Delivering and developing oral contraceptives for grey squirrels G. Massei, J. Lane, S. Beatham, R. Pinkham, K. Rickell, M. Gomm National Wildlife Management Centre APHA Royal Forestry Society meeting, National Memorial Arboretum 30 October 2018

Human-wildlife conflicts are increasing Damage to crops, forestry, property Disease transmission Impact on native species Road traffic accidents Livestock predation Worldwide human-wildlife conflicts are increasing. Traditional methods to manage these conflicts include shooting and poisoning. However, non-lethlal methods to manage overabundant populations of wildlife are increasingly advocated by the public.

Grey vs. red squirrel Grey squirrels are currently at around 2.5 million, red squirrel numbers have reduced from approximately 3 million to 300,000.

Impact of grey squirrels Reduce grey squirrel number to decrease impact on forestry Impact of grey squirrels Conservation of native species Impact on forestry through bark stripping (£10 million per year) affects tree growth and timber value may cause death of tree, hence costs of restocking increases the susceptibility of trees to various pathogens 36 species of UK trees are damaged by grey squirrels: beech, sycamore, oak, ash, maple, sweet chestnut, etc. Reduce grey squirrel number to decrease impact on forestry Grey squirrels are currently at around 2.5 million, red squirrel numbers have reduced from approximately 3 million to 300,000. Control for 12 yrs in a beech woodland decrease % of squirrel damage from 50% to 8% of trees

Public attitudes Acceptability of grey squirrel control (n=3758) Planting trees Contraception Kill traps Shooting Live capture (+ kill) Biol. control (pine marten) Warfarin poisoning Acceptability of grey squirrel control (n=3758) Blue + Red: very acceptable or acceptable 0% 20% 40% 60% 80% 100%

we need an oral contraceptive Novel immuno-contraceptives Vaccines inducing antibodies against proteins or hormones essential for reproduction GonaCon Gonadotrophin Releasing Hormone vaccine “single-dose” injectable vaccines from our US partners 75-100% animals infertile for 2-8 years Credible practical applications BUT animals must be captured In the last 15 years, novel single-dose immunocontraceptive vaccines have been developed that offer practical applications to manage wildlife. To deliver these injectable contraceptives, animals must be captured first, with significant cost in staff time. The availability of oral contraceptives would reduce staff time as these contraceptives could be delivered with relatively less effort to more animals. we need an oral contraceptive

Fertility control applications via injectable contraceptives Wild boar: 1 dose GonaCon stopped reproduction in 92% sows for 5-8 years Feral goats: 1 dose GonaCon reduced fertility by 83-92% for > 4 years California ground squirrels: 1 dose GonaCon reduced the reproduction by 91% (year 1) and 96% (year 2) Examples of successful applications of the immunocontraceptive GonaCon to wildlife. The contraceptive was delivered via a single injection. These studies showed that GonaCon is very effective and may cause infertiility for many years.

Novel formulation to increase effect of oral vaccine Oral immuno-contraceptive vaccine targeting GnRH Biology, behaviour, population dynamics APHA & collaborators developed a novel GnRH-based oral vaccine 60% rats fed the contraceptive did not breed (control rats: 10-30%) Contraceptive fed in a bait : reduced response to the vaccine Novel formulation to increase effect of oral vaccine The project is based on a previous study, carried out by our group and collaborators, where we developed an oral contraceptive that made 60% laboratory rats infertile. When the same contraceptive was delivered through a bait, the effectiveness was reduced. Hence we are working on a novel formulation of this vaccine.

Novel formulation to deliver an oral contraceptives Spores and pollen grains Pollen/spores: natural, renewable, commercially available Genetic material removed to obtain sporopollenin exine capsules (SPECs) Proven effective delivery of drugs such as fats, oils, vitamins, enzymes, hormones, ibuprofen We formed a partnership with a UK company that patented spores and pollen grains (referred to as “SPECs”) to encaspulate and deliver a wide range of drugs. When SPECs are fed to animals (or human volunteers), the passage rate of SPEC-encapsulated drugs through the alimentary canal is slower than that of non-encapsulated drugs, possibly due to the “spiky” morphology of the SPECs. As a result, drugs are delivered more gradually and their effects seem to last longer.

