assemblages of leaf miners between the different groups and timepoints. However, greater numbers were found in group B (sprayed in 2012) > group A (sprayed in 2013) > group C (untreated) during the first two samplings, and this result was significant at the first sampling. The authors concluded that the leaf miners were responding to the fewer numbers of the larger folivores but recommend a more detailed study of the effect of spraying B. thuringiensis var. kurstaki for oak processionary moth control on oak leaf miners.

75 Impact of OPM control methods on oak tree biodiversity | March 2018 As of 31st March 2018, there was no further published information available regarding the by-catch data collected during the Freed and Reeve (2014) study.

76 Impact of OPM control methods on oak tree biodiversity | March 2018 Family Scientific Vernacular Eriocraniidae Eriocrania subpurpurella Leaf miner (Dyseriocrania subpurpurella) Nepticulidae Ectodemia quinquella Leaf miner Ectodemia albifasciella Leaf miner Ectodemia heringi Leaf miner Stigmella ruficapitella Leaf miner Stigmella roborella Leaf miner Stigmella samiatella Leaf miner Stigmella basiguttella Leaf miner Tischeriidae Tischeria ekebladella Leaf miner Tischeria dodonaea Leaf miner Heliozelidae Leaf miner Bucculatricidae Bucculatrix ulmella Leaf miner Gracillariidae Caloptilia alchimiela Leaf miner Caloptilia robustella Leaf miner Acrocercops brongniardella Leaf miner Phyllonorycter harrisella Leaf miner Phyllonorycter quercifoliella Leaf miner Phyllonorycter messaniella Leaf miner Coleophoridae Coleophora lutipennella Leaf miner Coleophora flavipennella Leaf miner Coleophora ibipennella Leaf miner Coleophora kuehnella Leaf miner Psychidae Narycia duplicella White-speckled Bagworm Luffia ferchaultella Virgin Bagworm Yponomeutidae Ypsolopha sylvella Wood Smudge Ypsolopha ustella Variable Smudge Ypsolopha sp. Oecophoridae Carcina quercana Long-horned Flat-body (Peleopodidae) Oecophoridae Diurnea fagella March Tubic (Chimabachidae) Gelechiidae Carpatolechia decorella Winter Oak Groundling Teleiodes luculella Crescent Groundling terrella Cinerous Neb Psoricoptera gibbosella Humped Groundling adustella Dingy Dowd Tortricidae Pandemis corylana Chequered Fruit-tree Tortrix Pandemis cerasana Barred Fruit-tree Tortrix Pandemis heparana Dark Fruit-tree Tortrix Archips podana Large Fruit-tree Tortrix Archips crataegana Brown Oak Tortrix Archips xylosteana Variegated Golden Tortrix Syndemis musculana Dark-barred Tortrix Ptycholoma lecheana Brindled Tortrix Red-barred Tortrix Yellow Oak Button Tortrix viridana Green Oak Tortrix Acleris ferrugana Rusty Oak Button Eudemis profundana Diamond-back Marble Ancylis mitterbacheriana Red Roller

77 Impact of OPM control methods on oak tree biodiversity | March 2018 Zeiraphera isertana Cock’s-head Bell Gypsonoma dealbana Common Cloaked Shoot Spilonota ocellana Bud moth Strophedra nitidana Little Oak Piercer Pammene splendidulana Drab Oak Piercer Pammene giganteana Early Oak Piercer Pammene argyrana Black-bordered Piercer Pammene fasciana Acorn Piercer Pammene sp. unidentifed Cydia splendana Marbled Piercer Pyralidae Conobathra repandana (Acrobasis Warted Knot-horn repandana) Acrobasis consociella Broad-barred Knot-horn Phycita roborella Dotted Oak Knot-horn Lycaenidae Neozephyrus quercus (Favonius Purple Hairstreak quercus) Drepanidae Watsonalla binaria Oak Hook-tip Thyatiridae Polyploca ridens Frosted Green (Drepanidae) Geometridae Alsophila aescularia March Moth Hemithea aestivaria Common Emerald Cyclophora punctaria Maiden’s Blush Chloroclysta siterata Red-green Carpet Operophtera brumata Winter Moth Eupithecia abbreviata Brindled Pug Ennomos erosaria September Thorn Biston strataria Oak Beauty Agriopis leucophaearia Spring Usher Agriopis marginaria Dotted Border Erannis defoliaria Mottled Umber Cabera pusaria Common White Wave Notodontidae Drymonia ruficornis Lunar Marble Brown Lymantriinae Orgyia antiqua The Vapourer Lymantria dispar Gypsy Moth Noctuidae Orthosia cruda Small Quaker Orthosia cerasi Common Quaker Orthosia gothica Hebrew Character Lithophane ornitopus Grey Shoulder-knot Eupsilia transversa Satellite Conistra vaccinii The Chestnut Amphipyra berbera Svensson’s Copper Underwing Cosmia trapezina The Dun-bar Bena bicolorana Scarce Silver-lines Nycteola revayana Oak Nyceteoline Table 5. A list of all the species collected and identified by Freed and Reeve (2014) during their Richmond Park study.

78 Impact of OPM control methods on oak tree biodiversity | March 2018 Points to note from the Freed and Reeve (2014) study:

 2013 was a very poor year for numbers of moth larvae nationally (communicated within Freed and Reeve, 2014) and this is reflected in the reduced numbers/absence of some species known to be previously common and widespread within Richmond Park (including March Moth, Winter Moth, Mottled Umber, November Moth, Brindled Green and Green Silver-lines);  Spring 2013 was extremely cold and set back most lepidopteran emergence dates by three or more weeks (reported by Freed and Reeve, 2014);  Each sample took on average 2.5 hours to sort, count and store after collection (plus the time required for identification);  No baseline (pre-study) data is available for this study. The authors acknowledge that without pre-study baseline data, differences between the tree groups could have resulted from other reasons (e.g. differences in microclimate) even though the trends and significant effects observed are consistent with the expected result following spraying with B. thuringiensis var. kurstaki;  The Group B trees had only been sprayed once with B. thuringiensis var. kurstaki whereas the Group A trees had been sprayed three times;  The authors express concern that whilst B. thuringiensis var. kurstaki is sprayed onto the leaf canopy, it has the potential to drip off/be washed off the leaves leading to contamination of the understorey and herb layer below. Their study only investigated the impact on lepidopteran larvae within the leaf canopy. Many lepidopteran larvae are present in the understorey and herb layer (either continuously or as a proportion of their life cycle) feeding on grasses, herbs, leaf litter and dead wood. These include Nationally Scarce and IUCN Red Data Book (RDB) species such as Double Line Mythimna turca (Notable B) and Scythris potentillella (pRDB 1) (Freed and Reeve, 2014; see Appendix 2 for RDB classifications and criteria), the implication being that the impact of B. thuringiensis var. kurstaki spraying for Oak Processionary moth control on these species should be investigated.

