Master Thesis:

The Use of Wood Ants for Pest Control in Plantations

Speciale: Brug af Skovmyrer til Bekæmpelse af Skadedyr i Plantager

Jesper Stern Nielsen – 20107416 Department of Bioscience - Plant and Insect Ecology Aarhus University AU AARHUS May 2016 Master thesis by Jesper Stern NielsenUNIVERSITY 1

Data sheet:

Project: Master thesis in Biology Scope: 60 ECTS

English main title: The Use of Wood Ants for Pest Control in Plantations English manuscript titles: Manuscript 1: Transplantation of Formica polyctena Förster (Hym., Formicidae) into Plantation Crops for Biological Control Manuscript 2: Contribution from Wood Ants to Fruit Production and Control of Winter Moths in an Organic Apple Orchard

Danish main title: Brug af Skovmyrer til Bekæmpelse af Skadedyr i Plantager Danish manuscript titles: Manuscript 1: Transplantation af Formica polyctena Förster (Hym., Formicidae) til Biologisk Bekæmpelse i Plantager Manuscript 2: Bidrag fra Skovmyrer til Frugtproduktion og Kontrol af Frostmålere i en Økologisk Æbleplantage

Author: Jesper Stern Nielsen, 20107416 Institution: Aarhus University, Department of Bioscience – Plant and Insect Ecology

Supervisor: Joachim Offenberg

Delivered: 5th of May 2016 Defended: 31th of May 2016

Key-words: Biological control, Wood ants, Formica polyctena, Transplantation, Winter moth, Operophtera brumata, Fruit yield.

Foto – Front page: Wood ant which had captured a moth larva in an apple tree, at Æbletoften, Jutland 2015 (by Anne Aagaard Lauridsen and Jesper Stern Nielsen)

Number of pages: 107

Master thesis by Jesper Stern Nielsen 3

Table of Contents

Preface ...... 7 Summary ...... 7 Main findings ...... 7 Conclusion and suggestions for future studies ...... 8 Acknowledgements ...... 9 Manuscript 1: Abstract ...... 13 Resumé ...... 15 Dictionary ...... 15 Introduction ...... 17 Methods ...... 23 Results ...... 31 Discussion ...... 37 References ...... 43 Appendix 1 ...... 47 Manuscript 2: Abstract ...... 53 Resumé ...... 54 Dictionary ...... 54 Introduction ...... 55 Methods ...... 60 Study site ...... 60 Transplantation and maintenance of ants ...... 62 Treatments ...... 62 Data collection and arrangement ...... 63 Statistics ...... 65 Results ...... 67 Effect of winter moth larvae on leaf damage ...... 67 Effect of winter moth larvae on bud loss and apple yield ...... 68 Effect of ant activity on the number of winter moth larvae ...... 69 Effect of ant activity on leaf damage ...... 71 Effect of ant activity on apple buds and apple yield ...... 72 Effect of distance from nearest active nest on apple yield ...... 74 Ant distribution ...... 75 Observations on behavior ...... 77 Discussion ...... 81 Summary of results ...... 81 Effect of ants on yield ...... 81 Ant distribution ...... 83 Effect of ants on winter moth larvae and leaf damage ...... 83 Effect of ant presence in the apple orchard ...... 85 Problems with aphids ...... 86 Other remarks ...... 86 Conclusion ...... 87 References ...... 89 Additional thoughts, methods and tests ...... 93 Estimation of nest activity level ...... 93 Evaluating the effect of ants on winter moth larvae by differences in number, larvae weight and size 94 Evaluating effect of ants by looking at winter moth pupae ...... 95 Evaluating effect of ants by looking at the number of adult winter moths ...... 96 Evaluating the effect of ants on Monilia fructigena ...... 97 Other observations that was not used ...... 97 References ...... 98 Picture Gallery ...... 99 The Fragmentation and Transplantation Process ...... 99 Damage to Apple Trees ...... 102 Winter Moths ...... 103 Ants and Aphids ...... 104 Ant Deterrence and Predation ...... 105 Apple Trees ...... 106 The Other Stuff ...... 107

Master thesis by Jesper Stern Nielsen 6 Preface

This master thesis comprises the work of 60 ECTS, corresponding to a full year's work. The aim of the study was to examine the usability of woods ants for biological control purposes, and collect knowledge to form the basis for transplanting and examining effects by wood ants in plantations.

The project and thesis is supervised by Joachim Offenberg, Department of Bioscience – Plant and Insect Ecology, Vejlsøvej 25, 8600 Silkeborg, Denmark. The project was a part of AntAid (Project ID: 15729).

The thesis consists of four parts. Part 1 comprises a summary of the main findings of the two studies, a conclusions and suggestions for future studies. Part 2 includes two individual manuscripts that are written with a structure of a paper, but is more detailed than would be expected of publication-ready manuscripts. Part 3 includes an overview of thoughts, experiments, and field observations that was not included in the two manuscripts. And lastly, part 4 comprises a picture gallery.

The manuscripts deal with each their subject within the theme “The Use of Wood Ants for Pest Control in Plantations”. The first manuscript deals with the issue of whether it is possible to establish wood ant nests in different plantations, while the second manuscript deals with the effect of ants in a plantation.