SpECs for an oral contraceptive vaccine Proven vaccine delivery in SPECs fed to mice increased antibodies in mice antibody response lasted for up to 7 months a possible mechanism for oral vaccination

Developing and delivering oral contraceptives to control populations of grey squirrels Identify SPECs species Lab trials with rats to test SPEC-encapsulated oral vaccine Monitor bait uptake by squirrels (lab + field) Lab trials with grey squirrels Optimise bait uptake by squirrels Field pilot trials with squirrels to confirm efficacy Large-scale trials to monitor effects at population level Initiate trials for registration package Year 1 Year 2 Year 3+ 4 Year 5 S9 The project, which started in April 2017, will progress from laboratory trials with rats (to test various formulations of SPEC-delivered contraceptives) to captivity trials with squirrels and eventually field trials to test the effectiveness of oral contraceptives on local populations of grey squirrels. In parallel, we are developing 1. methods to monitor patterns of bait uptake by free-living squirrels, to maximise ingestion of baits containing the contraceptive and 2. methods to deliver the baits to grey squirrels only.

The project team Project leader Field studies coordinator 1 statistician Field studies coordinator Lab studies coordinator 2 lab technicians 2 modellers 4 field ecologists 2 lab ecologists Two teams in USA and France The Animal & Plant Health team comprises 14 staff in addition to collaborators in the UK, France and the US, the latter in charge of assembling the vaccine.

Year 1 results: testing oral vaccine in model species Tested SPECs of sunflower and club moss fed to rats 60% (n=12 of 20) had antibodies to oral vaccine but not sufficient to cause infertility 100% rats (n= 19) had antibodies to the injected vaccine with 95% sufficient to cause infertility We are testing a modified version of the squirrel-specific hoppers previously used to deliver warfarin. We have used Rhodamine B (RB) as a bait marker: RB appears as fluorescent bands in hair and whiskers of squirrels that ingested a bait. This marked can be used to quantify the proportion of a population exposed to oral contraceptives. More work in Year 2 on formulation of oral vaccine

Year 1 results: delivering contraceptives to grey squirrels Developed grey squirrel-specific hopper to: deliver baits that will contain oral contraceptives monitor patterns of bait uptake by individual squirrels Tested Rhodamine B (RB) to: monitor bait uptake in the population Pilot trial 51 grey squirrels trapped in 2 woods, PIT-tagged, released 48 bait hoppers with PIT-tag readers deployed We are testing a modified version of the squirrel-specific hoppers previously used to deliver warfarin. We have used Rhodamine B (RB) as a bait marker: RB appears as fluorescent bands in hair and whiskers of squirrels that ingested a bait. This marked can be used to quantify the proportion of a population exposed to oral contraceptives.

Year 1 results: delivering contraceptives to grey squirrels Bait hoppers Established how often squirrels visit hoppers > 93% PIT-tagged squirrels entered >1 hopper at least once in 4 days Rhodamine B After 4 days of RB-treated baits > 56% squirrels had RB bands in hairs We are testing a modified version of the squirrel-specific hoppers previously used to deliver warfarin. We have used Rhodamine B (RB) as a bait marker: RB appears as fluorescent bands in hair and whiskers of squirrels that ingested a bait. This marked can be used to quantify the proportion of a population exposed to oral contraceptives.

Year 1 results integrated with Defra study Model the effort required to eradicate grey squirrels via culling, contraception or culling + contraception We have developed a mathematical model to assess the impact of different methods of population control on squirrel numbers.

Year 1 results integrated with Defra study The results of the model suggest that culling, carried out using a minimum of 3-4 traps per hectare, can reduce drastically a local population of grey squirrels in a few years. However, this method has significant costs associated with staff effort as traps, that can be activated for several months, must be checked every day. Culling with different levels of effort

Year 1 results integrated with Defra study The model also suggested that integrated management, where culling is initially used to decrease the size of the population and is followed by the use of oral contraceptives, might be the most cost-effective method to reduce numbers of squirrels Culling in Year 1, followed by fertility control with different levels of efficacy

Highlights of Year 1 A method, based on SPECs, to encapsulate an oral vaccine Induced an immune response to an oral contraceptive vaccine A food hopper to monitor bait uptake patterns by individuals A bait marker (RB) to quantify the % of grey squirrels feeding on baits that will contain oral contraceptives Established captive colony of grey squirrels to test contraceptives We have developed a mathematical model to assess the impact of different methods of population control on squirrel numbers.

Scalable applications for oral contraceptives Oral contraceptives scalable to new species and contexts Non-native invasive species, incl. feral animals Over-abundant wildlife Where culling is illegal, unfeasible, undesirable To complement culling to control populations or their economic / environmental impact As public demand for non-lethal methods to manage human-wildlife conflicts increases, oral contraceptives could be applied to many species and contexts or used to complement other methods of population control

Non-lethal control : when? When lethal control is: illegal unacceptable unfeasible unsustainable environmentally hazardous ineffective BUT also complementary to lethal control

What next… Integrated Pest Management We have had spring traps for many years but we did not solve the problem Integrated Pest Management Sustained control over long period Less staff intensive More publicly acceptable Fertility control

…the squirrel team! Questions? With thanks to… giovanna.massei@apha.gsi.gov.uk