Multiple field studies have been performed in the USA (Miller, 1990a,b; Sample et al., 1996; Wagner et al., 1996; Strazanac and Butler, 2005 to name just a few) to assess the impact of Btk on non-target organisms in broadleaved forests; B. thuringiensis var. kurstaki (usually formulated as Foray® 48B) is aerially sprayed as one of the control options for gypsy moth (Lymantria dispar (L.)), a destructive, defoliating lepidopteran pest that feeds on a range of tree species, including oak.

One such study was conducted by Wagner et al. (1996), over three years, to assess the impacts on non-target Lepidoptera following a single 90 BIU/ha aerial application of Foray® 48B. Twenty hectare plots were used (five treated and five untreated), which were matched in terms of geography, plant species composition and density. These authors also assessed the persistence of insecticidal activity on sprayed foliage by collecting foliage samples at 2, 4, 24, 48 and 72 hours after spraying, feeding them to gypsy moth larvae in the laboratory for four days before switching to fresh untreated foliage, and monitoring for mortality. Two sampling methods were used to assess the impact of spraying on non-target Lepidoptera: branch tip foliage sampling (10-15 cm of the terminal shoot) and burlap band sampling (30 cm wide, 1-2 layers thick, attached at chest height to the trunks of scarlet and chestnut oak species) (Wagner et al., 1996). Foliar branch sampling was performed two days prior to spraying (pre-treatment), and again six days and twelve days after spraying (treatment samples). The following

79 Impact of OPM control methods on oak tree biodiversity | March 2018 year, branch samples were collected on three separate occasions, on dates that were considered to be phenologically comparable to the dates when samples were taken the previous year (post- treatment sampling); Burlap band sampling was performed at frequent intervals during May and June of each year (Wagner et al., 1996). No foliage samples were collected during the second year after treatment but burlap band sampling was performed during the month of May. Foliage samples were visually inspected; externally feeding larvae (i.e. those not in leaf rolls etc.), and larvae collected from under the burlap bands, were removed, placed individually into plastic vials with fresh foliage and raised in the laboratory for identification. Colour photography was also used to help with the identification of macrolepidopteran larvae. Microlepidopteran larvae identified to species level required rearing to adulthood. ANOVA and t-tests were used to compare the abundancies of larvae across the treatment and control plots. Sign tests were used to evaluate changes in abundancies of some species (Wagner et al., 1996).

The Wagner et al. (1996) study indicated that B. thuringiensis var. kurstaki insecticidal activity was highest (significantly so) on foliage collected two and six hours after spraying, as confirmed by mortality of gypsy moth larvae feeding on collected foliage. This result confirmed both deposition of B. thuringiensis var. kurstaki following spraying and perhaps rapid degradation of insecticidal activity (Wagner et al., 1996). The authors report that the single application of B. thuringiensis var. kurstaki had little impact on the total number of larvae collected by foliar sampling; abundance was lower, modestly so and mostly not significant, in the treated plots (Wagner et al., 1996). The authors suggest this was because more than 95% of the larvae collected in the treatment year samples were microlepidoptera that had already formed shelter (leaf rolls, folds, fascicles) before spray application, providing them with some protection from B. thuringiensis var. kurstaki. They report a decline in relative abundance of some leaf rolling gelechiids and tortricids was evident following treatment, but results were never statisitically significant. Other authors also hypothesise that microplepidopteran larvae are protected/able to avoid eating Btk-treated foliage by consuming leaf tissue within their shelter (Krieg and Langenbruch 1981; Sobczyk, 2014). However, Wagner et al. (1996) present very contrasting data from the burlap band sampling. The total number of larvae sampled from under the bands in the treated plots was four times lower than on the untreated plots, a difference that proved to be statistically significant. Several macrolepidoteran species (predominantly external feeders) found under the bands did show pronounced adverse treatment effects. Eleven of the twelve most common species were more than twice as likely to be recovered from the control plots. As the twelve most common species represented eleven genera within six different families this result indicates that a broad taxonomic range of species were affected by the B. thuringiensis var. kurstaki application (Wagner et al., 1996). The significant reduction (35%) in the total number of larvae found under the burlap bands in the treated plots continued into the first post-treatment year, and was mostly due to differences observed in the early sample dates during that year; only populations of the two most affected species remained significantly lower in this year. By the second post-treatment year no significant differences were observed in total numbers of larvae and only one of the affected species remained significantly less abundant in the treated plots.

Wagner et al. (1996) were able to draw a number of conclusions from their study:

80 Impact of OPM control methods on oak tree biodiversity | March 2018 1. Early instar larvae are likely to be more susceptible than late instar larvae (10 of the 11 susceptible species collected under the burlap bands would have been early to mid-instar larvae when the B. thuringiensis var. kurstaki spray was applied). 2. Most of the affected species were univoltine and had specialised feeding habits, preferring soft, spring oak foliage. 3. Species whose larvae are not active until June or later are likely to be unaffected by early-mid May application of B. thuringiensis var. kurstaki. The authors suggest that these species could benefit as a result of less defoliation by the pest insect and from leaf material of a higher quality (i.e. ample food source containing lower concentrations of inducible defence compounds). 4. Understorey lepidopteran larvae were also impacted with fewer larvae in the year of treatment statistically significant from the second sampling after treatment. 5. The authors found it very challenging to get a good representation of any species to allow for meaningful statistical analyses at the species level. They identified 128 species of Lepidoptera in total. For 24% of species only one individual was recorded, two individuals recorded for 13% of the species, three specimens recorded for 8% of the species and four individuals recorded for 9% of the species (i.e. 54% of all species were recorded on four or less occasions throughout the entire duration of the study). 6. Variance between the study plots made it difficult to demonstrate a treatment effect (Wagner et al., 1996). A low number (five) replicates were used but even this low number of replicates required 20 field and laboratory personnel to complete, and 10,000 larvae were collected in the treatment year alone. Increasing the number of plots can become prohibitively costly and time consuming. 7. Even with more replicates and larger samples the authors conclude that it would not have been possible to ascertain a treatment effect for the rarer taxa, indicating that studies such as these are not going to provide accurate information on the fate of uncommon taxa under such circumstances. The authors suggest that the only likely way of ascertaining the susceptibility and vulnerability of rare and endangered species, present at low densities, is to perform laboratory studies. 8. A single spring application of 90 BIU/ha B. thuringiensis var. kurstaki did not eliminate lepidopteran fauna in the treated areas; many species survived and rebounded quickly. However, with more than one application per year (Miller, 1990b) and/or treatment over successive years and/or larger treated areas a more significant impact may occur, and recovery may be slower as observed by Miller (1990b).