Summary

Main findings

The main findings in this study were:

1. It was possible to fragment and transplant Formica polyctena into highly different types of plantations, and establish them in artificial nests at the exact position of transplantation, despite the fact that movement was observed during the study. 2. The number of successfully established nests in August 2015 was 20 out of 24, corresponding to 83.3 %, while in April 2016 establishment success was 18 out of 24, corresponding to 75 %. 3. The achieved nest density in the three plantations was 20 nest/ha, 7.3 nest/ha, and 10 nests/ha, in the organic apple orchard, the organic Christmas tree plantation, and the conventional Christmas tree plantation, respectively. 4. Apple trees with high ant activity had an average apple yield of 5.1 apples per tree, which was more than twice as much as trees with no or low ant activity.

Master thesis by Jesper Stern Nielsen 7 5. No clear pattern of significance was seen from ant activity on winter moth larvae, apple bud loss, or leaf damage. 6. The distance from trees to the nearest active nest was significantly negatively correlated with the apple yield on each of the observation dates. 7. Observations regarding ant presence included: Escape behavior of winter moth larvae, predation by ants on winter moth larvae and other arthropods except spiders and harvestmen, as well as aphids being attended by ants both on apple trees and other plant species.

Conclusion and suggestions for future studies

This study shows that the wood ant F. polyctena can be transplanted into highly different plantations, where its ecosystem services are needed. The method used for transplantation resulted in nest establishment at the exact position of transplantation, and can thus be a guideline for future studies. The effect of the ants in an apple orchard has proven to cause an increase in yield, despite no measurable effect of ants on the underlying levels: leaf damage and number of winter moth larvae. However, the ants have been seen to predate and deter other arthropods, and this effect would be interesting to examine more thoroughly. Such study should include: a deeper look into the effect of ants on the number of winter moth larvae, the difference in weight or size of winter moth larvae between trees with high and no ant activity, and the effect of ants on fruit quality and yield weight. Also, more investigations on the distribution and number of ants in trees from transplanted nests are of importance. Furthermore, nest transplantations into other plantation types should be implemented and examined.

Master thesis by Jesper Stern Nielsen 8 Acknowledgements

A great thank you to Joachim Offenberg for the support, inspiration, and valuable feedback during preparations of the study and on the shaping of the manuscripts. I thank Mogens Gissel Nielsen for his help during excavation of ant nests, and for sharing his knowledge about wood ants. I thank Joachim Westergaard Lassen for great sparring in generating and evaluating ideas, and for help in the field. A great thank you to Anne Aagard Lauridsen and Jørgen Aagaard Axelsen for comments on the manuscripts and for idea evaulation. Also, thank you to Jørn Stern Nielsen for help with technicalities and comments on the manuscripts. Also, thanks to the forest ranger of Løvenholm for permission to use the area, and to the plantation owners Jens H. Petersen and Karin Alsgård Jensen, Bent Møller, and Steen Sørensen, for letting us use their plantations. I also thank Jens H. Petersen for sharing his great knowledge on apple growing, for his fantastic pictures of the transplantation, and for being welcoming on my many visits in Æbletoften. Thanks to Steen Borregaard, Steen Brock and Borregaard Bioplant for help in acquiring material and finance, and for exchange of ideas during the process. Thanks to Lise Lauridsen for help to find and get equipment, and for sharing her knowledge of data-collection in the field. Thanks to all who helped in the field with fragmentation and transplantation: Mogens Gissel Nielsen, Joachim Westergaard Lassen, Andreas Niebuhr, Maja Møholt, Klaus Berg, Line Dam-Hansen, and Steen Brock. Thank you for support from family and friends. Lastly, I would like to thank my girlfriend, Anne Aagaard Lauridsen, for her support and great patience during the whole process.

Master thesis by Jesper Stern Nielsen 9

Manuscript 1:

Transplantation of Formica polyctena Förster (Hym., Formicidae) into Plantation Crops for Biological Control

Jesper Stern Nielsen

Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark

Master thesis by Jesper Stern Nielsen 11

Photo by Jens H. Petersen

Transplantation of Formica polyctena Förster (Hym., Formicidae) into Plantation Crops for Biological Control

Jesper Stern Nielsen

Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark

Abstract

1. Predaceous ants can be used as a biological control (biocontrol) agent against arthropod pests as an alternative to chemical pesticides. Wood ants of the Formica rufa group possess many great qualities for biocontrol purposes and have thus in many cases been transplanted into areas to control pests. 2. Fragmentation was carried out on three donor nests of the species Formica polyctena in the spring 2015, and the fragments were transplanted into an organic apple orchard (TS1), an organic Christmas tree plantation (TS2), and a conventional Christmas tree plantation (TS3). The percentage of successfully established nests was in August 2015 83.3 distributed on 62.5, 100, and 87.5 in TS1, TS2, and TS3, respectively. The percentage of successfully established nests was in April 2016 75 distributed on 62.5, 100, and 62.5, in TS1, TS2, and TS3, respectively. The achieved nest density in the three plantations was 20 nest/ha, 7.3 nest/ha, and 10 nests/ha, in TS1, TS2, and TS3, respectively. 3. The establishment success shows that it is possible to transplant F. polyctena into highly different types of plantations. Unlike most other studies nest establishment was achieved at the exact position of transplantation, despite movement was observed during the study.