Other studies within broadleaved forests in the USA report similar results following an assessment of the impacts of aerial spray applications of B. thuringiensis var. kurstaki on non-target arthropods. For instance, Sample et al. (1996) conducted a four year study (one year of pre-treatment, the treatment year (one application of B. thuringiensis var. kurstaki (14.4 BIU/ha)), and two years of post-treatment sampling). Plots were 20 hectares and six replicates were used. Both abundance and species richness of non-target Lepidoptera were reduced, relative to the untreated plots, following application but was relatively short term (less than a year). In contrast, Miller (1990b) report on a three year study (one treatment year followed by two post-treatment years) monitoring non-target leaf-eating Lepidoptera on Garry Oak (Q. garryana) in the USA, following three applications of B. thuringiensis var. kurstaki in

81 Impact of OPM control methods on oak tree biodiversity | March 2018 the spring of the treatment year. Miller (1990) reports that total numbers of Lepidoptera in the treatment plots were significantly reduced in the treatment year, and in the first post-treatment year however, the species richness was significantly reduced in the treatment plots in all three years of the study, indicating that species richness had not recovered after two full post-treatment years.

In 2005, Strazanac and Butler reported on an extensive seven year study to assess the impact of B. thuringiensis var. kurstaki (and Gypchek® and Entomophaga maimaiga, also used for gypsy moth control) on non-target organisms. This study was conducted in mixed broadleaved-pine forests, over a large geographical area, in the Central Appalachians, USA, and included two years of pre-treatment baseline, two consecutive years of treatment and three years of post-treatment evaluation on arthropods, birds and salamanders. This project came about when the Appalachian Integrated Pest Management (AIPM) project for gypsy moth identified several gaps in the data concerning the impacts of different treatments on non-target organisms; use of B. thuringiensis var. kurstaki (Foray® 48F) and diflubenzuron (Dimilin®) ranked the highest with regard to this concern. The project had several aims (Strazanac and Butler, 2005):

1. To collect baseline data on Lepidoptera and other selected arthropods (herbivorous, predatory and parasitic), songbirds, and terrestrial and aquatic salamanders. 2. To evaluate the impact of two sequential annual applications of B. thuringiensis var. kurstaki (Foray® 48F), on the above arthropod communities, and selected insect pollinators, and to analyse the impact of any changes within the arthropod community on songbirds and terrestrial salamanders. 3. To identify the best indicator species/communities amongst the arthropods to use for evaluating impacts of B. thuringiensis var. kurstaki. 4. To develop recommendations to minimise the impact on non-target species.

The design of the research project included a number of elements deemed to be important (Strazanac et al., 2005a):

 Two years of pre-treatment – to establish baseline data enabling comparison of treatment and post-treatment data;  Two consecutive years of treatment at the highest allowed dose – representing the (then) current methods for suppressing major outbreaks or new isolated infestations of gypsy moth;  Three years of post-treatment sampling – to monitor rebounds and recovery of B. thuringiensis var. kurstaki sensitive populations, and to identify impacts on long term population dynamics;  Large study plots (200 hectares) – to minimise the impacts of localised weather conditions and to reduce the influence of migration from outside treatment plots on sampling results;  Five sampling methods over a wide geographic area – to investigate a large number of species and additional environments;  Natural enemies and competitors of the B. thuringiensis var. kurstaki sensitive groups were surveyed – to investigate the recovery of natural enemies, impacts on feeding guilds, and the release of competitors of the B. thuringiensis var. kurstaki sensitive species;

82 Impact of OPM control methods on oak tree biodiversity | March 2018  Data collected on the physical attributes, weather and vegetation of the sites – to help contribute to an ecosystems approach;  Long annual sampling periods – to monitor adult stages of univoltine species and the population dynamics of multivoltine B. thuringiensis var. kurstaki sensitive species, and their competitors and natural enemies.

The Strazanac and Butler (2005) study used 200 hectare study plots, sited within three blocks and divided between two locations. Gypsy moth was absent from these areas at the beginning of the study, and once it arrived in the areas remained at low levels. Six plots were treated with B. thuringiensis var. kurstaki and six were untreated (control); treatments were applied in a randomised block design based on vegetation. Within each plot, a 30 hectare subplot (600 m x 500 m) was used for the monitoring of arthropods, birds and salamanders, and other data acquisition. The non-target organisms that were assessed included lepidopteran larvae that may receive a direct effect (specifically the macrolepidoptera, which are usually exposed on foliage), competing herbivorous sawfly larvae, predators such as ground beetles and spiders, parasitic wasps and flies, selected song birds, and salamanders. Extensive data was collected for each site and included soil characteristics, climate and weather, and vegetation analysis (Strazanac et al., 2005b). Foray® 48F was applied (40 BIU/acre in the spring, following leaf bud-break when white oak (Quercus alba L.) leaves were approximately 25% of full size (about 3cm) (Rastall et al., 2005). Coverage was confirmed with the use of water-sensitive spray cards distributed within the plots. Foliage samples were taken and analysed by ELISA to determine the concentration of B. thuringiensis protein toxins in relation to the leaf surface; analysis confirmed that concentrations ranged from non-detectable levels to 171 ng/cm2 with only 9.9% of samples (equates to 11 samples) being less than 20 ng/cm2 (Rastall et al., 2005).

Sampling methods included the use of malaise traps, pitfall traps, canvas band traps, light traps (to sample nocturnal moths) and foliage sampling (Strazanac et al., 2005a). Fifteen weekly samples along established transects were collected during each year of the study covering the weeks before and after gypsy moth caterpillars and adults were present, from within two vegetatively diverse areas of the sampling subplots. Results were analysed with mixed model ANOVA as a randomised complete block design, with post-treatment years as repeated measures and pre-treatment years combined as a covariate. Analyses included testing for effects of treatment, year and the interaction between the two. Non-normal data was log transformed. When appropriate, non-target organisms were grouped by plot, sampling method, year or date (Strazanac et al., 2005a). The intention was to examine species of lepidopteran larvae and other similar arthropods (e.g. sawfly larvae) that would likely be directly affected, determine how much post-treatment time was required for them to return to pre-treatment abundance levels, and to infer indirect effects on predators, parasitoids and species that compete with resources.

Lepidopteran larvae with known phenologies most similar to gypsy moth larvae were identified to species level. Larval counts were grouped as (1) present during treatment (2) present shortly after or (3) not present during the treatment efficacy period; this allowed for analysis of direct impacts and elimination of any potential fluctuations in sample numbers across plots over time that are not directly influenced by treatment. Certain macrolepidopteran species were collected each season and reared in the laboratory to establish relationships with hymenopteran parasitoids. Parasitoids were identified to species level, percent parasitism was calculated and the recovery of parasitoids that attack non-

83 Impact of OPM control methods on oak tree biodiversity | March 2018 target species investigated (Strazanac et al., 2005a). It was considered important to identify the tachinid flies collected during sampling to species level because some will attack a variety of lepidopteran species, others will parasitise larvae not present at treatment application, and some of the most commonly collected do not attack lepidopteran larvae at all (Strazanac et al., 2005a). Predators (carabid beetles, spiders and pentatomid stinkbugs with predatory habits) were identified to species level and monitored for fluctuations. Sawfly larvae were also included in the study because they share similar foliage feeding habits with the macrolepidoptera and are predated upon/attacked by many of the same predators/parasitoids that attack lepidopteran larvae. Early literature suggests that some commercial preparations of B. thuringiensis were toxic to sawfly larvae however, this is thought likely to be caused by an exotoxin present in the product rather than directly caused by the B. thuringiensis endotoxins/spores themselves (Smirnoff and Berlinguet 1966). Arthropods considered less likely to be affected were generally analysed at family level and were grouped either by taxonomic group or ecological niche (Strazanac et al., 2005a).

The authors draw a number of conclusions from their study as detailed below (Strazanac et al., 2005a):

1. The two years of baseline counts not only provided a baseline to compare year to year changes on the study plots, they also gave an idea of normal relative counts among the plots. Pre- treatment counts were used as covariants in the analyses to compare relative plot counts grouped by treatments with the baseline plots grouped in the same way. 2. The impacts of any localised weather conditions were minimised by spreading the plots over two areas. 3. Direct negative impacts were seen when data for the macrolepidopteran larvae that were expected to be sensitive to treatment timing were pooled; declines in abundance were observed during the two treatment years, but a significant rebound was observed for this group of larvae beyond “normal” baseline counts in the second post-treatment year. The tortricid and gelechiid families of microlepidoptera also had lower counts on B. thuringiensis var. kurstaki plots during the treatment years but the difference was only significant for the Tortricidae; recovery occurred during the second post-treatment year. 4. During the study, a total of 608 species of macrolepidoptera were identified from oak, hickory and maple, using foliage sampling, canvas bands sampling and light traps (moths). 5. The total number of oak-associated lepidopteran larvae was significantly lower in the treatment years and first post-treatment year in the treated plots compared with the control plots. 6. No impact was observed on the lepidopteran larvae that feed on leaf litter (Herminiinae), suggesting that aerial application does not increase the longterm pathogen load in the leaf litter. 7. Univoltine species of Lepidoptera had greater and longer lasting decreases in numbers as a result of B. thuringiensis var. kurstaki treatment, with multivoltine species recovering faster. 8. Light traps were used to sample adult moths to include additional species and increase sample sizes. However, results from light trap catches should be interpreted carefully – most moths are strong fliers so results should be interpreted with regard to their flight range. 9. Significant decreases in adult moth catches were seen in the second treatment year for species whose larvae would be sensitive to the B. thuringiensis var. kurstaki treatment timing.

84 Impact of OPM control methods on oak tree biodiversity | March 2018 10. No direct (e.g. toxicity) or indirect (e.g. release from competition) effects were observed on sawfly (Symphyta). However, the authors acknowledge that they had limited biological knowledge and taxonomic skills for this group of arthropods. 11. Negative indirect effect trends were observed in parasitoids flies (Tachinidae) and wasps (Ichneumonidae: Ichneumoninae and Braconidae: Microgastrinae) that specialise in lepidopteran larvae. In the case of one tachinid fly, this effect was significant and observed in the second year of treatment and first post-treatment year. 12. Generalist predators such as spiders, and most carabid beetles, were not significantly impacted (negatively or positively) when lepidopteran larvae numbers fell on the treated plots. One exception was observed; numbers of a carabid species reported to selectively prey on caterpillars significantly declined but the treatment and post-treatment counts had to be combined to demonstrate this effect. Weak trends were observed for other caterpillar or caterpillar-like larvae specialists on the treated plots. Similar results were obtained for the predatory stinkbugs that specialise on caterpillar/caterpillar-like larvae. A trend towards lower abundance was observed when numbers of caterpillar larvae fell but was only significant when treatment and post-treatment years were combined and was dependent on sampling method. Numbers recovered when caterpillar numbers recovered. 13. Reductions in natural enemies brought about by the reduction in spring lepidopteran larvae did not appear to release other potential defoliators from competition. 14. Populations of primary foliage consumers can be quite robust, as evidenced by record low temperatures in the spring of the first treatment year followed by application of B. thuringiensis var. kurstaki. 15. Macrolepidopteran adults and many of their specialised natural enemies, which are often strong flyers or easily blown on the wind, can migrate from adjacent areas quickly allowing re- establishment of populations and host/prey relationships.

Point to note from the Strazanac and Butler (2005) study:

 By the end of the study, in excess of two million insects and spiders had been collected and sorted;  Two severe weather events did affect all plots impacting on total abundance counts – a cold period in the early spring of the first treatment year and a drought during the summer of the first post-treatment year;  Factors outside of the manipulated variables affect sample sizes; the assumption is that these factors were felt equally among all study plots. Highly varied terrain and inherent differences between the plots added a level of complexity in terms of interpreting the results;  Total counts for lepidopteran larvae should be avoided so that non-treatment factors do not influence the analyses, species should be grouped according to presence at treatment, shortly after, or not present (Strazanac et al., 2005a);  The authors (Strazanac et al., 2005a) describe apparent trends for individual species but very few statistically significant differences were observed at species level. Many of the results appeared difficult to interpret due to fluctuations in sample size on a year by year basis.

In addition to studying the impact of B. thuringiensis var. kurstaki application on invertebrate biodiversity, the Strazanac and Butler (2005) study also extensively investigated the wider biodiversity

85 Impact of OPM control methods on oak tree biodiversity | March 2018 impacts of B. thuringiensis var. kurstaki application on birds (dependent on lepidopteran larvae for their own survival and feeding of their young) and salamanders (play a vital role in the forest ecosystem). The reader is directed to Strazanac and Butler (2005), Bioscan (2014), Ecosulis (2014) and other available publications reporting on the wider biodiversity impacts of B. thuringiensis var. kurstaki spray applications for more information.

One study (Beck et al., 2004) reports on a direct comparison of the effects of B. thuringiensis var. kurstaki (DiPel® ES) and diflubenzuron (Dimilin® 25WP) on soil invertebrates in a mixed deciduous forest (mainly oak and hornbeam interspersed with beech) in Germany (Upper Rhine Valley). Four study plots (200 m2) with comparable vegetation and soil characteristics were established. One was left untreated (control), one was treated in the spring with 25 g/ha diflubenzuron with a further 25 g/ha treatment in the autumn (to simulate diflubenzuron reaching ground level with leaf litter; DIM1), and the third plot was treated with a spring application of 33.2 g/ha B. thuringiensis var. kurstaki. These concentrations represented 50% of the amount of each product used during aerial application of the insecticides. The final plot was treated with a 10x concentration of diflubenzuron (250 g/ha; DIM10). The products were applied directly to the forest floor. Statistical correlations between treatments and effects could not be established due to a lack of replication; similarity in the fauna at each plot was therefore compared using the Renkonen-Index. Deviations were interpreted as a response to treatment if numbers in the potential effect phase were different to those in the other phases in the same plot, and to the untreated plot in the same phase (Beck et al., 2004).