Key words: Transplantation, Fragmentation, Wood ants, Formica polyctena, Biological control.

Master thesis by Jesper Stern Nielsen 13

Resumé

1. Brug af myrer til biologisk bekæmpelse af arthropod-skadedyr er et alternativ til brugen af kemiske pesticider. Skovmyrer fra Formica rufa-gruppen har mange egenskaber, der gør dem brugbare til biologisk bekæmpelse, og de er derfor i flere forsøg blevet transplanteret til nye områder for at bekæmpe skadedyr. 2. Fragmentering blev udført på 3 donortuer af arten Formica polyctena i foråret 2015, og fragmenterne blev transplanteret ind i en økologisk æbleplantage (TS1), en økologisk juletræsplantage (TS2) og en konventionel juletræsplantage (TS3). I august 2015 var 83,3 % af tuerne succesfuldt etableret, fordelt som 62,5, 100 og 87,5 % i henholdsvis TS1, TS2 og TS2. I april 2016 var 75 % af tuerne succesfuldt etablerede, fordelt på 62,5, 100 og 62,5 % i henholdsvis TS1, TS2 og TS2. Den opnåede tæthed af tuer i de tre plantager var henholdsvis 20 tuer/ha, 7,3 tuer/ha, og 10 tuer/ha, i TS1, TS2 og TS3. 3. Transplantation- og etableringssuccesen viste, at det er muligt at transplantere F. polyctena ind i tre vidt forskellige typer af plantager. Modsat størstedelen af andres forsøg lykkedes det at etablere tuerne på præcist det sted, hvor de blev placeret ved transplantationen, selvom flytning blev observeret under studiet.

Dictionary

Due to the structural complexity of Formica polyctena colonies, some terms used in this paper are here defined:

Nest: Referring to a single dome within a colony.

Colony: A community of ants that are socially connected and live close together in one or several nests. Resources, individuals and brood are exchanged via connecting trails (Definition inspired from Bugrova & Reznikova, 1990; and Oxforddictionaries.com, accessed 23-03-2016).

Polydome colony: Ant colonies that inhabit several spatially separated, but socially connected nests (Ellis & Robinson, 2015).

Polygyne: The condition of having more than one egg-laying queen in a colony (Oxforddictionaries.com, accessed 23-03-2016).

Budding: Formation of new nests by colony splitting (Mabelis, 1994).

Master thesis by Jesper Stern Nielsen 15

Introduction

Arthropod pest can cause great damage to agricultural plant production. Tree defoliation, deformation of branches or conifer needles, damage to flowers and buds, and loss of yield are some of the negative effects caused by arthropod pests, which can have a large economic impact for the owners of orchards and plantations (Müller, 1956; Lind et al., 2003; Wesolowski & Rowinski, 2006; Chang et al., 2012; Tesanovic & Spasic, 2013; De Ros et al., 2015). Efforts to limit this impact have included the use of chemical pesticides, which have been effective, but are now facing challenges in terms of pesticide resistance, more strict environmental legislation, and reduced development of new chemical compounds for pest control (Hajek, 2004). In addition there is an increased concern about the negative side-effects of pesticides on humans, animals and on the environment, and therefore a demand for a non-chemical environmentally sustainable solution (Hajek, 2004). Biological control (biocontrol) is a solution where living organisms are used to control pests and is based on the idea of using nature’s own agents to control the pests (Hajek, 2004). A promising agent for this purpose is ants.

Records from 324 B.C. are some of the first to describe habitat manipulation to increase the natural population of the weaver ant Oecophylla smaragdina in citrus trees controlling large boring beetles and caterpillars (Way & Khoo, 1992; Hajek, 2004). The effect of ants has since then been studied in a wide array of experiments, including both direct predatory effects and indirect effects on their surroundings. Some of the very positive effects of the presence of ants include: better tree growth (Whittaker & Warrington, 1985), higher yield (Peng & Christian, 2008), less leaf damage (Skinner & Whittaker, 1981; Warrington & Whittaker, 1985; Karhu, 1998; Rosumek et al., 2009), a lower number of Lepidoptera larvae (Skinner & Whittaker, 1981), antibacterial and antifungal effect (Chapuisat et al., 2007), soil improvement and nutrient cycling (Way & Khoo, 1992), and nutrient deposition from ant manure to host trees (Pinkalski et al., 2015; Vidkjær et al., 2015). The effects of ants are unfortunately not only positive. Negative effects include increased number of damaging aphids (Hemiptera sp.) (Karhu, 1998), nuisance for farm workers in form of biting (Peng & Christian, 2008; Offenberg, 2015), and predation on or deterrence of beneficial insects like pollinators or other predators (Tsuji et al., 2004; Assunção et al., 2014). Fortunately solutions are available to eliminate or reduce many of the negative effects (Offenberg, 2015), and the many positive qualities of ants thus seems to make the negative ones negligible.