Three different sampling methods were used: collection of leaf litter, extraction of earthworms by electricity, and soil and litter sampling using a split-corer down to 10 cm of the mineral soil. Core samples were divided vertically into the organic layer and two layers of mineral soil for analysis (0-5 cm and 5-10 cm) (Beck et al., 2004). Except for the juveniles, earthworms, gamasid mites and enchytraeids were identified to species level; oribatid mites were identified to family level (except on two sampling occasions when they were identified to species level); the collembolans were classified to five taxonomic families, with the dominant species Folsomia quadrioculata measured and classified into four size and age groups (Beck et al., 2004). Pre-treatment sampling was performed for 10 months prior to treatment, and sampling continued for one year post-treatment; samples were taken every two months.

These authors measured the diflubenzuron concentration in the litter and humus layer of the soil and found average recovery rates to be 103% of the applied amount. The majority of the dose remained in the litter layer with a small amount penetrating into the upper soil mineral layer after 14 days. No diflubenzuron was detected in the DIM1 plot after two months with an estimated half-life between 10 and 20 days; however, a slower decomposition of the product occurred following the second autumn application to this plot (Beck et al., 2004). In contrast, diflubenzuron (1 mg/kg) was detectable in the litter layer of the DIM10 plot for six months. Neither B. thuringiensis var. kurstaki or diflubenzuron appeared to have any detrimental effect on earthworms. Likewise, overall potworm (Enchytraeid) abundance did not appear to be affected, although species within the genus Enchytraeus did appear to be affected by diflubenzuron (Beck et al., 2004). These authors demonstrate a clear reduction in overall abundance of the Gamasina, as well as reductions in some single species of oribatids and predatory Gamasida, and to the isotomid Collembola F. quadrioculata in response to diflubenzuron treatment at the lower dose tested (thought to represent a relevant dosage of the litter

86 Impact of OPM control methods on oak tree biodiversity | March 2018 following standard application rates). In the case of the Gamasida and Collembola, this was still evident in the post-treatment recovery phase, but in all cases signs of recovery were observed (Beck et al., 2004). The application of B. thuringiensis var. kurstaki did not have any detrimental impacts on the soil fauna, the only noticeable effect being the reduction in dominance of one Gamasida species, Veigaia nemorensis (Beck et al., 2004). The authors therefore concluded that side effects on soil invertebrates following application of diflubenzuron should be monitored with the isotomids within the Collembola, whereas effects of B. thuringiensis var. kurstaki may be monitored by observing the species composition of the Gamasida. These authors also indicate that no other studies demonstrated a negative effect on any taxa of soil invertebrates when B. thuringiensis var. kurstaki was applied at relevant concentrations.

5.2 Studies with diflubenzuron

Diflubenzuron was approved for use in the USA in 1976 for the control of gypsy moth. Studies have been conducted to assess the non-target impacts of diflubenzuron in forest ecosytems.

In 1997, Butler et al. report on an extensive study assessing the non-target impacts of diflubenzuron treatment on canopy arthropods in closed deciduous watersheds in a central Appalachian Forest in the USA. This study included three years of pre-treatment sampling, one year of treatment followed by two years of post-treatment sampling. Two treatment plots (44.3 ha in total) were aerially sprayed with Dimilin® 4 L at a dose of 35.1 g active ingredient (AI)/ha, and two control plots (totalling 62.9 ha) remained untreated. Sampling was conducted throughout May to August and consisted of burlap bands and foliage sampling (Butler et al., 1997). Samples were taken from seven species of trees including red and white oak. Bands were checked on a weekly basis and all arthropods removed. The foliage was sampled by removing 25 cm branch tips, using a pole pruner, from the low to mid canopy and was initiated soon after leaf expansion each spring; two samples were taken from each tree species, at each plot. Samples were placed in plastic bags and returned to the laboratory for identification. For both sampling methods, macrolepidoptera were identified to species level, with rearing to adulthood if necessary, whereas all other arthropod specimens were identified to family level. Arthropod abundance for the foliage samples was standardised to 50 g dry leaf weight. ANOVA was used to test for treatment differences each year between the control and treated plots, but between-year comparisons were not made due to high variation in both abundance and diversity in different years. Numbers of mircolepidoteran larvae were too low for analysis, as were the numbers of spiders and orthopteroids on the foliage (Butler et al., 1997).

The authors (Butler et al., 1997) report on species diversity data. Burlap band sampling identified a significant reduction in both mean total arthropod diversity at family level (not including macrolepidoptera), and macrolepidopteran diversity, as a result of Dimilin® application in the year of treatment. This contrasts slightly with the results from the foliage samples. These indicated a reduction in family level diversity (not including the macrolepidoptera) in the post-treatment year only, and no significant reductions in macrolepidopteran diversity were observed (only a trend towards reduction in the year of treatment).

87 Impact of OPM control methods on oak tree biodiversity | March 2018 Butler et al. (1997) also report on arthropod abundance. Burlap band sampling indicated that Dimilin® did not appear to impact on total arthropod abundance (excluding macrolepidoptera), or on the abundance of the most common taxa (excluding macrolepidoptera) caught under the bands: Phalangida, Formicidae, Agelenidae, Diplopoda, , Theridiidae, Chilopoda. A trend was observed for a reduction in Carabidae and Gryllacrididae in the treated plots for both post-treatment years, but this was not statistically significant (Butler et al., 1997). In contrast, significant reductions were observed in total arthropod abundance (excluding macrolepidoptera) in the foliage samples in the treatment year and the second of the two post-treatment years. When data for the coleopterous herbivores (Curculionidae and Chrysomelidae) were pooled, a significant reduction in their numbers was observed during the treatment year, and trends in reduction observed during the two post- treatment years (although numbers of these taxa were also higher on the control plots in one of the two pre-treatment years). Whilst some trends towards reductions in abundance for other taxa were noted, including sapfeeders (Aphididae, Miridae, Membracidae, Eriosomatidae), predatory thrips (Phlaeothripidae) and bark lice (Psocidae and Polypsocidae), within the treatment year at least, they were not statistically significant (Butler et al., 1997). In terms of macrolepidopteran abundancy, burlap band sampling, indicated a significant reduction within the year of Dimilin® application, with only one of the 86 species caught appearing to be unaffected (Butler et al., 1997). Larvae of the one unaffected species would have been in a late developmental stage at the time of Dimilin® application. In contrast, the foliage sampling demonstrated reduced macrolepidopteran abundancy during the year of treatment and both post-treatment years, but differences were only statistically significant for the first post-treatment year (Butler et al., 1997).

The Butler et al. (1997) publication formed part of a wider study into the effects of diflubenzuron on non-target organisms in broadleaved forests in the north east USA, edited by Reardon (1995). As well as canopy arthropods, the study investigated the potential impacts upon other terrestrial organisms: pollinating insects, invertebrates and microorganisms within the leaf litter and soil, and terrestrial salamanders, along with potential impacts upon aquatic organisms: fungi, macroinvertebrates and aquatic salamanders. The reader is directed to Reardon (1995) for further information.

5.3 Studies with deltamethrin

No relevant literature has been found on the impacts upon biodiversity following application of deltamethrin to trees and forest environments.

5.4 Impacts of no treatment on non-target invertebrates

Decision makers should be mindful that in circumstances when a defoliating pest is not controlled, and severe defoliation occurs as a result, non-target effects will be observed both at the overstorey level and the understorey level (e.g. as a result of desiccation because of lack of shading) (Scriber, 2004). Both leaf chewing and sap-sucking native species, which feed on oak in the presence of oak processionary moth, may be left with insufficient, or poor quality, food resource (Sobczyk, 2014). References cited within Scriber (2004) provide evidence of altered leaf quality in poplar that has been

88 Impact of OPM control methods on oak tree biodiversity | March 2018 severely defoliated and also report that slowed growth and/or smaller pupae and/or reduced fecundity were observed in eight lepidopteran species feeding on these trees not only in the same year that defoliation occurred but also 2-3 years post-defoliation (Scriber, 2004). Consideration should also be given to the potential rise in populations of generalist natural enemies, such as parasitic tachinid flies, that may build up in response to a high pest population, and then may parasitise native lepidopteran larvae when the pest species is not available (e.g. at later points in the year) (Kudrna, 2001; Scriber, 2004). On the otherhand, more specialist parasitoids such as those with a very narrow host range or searching habits, will reduce in numbers the following year (Kudrna, 2001).

Sample et al. (1996) report on the impacts on native Lepidoptera when no control method is used, and therefore trees are defoliated, in mixed broadleaved forests in the USA where gypsy moth is present. A four year study (one year of pre-treatment, the treatment year, and two years of post- treatment sampling) was conducted on 20 hectare plots. Three treatments groups were compared, each with six replicates (1) no control application, gypsy moth present (i.e. defoliation) (2) application of B. thuringiensis var. kurstaki, gypsy moth absent (3) application, gypsy moth present. When used, B. thuringiensis var. kurstaki was applied once at a dose of 14.4 BIU/ha. Both species richness and abundance of some species of Lepidoptera (larvae and adults) were reduced in the plots that were not treated and hence suffered from defoliation. The authors concluded that although the short-term impacts (less than one year) of spraying with B. thuringiensis var. kurstaki were negative on non-target Lepidoptera, the long-term effects of B. thuringiensis var. kurstaki treatment, i.e. a successful reduction in pest defoliators, was likely to be beneficial for some of the native non-target species.

Summary

1. Freed and Reeve (2014) report strong and consistent differences between matched stands of oak corroborating the expected result of spraying with B. thuringiensis var. kurstaki. Strong evidence of reduced taxonomic biodiversity and abundance of non-target Lepidoptera in trees sprayed during the sampling year compared with untreated control trees is reported. The first May/June sampling point also provided evidence that biodiversity and abundance of some species remained lower in trees that had been sprayed the previous year compared with untreated trees. Note, however, that no baseline data was collected in this study.

2. Other studies following application of B. thuringiensis var. kurstaki (aerially sprayed plots in the USA) also report reduced taxonomic biodiversity and abundance of non-target Lepidoptera in sprayed plots during the sampling year compared with untreated plots. Recovery begins in the first post-treatment year (Miller, 1990b; Sample et al., 1996; Wagner et al., 1996; Strazanac et al., 2005a). However, reduced species richness was still observed in one study after two full post-treatment years; this was following three spring applications of B. thuringiensis var. kurstaki in a single treatment year (Miller, 1990b).

3. Lepidopteran communities could go through a process of readjustment in the years following B. thuringiensis var. kurstaki spraying, such that whilst biodiversity starts to recover, and numbers of individual species might increase, it might not necessarily reflect the species and numbers that were present before treatment. In other words, new species might become

89 Impact of OPM control methods on oak tree biodiversity | March 2018 more abundant as they take advantage of less competition from other defoliators/new available niches, and other species will decline if they are slow at recolonization resulting in community structure that represents primary colonising species (Freed and Reeve, 2014).

4. Spring defoliators, feeding at the time of, or shortly after application of B. thuringiensis var. kurstaki, and weak-flying species are likely to be the most affected, and take the longest time to recover, following application of B. thuringiensis var. kurstaki to control oak processionary moth (Strazanac and Butler, 2005; Freed and Reeve, 2014).

5. Numbers of leaf miners may increase after spraying with B. thuringiensis var. kurstaki for control of oak processionary moth because they will face less competition from larger foliovores (Freed and Reeve, 2014). They could also be afforded some protection from B. thuringiensis var. kurstaki due to the nature of their feeding habits.

6. Photography and rearing of larvae to adulthood both appear to be necessary if identification of all Lepidoptera to species level is required (Wagner at al., 1996; Freed and Reeve, 2014).

7. Application of diflubenzuron treatment (aerially sprayed plots in the US) had significant adverse effects on total canopy arthropod diversity and abundance, with numbers of macrolepidotera and beetles significantly reduced for more than two years after application. Significant reductions in total arthropod diversity, but not abundancy, were also observed within the taxa that move up and down the tree (Butler et al., 1997).

8. The chances of detecting pesticide treatment effects at the species level are low when arthropod density, sampling intensity and replicate numbers are low. Even when these factors are increased it is difficult to show statistical significance because of high variation between samples.

9. The impacts of “no control” on non-target invertebrate species should also be considered for defoliating pest species because severe defoliation can be detrimental to species diversity and abundancy (Sample et al., 1996; Glare and O’Callaghan, 2000; Scriber, 2004; Sobczyk, 2014).

90 Impact of OPM control methods on oak tree biodiversity | March 2018 Conclusions

The native oak trees of the UK (Q. robur and Q. petraea) support a diverse array of species; over 2000 species of mosses, lichens, fungi, invertebrates, birds and mammals. Included in this statistic are a large number of invertebrate species, estimated to be in excess of 1000 species associated with Q. robur and in excess of 800 species associated with Q. petraea (excluding Nematocera) in the canopy. Of these, there are in excess of 100 macrolepidoptera species and in excess of 80 species of microlepidoptera, with the Psocoptera, Hymenoptera, Diptera, Hemiptera and Heteroptera also making up a significant proportion of the invertebrate fauna (in terms of ordinal numbers) within the canopy. It is clear that any method deployed within the oak tree ecosystem, to control pestiferous insects, can potentially impact on a large and diverse range of associated species and therefore the impact of such control methods on the biodiversity and abundance of these non-target species needs to be assessed.