The behavioral qualities of ants make them very suitable as biocontrol agents. They have a social way of life that normally includes a large worker force, whose aggressiveness towards many other organisms allows the ants to be positioned on a high trophic level (Gridina, 1990; Way & Khoo, 1992). Also, the ants are generalist feeders and thus not limited to a single type or life stage of prey, which makes them

Master thesis by Jesper Stern Nielsen 17 suitable in deterring or preying upon a wide variety of insect pests (Gridina, 1990; Paulson & Akre, 1992b; Way & Khoo, 1992; Mabelis, 1994). Ants are difficult to satiate because of the high number of workers, queens, and brood in their colonies that requires a large and continuous amount of food (Paulson & Akre, 1992b; Offenberg, 2007). Especially the queen(s) and brood need large amounts of protein, which they can get from gathered prey (e.g. pest insects in a plantation) (Paulson & Akre, 1992b; Offenberg, 2007). Because ants can store gathered food within a nest for later use, they do not respond on satiety but on prey density (Way & Khoo, 1992). If prey is absent or prey density is low the ants can still remain abundant because they can cannibalize their own brood or switch to another type of prey that is more abundant (Lafleur, 1941; Risch & Carroll, 1982; Paulson & Akre, 1992b; Way & Khoo, 1992). This also allows the ants to stay in an area for a long time. In addition to the behavioral qualities, ants possess the quality of being adaptive to the surrounding environment (Way & Khoo, 1992). They show high ecological flexibility, which makes them well-distributed and abundant in many types of habitats (Pisarski & Czechowski, 1990; Douwes et al., 2012). Also, they remain more or less stationary, due to their lack of wings on workers, but are able to spread into other areas through winged mated queens (Mabelis, 1994).

In northern Eurasia wood ants of the Formica rufa group are particularly suitable as biocontrol agents, because they are the dominant invertebrate predator in woodland and have no significant ant competitors (Savoläinen & Vepsäläinen, 1988; Way & Khoo, 1992). The wood ants are primarily found in the edge of conifer woods where they live in mound-shaped nests constructed by conifer needles, twigs, and other litter (Douwes et al., 2012). Wood ants are typically active from early spring (approximately March) when the ants awake from hibernation and till autumn (approximately October) where they again go into hibernation (Rosengren et al., 1979; Douwes et al., 2012). A minimum foraging level is observed at about 6°C though traffic has been observed from a nest at even lower temperatures (Rosengren et al., 1979). With a thermal activity threshold that is consistent to the thermal activity threshold of the majority of other arthropods, wood ants have the potential of subduing and collecting large numbers of arthropod pests. Surveys and estimates indicate that ants from a medium sized wood ant nest that include approximately 100,000 foragers can collect somewhat between 64,000 and 8,000,000 food items per year (Adlung, 1966). Though the great majority of the collected items include pieces of chitin and carrion, or insects that have not been killed by the ants, which is of no direct interest from a biocontrol perspective, the contribution to removal of pest insects is still present (Rosengren et al., 1979). Rosengren et al. (1979) estimated that 3200 Lepidopteran and sawfly larvae were carried back to their wood ant study nest each day in late June and early July, which shows the potential of ants to control defoliators and other damaging arthropods. Though wood ants also remove beneficial arthropods (Adlung, 1966), their potential in controlling arthropod pests makes them appealing as biocontrol agents.

Master thesis by Jesper Stern Nielsen 18 Despite the ability of ants to spread naturally to other areas a more rapid and targeted introduction of the ants for biocontrol purposes could be desirable. Transplantation is a way of artificially moving ants to areas where their ecosystem services are needed, and such introduction enables the ants to be used in other types of habitats than they normally occur in. In plantations and orchards pests can be present in high densities and the predator to pest-ratio is often low. A transplantation of ants can increase this ratio and may rapidly secure an effective density of predators (e.g. ant workers) to control the pests. The issue on whether transplantation is bio-pollution should of cause be considered, but the transplantation of native species to man-made plantations and orchards will most likely not be of large concern.

Transplantation of wood ants for pest control purposes has been practiced and studied for several years. In Germany, the first description of experiments on the artificial founding of red wood ant colonies for biocontrol purposes dates back to 1862, but a method for large scale founding was not developed and described until 1926 by Cantzler and 1938 by Gösswald (Wellenstein, 1973). A description of how to found wood ant nests artificially was given by Gösswald (1951 in Wellenstein, 1973). The method comprise the removal of nest material including workers and queens from a donor nest in March or April, transport them in 50-100 L containers, and pour the nest material with ants out on an old tree stump at a sunny forest road edge or in a clearing, after which the ants would build a new nest close to the place of release (Wellenstein, 1973). Several transplantation experiments have been conducted in Germany by Gösswald and Cantzler and in Italy by Pavan (Wellenstein, 1973; Gösswald, 1990), but these have not been documented in English, and the linguistic barrier has caused their tremendous works to get less attention than they deserve. To mention some of their experiments, Gösswald transplanted twenty-five 200 L fragments of Formica lugubris into the Gramschatzer Forest, Germany in 1965 (Gösswald, 1984). Of the 25 fragments, 22 (88 %) plus a number of buddings survived until 1973 (Gösswald, 1984). An example of transplantation by Pavan (1966 in Gösswald, 1990) is the transplantation of nests of F. lugubris from the Alps to Apennine Mountains and to the island of Sardinia, Italy. The success rate is to my knowledge unknown. Wellenstein (1973) has in the years from 1952 to 1973 succeeded in founding 890 new ant nests (primarily Formica polyctena) in very different forest biotopes in south-west Germany by transplantation. The transplantation method followed the method by Gösswald (1951 in Wellenstein, 1973), and the ants constructed nests close to the place of their release. Most nests were transplanted in spring, because other experiments showed that nests founded in the summer did not thrive and died off within a few years (Wellenstein, 1973). Bradley (1972) transplanted nests of Formica obscuripes into three young jack pine plantations in Manitoba, Canada in 1969. The transplantation was done by digging up nest material and ants and put them in a large plastic bag. The surrounding sand was put in separate bags. At first all the content was placed in a hole, roughly the same size as the content of bags from one nest, but this resulted in large numbers of buried ants. In