Three chemical control methods are available for the control of the invasive oak processionary moth: the biopesticide B. thuringiensis var. kurstaki (formulated as DiPel® DF), the insect growth regulator diflubenzuron (formulated as Dimilin® Flo) and the broad-spectrum synthetic pyrethroid deltamethrin (formulated as Bandu®). Of these three chemicals, B. thuringiensis var. kurstaki is considered the least toxic. Products containing B. thuringiensis var. kurstaki as the active ingredient have been classified as non-toxic towards mammals, birds, earthworms and honey bees, with moderate toxicity towards aquatic life, and without long lasting effects to the environment. In contrast, diflubenzuron is highly toxic to aquatic life, with long lasting effects, and deltamethrin is highly toxic to mammals, aquatic life and honey bees; both are classified as dangerous to the environment.

Deltamethrin is a broad-spectrum contact and stomach action insecticide, with the capacity to kill a broad range of insect species, across several insect Orders, very quickly. Diflubenzuron has the ability to disrupt chitin synthesis in all arthropod species within the classes: Insecta, Arachnida, Crustacea and Myriapoda. Immature stages of a wide range of invertebrate taxa are therefore at risk, although many species are not susceptible; lepidopteran, coleopteran and dipteran species are particularly susceptible. Bacillus thuringiensis var. kurstaki on the otherhand is not a broad-spectrum insecticide but has more targeted impacts. It was originally thought to be active towards lepidopteran species only. It is certainly the case that a great many lepidopteran species in a variety of ecosystems are susceptible to B. thuringiensis var. kurstaki. There is little doubt therefore, that non-target Lepidoptera will be adversely impacted if B. thuringiensis var. kurstaki is applied to oak trees to control oak processionary moth, particularly those whose larvae feed within the oak tree canopy at the same time as oak processionary moth, and that will therefore be present at the time of any application of B. thuringiensis var. kurstaki to control oak processionary moth (a list of these species is provided in Appendix 1). Adverse impact will manifest itself in the form of larval mortality resulting, at the very least, in fewer numbers of lepidopteran larvae within the oak tree canopy and a reduction in species richness (i.e. the number of different species present). This is confirmed by numerous field studies indicating declines in lepidopteran species richness and abundance following applications of B. thuringiensis var. kurstaki; for example, to control gypsy moth in the USA. These same studies indicate that species richness and abundance begin to recover during the first post-treatment year, although species richness may still be reduced even after two full post-treatment years and may not reflect the composition of species present prior to spraying. It is considered that spring defoliators, species that

91 Impact of OPM control methods on oak tree biodiversity | March 2018 are univoltine or weak fliers will be most affected and take the longest time to recover. The authors cannot infer any conclusions on impacts that may occur following ingestion of sublethal quantities of B. thuringiensis var. kurstaki by non-target larvae because there is insufficient data in the literature on sublethal effects. Such sublethal impacts may include for example, lower reproductive ability, slower development, and greater likelihood of predation/parasitism.

Whilst it was originally thought that B. thuringiensis var. kurstaki was only active towards lepidopteran species, some literature suggests that this may not be the case. Data presented within Glare and O’Callaghan (2000) suggest that some species of Coleoptera, Dictyoptera, Diptera, Hemiptera, Hymenoptera and Isoptera (as well as some species of ticks, lice and nematodes) are susceptible to the product DiPel®. This information has come from relatively old literature (before the year 2000) and therefore presumably involved the use of old formulations. Formulations may well have advanced and been improved upon since these reports. For instance, early formulations also sometimes contained an exotoxin, which is removed from present day formulations. In addition, cross-Order activity has been reported for three of the δ-endotoxins known to be produced by B. thuringiensis var. kurstaki strain ABTS-351 (Cry1Ab, Cry1Ac and Cry2Aa) across a total of five insect Orders. However, data on cross-Order/cross-Class activity of the δ-endotoxins must be interpreted with caution for a variety of reasons, such as the dose used, how the product/toxin was delivered in the assay system, and the biological relevance of any observed toxic response. It has been suggested that the only Cry1Ab, Cry1Ac or Cry2Aa cross-Order activity to be validated is in relation to the aphid, A. pisum.

Decision makers should also consider that a “no control” approach is likely to have some impact on non-target invertebrates, particularly when the pest involved is a defoliator. Defoliation of oak would lead to a reduction in both the quantity and quality of the food resource available for all the primary trophic feeders on oak, which in turn would no doubt have knock on effects further down the food chain. A large pest population may also result in the build-up of large populations of generalist predator and parasitoid species that may predate/parasitise native species when the pest species is not around. Whichever control method is used, some impact on non-target invertebrates will occur. It is a question therefore of minimising that impact.

Finally, this literature review is not exhaustive. It does not by any means cover all aspects of the biodiversity associated with oak trees, focussing instead on the lepidopteran fauna within the tree canopy. Thus, the impacts of oak processionary moth control measures on this wider biodiversity cannot be fully understood from this review.

92 Impact of OPM control methods on oak tree biodiversity | March 2018 Recommendations

It must be noted that this document is a literature review, and as such the information contained within it is gathered from scientific publications, grey literature and information available on the internet rather than from any practical experience on the control and management options of oak processionary moth by the authors.

Following review of the literature, the authors would recommend consideration of the following aspects for assessing the impacts of oak processionary moth control on non-target invertebrate biodiversity in oak trees.

1. Evidence from the literature would suggest that of the three active ingredients under consideration (B. thuringiensis var. kurstaki, diflubenzuron and deltamethrin), it is likely that B. thuringiensis var. kurstaki will have the lesser impact on non-target organisms and oak tree invertebrate biodiversity; therefore from this point of view (i.e. invertebrate biodiversity) B. thuringiensis var. kurstaki may be the better insecticidal control option to use for a one year- only spray application to control oak processionary moth.

2. Evidence from the literature would suggest that both numbers of lepidopteran larvae and species richness will begin to improve in the first post-treatment year after a one-year spray application of B. thuringiensis var. kurstaki. However, it is important to note that the returning species richness following application may not be representative of the assemblage of species that were present on the tree prior to treatment. There is evidence in the literature to suggest that the assemblage of species will change as for example, some will be able to take advantage of less competition, previously occupied niches that become available, and the ability of species to be able to re-populate (dependent on how far away other population sources are and the ability of the species to naturally disperse or migrate into new areas). Presently there is insufficient detailed information available on the full recovery of lepidopteran species following a one-year treatment of B. thuringiensis var. kurstaki and therefore further research is required utilising a longer post-recovery period (minimum of three years).

3. There is limited evidence in the literature (and none from the UK) regarding the impacts of B. thuringiensis var. kurstaki application over a period of more than one year, whether it be applied for example, in two or more consecutive years or in alternate years and therefore no conclusions can be drawn on the impact on oak tree biodiversity when B. thuringiensis var. kurstaki (or any other insecticide) is used for controlling oak processionary moth under these circumstances. In the one study where spraying over two consecutive years was investigated, recovery in species richness and abundance were observed in the second post-treatment year (Strazanac and Butler, 2005). Further research is required to investigate the impacts of B. thuringiensis var. kurstaki application under different circumstances, such as two or more years of consecutive application or alternate years of application, coupled with a minimum of three years post-treatment monitoring following on from the year of final spray application.