Master thesis by Jesper Stern Nielsen 19 later transplantations this was avoided by pouring the sand in a shallow ring on the ground, around a hole only deep enough to remove ground cover, where the rest of the nest material was placed. Bradley (1972) experienced that some nests did not move from their exact position of transplantation in a young jack pine plantation, but experienced that others moved and merged both in that plantation and in older jack pine plantations. Finnegan (1975) succeeded in transporting F. lugubris from Italy to Quebec, Canada, and transplant it in a mixed conifer forest in 1971. The transplantation involved approximately 1.3 million worker ants and 2000 mated queens that were shipped with little nest material in well-ventilated plastic barrels. In Canada the nest material was separated from the ants and was autoclaved, while the ants were released around 6 tree stumps, each separated by approximately 50 m and covered with dead branches and pine needles (Finnegan, 1975). The nests were abandoned within 3-4 days, and about 35 small, natural nests were formed. Finnegan (1975) experienced great fluctuations in colony numbers and positions as the ants in September had re-grouped into 5 large nests and 10 smaller nests, which overwintered successfully. In the summer 1972 re-grouping again occurred and resulted in 3 large nests and 2 of a medium size by October. No change in nest number occurred in 1973, but an increase in nest sizes was observed. Finnegan (1975) also transplanted another colony of F. lugubris from Italy to a pine stand in Quebec, Canada, in 1973, which resulted in the establishment of 40 nests in the first growing season, and after re-grouping resulted in 32 nests in October, 1973. A follow-up on the transplantations by Finnegan (1975) was made by Storer et al. (2008). 34 years after the transplantation 114 nests of varying size were found in the transplant area, of which 21 were abandoned. Storer et al. (2008) also estimated that the population size of the transplanted ants had increased from the start estimate of 1.3 million ants based on nest volume to an estimate of 8.1 million ants. Wilkinson et al. (1980) made 5 transplantations of Formica integra nests to a pine and oak site near Florida, USA in 1973. The transplanted nests were created artificially from sterilized nest material, placed over a partially-rotted oak wood that was positioned in a plastic arena, inside a 2x2x2 m woven fabric cage. Plexiglas tubes with metal screens led from the arena to the ground outside the cage, and prevented the access of larger insects. Unfortunately, all transplanted F. integra ants was killed by carpenter ants Camponotus abdominalis floridanus within periods of 5 days to 14 weeks. Pisarski & Czechowski (1990) transplanted nests of F. polyctena to Gorce National Park, Poland, to increase the resistance of the local forests to any future pests. Nests were transplanted with a distance of 20-30 m over several years. Nests transplanted in the same year often merged, while nests from the same colony, but transplanted in different years did not merge. Another type of transplantation was carried out by Campbell et al. (1991), who experimented with designing a nesting box for transplantation of Formica exsectoides. On each of 3 dates, a cohorte of 4 ant nests were transplanted into a jack pine plantation in nesting boxes (changing in design according to observations), but all nests only survived in 3 to 7 weeks or were observed

Master thesis by Jesper Stern Nielsen 20 abandoning the nesting boxes and establish at new locations. Paulson & Akre (1992a) also used nesting boxes and succeeded in transplanting the wood ant species Formica neoclara into a pear orchard in Wenatchee, Washington. 22 out of 27 transplanted nests were successfully established outside but within 4 m of the nesting boxes, and two years after introduction the contribution from the ants to control pests were seen (Paulson & Akre, 1992a). Czechowski & Vepsäläinen (2009) managed to artificially establish wood ant colonies on several islands in the Tvärminne archipelago, Finland. One nest of Formica polyctena managed to survive on one of these islands for 22 years, despite living conditions that probably are close to the limit for the species. Sorvari et al. (2014) transplanted 26 nest fragments of Formica aquilonia into forest areas in the Laukaa-Konnevesi region of Central Finland. The fragments consisted of 400 L of nest material that was taken from the core of 26 donor nests without causing severe damage to the nests, and were transported in plastic buckets to 26 study stands. Despite re-grouping and relocation, 20 out of the 26 transplanted nests survived. Despite many cases of transplantations, few have managed to establish nests at the exact position of transplantation. An exception is the transplantations by Bradley (1972), who successfully transplanted and established non-fragmented nests. Different Formica species were used in the mentioned cases of transplantations, but for several reasons F. polyctena was chosen for this experiment.

The wood ant F. polyctena is a native species to Denmark (Douwes et al., 2012) and has many structural and reproductive qualities that make it suitable for transplantation and biocontrol purposes in plantations and orchards. F. polyctena are polygynous and polydomous which enable it to have several related nests in an area (Mabelis, 1994). The multiple queens in a nest make the colony less vulnerable to the loss of a single queen (Way & Khoo, 1992), but also allow an effective production of workers which decreases the time for the ants to reach beneficial population densities (Paulson & Akre, 1992a; Way & Khoo, 1992). Ants from a F. polyctena colony can potentially exploit an extensive area where they can control and deter pests without experiencing intraspecific competition between the nests (Lafleur, 1941; Gridina, 1990; Way & Khoo, 1992; Ellis & Robinson, 2014). The relatedness also results in division of gathered resources between nests by trails (Finnegan, 1975), which together with the feeding behavior of the ants could favor an effective removal of pests. Colonies of the F. polyctena stay more or less in the same area due to absence of wings on workers, but can spread by budding and thus contribute to a higher density of nests in a transplantation area (Mabelis, 1994). Also, the F. polyctena species show high ecological flexibility and can adapt to many types of habitats (Pisarski & Czechowski, 1990), which gives it great potential for transplantation into different habitats for biocontrol purposes.

Master thesis by Jesper Stern Nielsen 21 The many qualities of F. polyctena make it a desired agent to control pests in different plantations and orchards. The aim of this study was thus to test whether it was possible to fragment, transplant and successfully establish nests of F. polyctena in three highly different types of plantations. A further purpose of the study was to observe the movement of ants after transplantation, and the number and density of successfully established nests. The number of successfully established nests was assessed in August 2015 and in April 2016. Further, a method for transplanting the wood ant F. polyctena successfully is presented.

Master thesis by Jesper Stern Nielsen 22 Methods

In April 2015 two F. polyctena colonies were chosen as donor colonies for transplantation. Both colonies were situated in a conifer forest at Løvenholm, East Jutland, Denmark, but had no contact with each other. Each colony consisted of multiple adjacent nests connected by ant trails. The species identity of the colonies was confirmed by the grouped arrangement of multiple nests, the content of several ovipositing queens in each nest, and the morphological trait of a sparse number of erect hairs on the head and alitrunk of the ant workers (Adlung, 1966; Collingwood, 1979; Bugrova & Reznikova, 1990; Douwes et al., 2012).

The 27th, 28th and 29th of April, 2015, a total of three nests were fragmented and transplanted from the two donor colonies into three different types of crop plantations: an organic apple orchard, an organic Christmas tree plantation, and a conventional Christmas tree plantation. The three donor nests were chosen by their size (Medium sized nests) and their highly visible activity level. One donor nest was fragmented and transplanted into one plantation on each of the dates. Ten days before the fragmentation eight tree stumps were placed on each donor nest for the ants to leave scents. These tree stumps were later used in the creation of artificial nests.

The fragmentation of a donor nest was done by first digging up the upper part of the nest in horizontal layers with shovels and distributing each layer into eight 90 L tubs (Work>it by Millarco®). The content of each tub (70-90 L of nest material) represented one fragment that would serve as nest material in the construction of the artificial nests. When the lower layers of sandy soil were reached, these layers were distributed into smaller buckets, assuring that there was approximately 20 L sandy soil or more for each fragment. Ovipositing queens, eggs, and larvae were primarily found in the lower sandy soil layers, while pupae, mated but non-ovipositing wingless queens, and winged queens were found in the middle and upper layers of the nest (figure 1). Workers were found in all layers and furthermore large numbers of resin lumps were found in all layers. A search for ovipositing queens was made by searching the sandy soil in trays thoroughly with tweezers. Ovipositing queens found were kept in closed plastic cups with moist moss to keep humidity. Buckets and tubs were covered with tulle to increase air replacement. The tulle was fastened with an elastic strap. This was done to reduce the self-damaging effects of the ants’ chemical defense of formic acid (Mogens Gissel Nielsen, Personal communication). Tubs and buckets were transported to the transplantation sites on a trailer.

Master thesis by Jesper Stern Nielsen 23 Conifer needles Pupae

Winged queens

Mated wing-less queens Resin

Larvae Ovipositing queens Eggs Larvae

Tree stump

Figure 1: Sectional view of a F. polyctena nest (Modified from Gösswald, 1989). Mated wing-less queens, winged queens and pupae are found primarily in the upper part of the nest, while ovipositing queens, eggs and larvae are found primarily in the sandy soil or in the tree stump.