4. There is currently no published data on the impact of B. thuringiensis var. kurstaki application to control oak processionary moth on other non-lepidopteran species of invertebrates that

93 Impact of OPM control methods on oak tree biodiversity | March 2018 may be present within the oak canopy at the time of application, or within the vicinity of the treated trees (for example at ground level), or for species that are not associated with oak at the primary trophic level but are dependent on oak tree feeders to complete their life cycle (for example parasitoid wasps that parasitise the lepidopteran larvae normally present in the oak tree canopy). Evidence from studies in the USA would suggest that parasitoid species can be adversely indirectly affected following application of B. thuringiensis var. kurstaki. Investigations into the wider impacts of B. thuringiensis var. kurstaki application are required. For any future study, all ‘by-catch’ found on foliage sampled for lepidopteran larvae should be collected and preserved such that the ‘by-catch’ species can be identified, and data analysed. Ideally, further methods for assessing impacts on non-target species should be considered. Possibilities include the collection of lepidopteran larvae from the field and rearing in the laboratory to assess impacts of parasitism by parasitic wasps and flies; and/or other forms of sampling besides branch sampling and sampling at different (height) levels, to sample a larger range of species that could potentially be affected.

5. The ‘by-catch’ data collected in the Freed and Reeve (2014) Richmond Park study has not been published. Further useful information regarding the impacts on oak tree biodiversity following application of B. thuringiensis var. kurstaki within the UK could be obtained if this data were to be made publicly available. This could influence the design of any future research project.

6. Indirect effects could also potentially occur on non-invertebrate species following application of B. thuringiensis var. kurstaki (e.g. the effect on species of birds that rely heavily on lepidopteran larvae present in the spring to feed their young, to name just one example). Such wider effects on the impact of oak tree biodiversity were out of the scope of this literature review. Literature is available that reports on such effects (not necessarily specifically in relation to oak processionary moth control nor necessarily from studies conducted in the UK) and this can be consulted by the reader. This aspect of oak tree biodiversity could also be reviewed separately if deemed appropriate.

7. “No control” may also negatively impact on oak tree invertebrate biodiversity if oak processionary moth larval feeding results in severe defoliation. It is recommended that the impacts of B. thuringiensis var. kurstaki treatment versus the impact of no treatment on oak tree biodiversity be investigated in an area where oak processionary moth is present and could therefore result in defoliation if no control action is taken.

8. No conclusion can be drawn as to which control option is most suitable in areas where rare species of Lepidoptera exist because insufficient evidence exists to determine the implications of using B. thuringiensis var. kurstaki in areas where rare species exist. Nest removal is therefore the most appropriate method to use to control oak processionary moth in such areas until more data becomes available for an informed decision on chemical control to be made.

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105 Impact of OPM control methods on oak tree biodiversity | March 2018 Appendix 1. List of Lepidoptera whose larvae feed on oak at some point between April and June

NB. This list is not likely to be exhaustive.

Cymatophorina diluta Polyploca ridens Watsonalla binaria Carpatolechia decorella Psoricoptera gibbosella Stenolechia gemmella Agriopis leucophaearia Agriopis marginaria Alsophila aescularia Apocheima hispidaria Biston strataria Chloroclysta siterata Comibaena bajularia Deileptenia ribeata Ennomos erosaria Erannis defoliaria Eupithecia abbreviata Eupithecia dodoneata Eupithecia irriguata Hemithea aestivaria Hypomecis roboraria Operophtera brumata Selenia tetralunaria Malacosoma neustria Neozephyrus quercus Lymantria monacha Orgyia recens Acronicta auricoma Amphipyra berbera Amphipyra pyramidea Brachionycha (Asteroscopus) sphinx Catocala promissa Catocala sponsa Cerastis leucographa Griposia (Dichonia) aprilina Dicycla oo Dryobotodes eremita Lithophane ornitopus Lithophane semibrunnea

106 Impact of OPM control methods on oak tree biodiversity | March 2018 Bena bicolorana Conistra vaccinia Cosmia trapezina Orthosia cerasi Orthosia cruda Orthosia incerta Orthosia gothica Orthosia miniosa Anorthoa (Orthosia) munda Paracolax tristalis Meganola strigula Nycteola revayana Peridea anceps Carcina quercana Diurnea fagella Diurnea lipsiella (D. phryganella) Acrobasis repandana (Conobathra repandana) Phycita roborella Acleris ferrugana Acleris literana Aleimma loeflingiana Archips crataegana Archips xylosteana Eudemis profundana Gypsonoma dealbana Pammene argyrana Pammene giganteana (P. inquilina) Pammene splendidulana Pandemis cerasana Pandemis corylana Ptycholoma lecheana Tortricodes alternella Tortrix viridana Zeiraphera isertana Ypsolopha alpella Ypsolopha lucella Ypsolopha parenthesella Ypsolopha sylvella Ypsolopha ustella Hepialus humuli (feeds underground on roots) Cossus cossus (trunk feeder)

Leaf miners: Coleophora currucipennella

107 Impact of OPM control methods on oak tree biodiversity | March 2018 Coleophora flavipennella case bearer Coleophora ibipennella Coleophora lutipennella case bearer Coleophora kuehnella case bearer Eriocrania subpurpurella Acrocercops brongniardella Phyllonorycter harrisella Phyllonorycter messaniella Povolnya leucapennella Stigmella atricapitella Stigmella basiguttella Stigmella roborella Stigmella ruficapitella Stigmella samiatella Stigmella suberivora Argyresthia glaucinella

108 Impact of OPM control methods on oak tree biodiversity | March 2018 Appendix 2. IUCN Red Data Book (RDB) categories and criteria

 RDB 1 – species appear in the Red Data Book and are categorised as endangered;  RDB 2 – species appear in the Red Data Book and are categorised as vulnerable;  RDB 3 – species appear in the Red Data Book and are categorised as rare;  RDB K – species appear in the Red Data Book but the status is unknown, although they are thought to be rare;  pRDB 1 – species are likely to appear in the Red Data Book and be categorised as endangered;  pRDB 2 – species are likely to appear in the Red Data Book and be categorised as vulnerable;  pRDB 3 – species are likely to appear in the Red Data Book and be categorised as rare;  N – species are nationally notable and have been recorded in 16-100 ten kilometre squares in Great Britain.  Na – species are nationally notable and have been recorded in 16-30 ten kilometre squares in Great Britain;  Nb – species are nationally scarce and have been recorded in 31-100 ten kilometre squares in Great Britain.

109 Impact of OPM control methods on oak tree biodiversity | March 2018