The first transplantation site (TS1) was an organic apple orchard called “Æbletoften” situated near Tirstrup, East Jutland, Denmark (56° 18' 36.9"N, 10° 41' 28.1"E). The orchard covered an area of approximately 1.3 ha and included about 3000 row-arranged trees of 11 apple varieties. The transplantation area covered about 0.25 ha (figure 2) and included 9 rows of 5 different apple varieties, separated by a distance of 3.5 m between rows. A total of 425 approximately seven years old trees was included in the rows in the transplantation area, and were fixed on wires with a distance of 1 m between trees in a row. Crab apple varieties (Malus sp.) were positioned at 14 random positions in the study area and were used for pollinators. No herbicides or insecticides were used, but the area between the trees was hoed mechanically every week or every second week and the grass between the apple tree rows were mowed occasionally. Due to vegetation strips between and around the rows the height of vegetation and the species composition varied. The most common herbs and grasses were meadow-grass (Poa sp.), dandelium (Taraxacum sp.), orchard grass (Dactylis glomerata), couch grass (Elytrigia repens), common tansy (Tanacetum vulgare), cow parsley (Anthriscus sylvestris) and creeping thistle (Cirsium arvense). The

Master thesis by Jesper Stern Nielsen 24 difference in vegetation structure allowed both shaded paths and paths with plenty of sun. Forests to the west and east of the plantation provided wind cover, but from south only little wind cover was provided by the apple trees themselves. Despite the area being drained by drainpipes, standing water was sporadically found in more rainy months (September to November), and potentially caused flooding risk inside the ants nests. No conifer trees were present in the transplantation area.

TS1-N3 TS1-N7

TS1-N2 TS1-N4

TS1-N8 TS1-N5 TS1-N1 TS1-N6

Figure 2: Transplantation site 1 – Organic apple orchard – “Æbletoften”. The ant nests are situated in vegetation strips between and surrounding the nine rows of apple trees. A forest to the East and West provided wind cover, but from south only little wind cover was provided by the apple trees. Asterisks indicate the positions of the artificial transplant nests.

The second transplantation site (TS2) was an organic Christmas tree plantation called “Lilleheden” situated near Veng, Central Jutland, Denmark (56° 06' 11.7"N, 9° 54' 13.3"E). The plantation covered an

Master thesis by Jesper Stern Nielsen 25 area of approximately 3 ha and was divided into different sections. Four sections covering about 1.1 ha were chosen for the transplantation experiment (figure 3). Section 1 and 2 were primarily Noble fir (Abies procera) and Nordmann fir (Abies nordmanniana) of different ages (heights 0.2-6 m), while section 5 and 6 were mainly Norway spruce (Picea abies) of different ages (Tree heights: 0.2-6 m). A random distribution of the trees allowed both plenty of sunlight and shaded paths on the ground. Wind was reduced by the random distribution of tall and low trees plus a number of tall trees (6 m plus) that surrounded the plantation. Neither insecticides nor herbicides were used, but sheep were used for grazing. The dominating herbs and grasses were red fescue (Festuca rubra), perennial rye-grass (Lolium perenne), spear thistle (Cirsium vulgare), and common nettle (Urtica dioica). Standing water was not observed in the vicinity of the nests.

TS2-N3 TS2-N1

TS2-N7 TS2-N8

TS2-N4 TS2-N6 TS2-N2 TS2-N5

Sect. 1 Sect. 2 Sect. 3 Sect. 4 Sect. 5 Sect. 6

Figure 3: Transplantation site 2 - Organic Christmas tree plantation – “Lilleheden”. The area is divided into six sections. Section 1 and 2 were mainly Noble fir (Abies procera) and Nordmann fir (Abies nordmanniana), while section 5 and 6 mainly were Norwegian spruce (Picea abies). Tall trees provide wind cover from South, East and West. Asterisks indicate the positions of the artificial transplant nests.

Master thesis by Jesper Stern Nielsen 26 The third transplantation site (TS3) was a conventional Christmas tree plantation situated near Beder, Jutland, Denmark (56° 03' 15.9"N, 10° 14' 05.6"E). The plantation area covered approximately 1.6 ha of 2-3 years old (Tree heights: 0.3-1.6 m) Nordmann fir planted in squared patterns with a 1.1 m distance between trees. Herbicides (DFF® by Bayer CropScience, and Roundup® by Monsanto) and insecticides (Mospilan® by Nordisk Alkali) were normally used in the area, but during the study period no insecticides were used in the 0.5 ha transplantation area (figure 4). The species composition and vegetation height were homogenous and were dominated by common horsetail (Equisetum arvense), though other species as common bent (Agrostis capillaris) and creeping thistle (C. arvense) were present. The small trees and low vegetation allowed plenty of sunlight for the ants, but shaded paths were also available under the vegetation. The area had tall trees to the north and the east, but was more or less exposed to wind from south and west. Standing water was not observed in the vicinity of the nests.

TS3-N8 TS3-N7

TS3-N6 TS3-N5 TS3-N1 TS3-N2 TS3-N3 TS3-N4

Figure 4: Transplantation site 3 - Conventional Christmas tree plantation – near Beder (56° 03' 15.9"N, 10° 14' 05.6"E). The tree species was Nordmann fir (A. nordmanniana) planted in squared patterns with a 1.1 m distance. Old conifer trees gave shelter from wind from North and East, but not from South and West. Ant nests were positioned between trees. Asterisks indicate the positions of the artificial transplant nests.

Master thesis by Jesper Stern Nielsen 27 Transplantation of a nest into a plantation was constructed the same way in all three plantations; eight positions were chosen as transplant positions in each plantation (figure 2, 3, and 4). A transplant position was chosen by the following criteria (inspired by Kilpeläinen et al., 2008; Sorvari et al., 2014): 1) Sunlight exposure available, 2) Low risk of standing water (relatively high elevation), 3) No other ant nests visible at a distance of 3 m (unfamiliar wood ant nests were not observed in the plantations). In TS1 artificial transplant nests were positioned with a distance of approximately 15-20 m, except for two nests that were positioned at a distance of approximately 10 m due to soil conditions. In TS2 artificial transplant nest positions had a distance of minimum 25 m. In TS3 the distance between artificial nests was approximately 15-20 m. At the chosen transplant position a hole with a diameter of approximately 50 cm and a depth of 15-25 cm was dug. Sandy soil from the fragmentation was placed in the bottom of the hole for drainage. One of the tree stumps placed on the donor nest ten days before fragmentation was placed on the sandy soil, and the nest material and ants from a 90 L tub was gently poured over the piece of wood (figure 5). It was avoided to shake the tub too much, as this would squeeze the ants and make it difficult for them to settle the needles properly in the new transplanted artificial nest. Two to four of the ovipositing queens kept in plastic cups were added to each artificial nest two-three hours after transplantation, when a nest structure with ant entrances were observed in the transplanted artificial nests. This allowed the queens to hide inside the nest after being examined by the workers. Artificial transplant nests in TS3 experienced strong wind and some rain events the days after transplantation, but subsequently seemed to recover.

During the first week each artificial transplant nest was provided sugar dough (Ambrosia®, 85 % sucrose, Nordzucker) and water next to the nests to assure easy access to sugar in order to facilitate establishment (figure 5). The sugar dough was served in perforated cash boxes fixed to the ground by pegs to avoid disturbance from badgers (Meles meles), deer (Cervidae) etc., and the water was served in a plastic container with a piece of bark to secure ease and safe access. After one week the eight artificial transplant nests in the organic apple orchard (TS1) were fed with sugar dough in small tubes in the apple trees, and two nests in each of TS2 and TS3 were fed in small tubes in the Christmas trees (as a part of another experiment by Lassen, 2015). The rest of the nests continued to be fed in the cash boxes during the spring and summer. On the 16th of June and on the 11th of October 2015 each artificial transplant nest were provided one sack of extra conifer needles from dried cut-off branches of Christmas trees, next to the nest. In TS2 problems were experienced with birds spoiling the nests. To avoid this, chicken wire nets were used to cover the nests, which kept birds from disturbing the nests but allowed ants to move freely and to expand the nest size.

Master thesis by Jesper Stern Nielsen 28

Figure 5: Sketch of the artificial transplant nest setup.

The activity in the artificial transplant nests was recorded during the annual active period of the ants, from May to October, to evaluate the movement of ants after transplantation (mainly looked at in the apple orchard). The activity in the nests was evaluated by visually estimating activity levels of ants on the surface of the artificial transplant nests. The activity levels were rated as categories: 0: No activity (No ants visible), 1: Low activity (1-20 ants visible), 2: Medium activity, 3: High activity. The categories medium and high activity were differentiated by whether the ants in average had a density of approximately under or over 10 workers per 100cm2. Due to the spatial difference in where ants were situated on the nests, and due to a typical high number and speed of ants on the nests the assessment method was somewhat arbitrary and depended on the subjective assessment of the observer. In this experiment these assessments were made exclusively by the same observer. It was attempted to make the visual activity evaluation between 12.00 and 13.00 on days with sunshine, to enhance the likelihood of a standard evaluation, though such one cannot be fully accomplished due to variation in many environmental factors. Activity was evaluated without interference with the nests, unless no activity was observed. If no activity was observed the nest was tapped with a pencil to attract any present ants. If no activity was then observed, the nest was considered as a category 0. The movement of ants after transplantation was examined for TS1, TS2 and TS3 by plotting the activity levels (category level 0 to 3) against the dates of observations in a graph. No ants in TS1 were observed to establish outside of the artificial transplant nests. Activity levels in TS1 were assessed 16 times during the ant’s activity season (01-05-2015, 10-05-2015, 13- 05-2015, 22-05-2015, 27-05-2015, 04-06-2015, 11-06-2015, 21-06-2015, 28-06-2015, 04-07-2015, 16-07- 2015, 23-07-2015, 23-08-2015, 02-09-2015, 15-09-2015, 11-10-2015). Observations were only carried out three or four times during the season in TS2 (12-05-2015, 21-05-2015, 03-06-2015, and 12-08-2015) and TS3 (02-05-2015, 19-05-2015 and 12-08-2015) and the movement not followed as intensely as in TS1.

Master thesis by Jesper Stern Nielsen 29 To evaluate whether ants were short-term established (active in the first season after transplantation), activity levels of artificial transplant nests were recorded the 12th of August in TS2 and TS3, and the 23rd of August in TS1, 2015. Activity levels of the individual nests from the given days were interpreted as not established (category 0 and 1) or successfully established (category 2 and 3). This was done because previous experience showed that nests with low activity not necessarily are well-established (ants on the nest surface does not necessarily mean activity in the nest, Nielsen, 2015). Therefore, only nests that showed medium or high activity were considered successfully short-term established.

To evaluate whether ants where long-term established (active for more than one season) the activity of artificial transplant nests were recorded the 1st of April for TS2 and TS3 and the 8th of April for TS1, 2016. Again, activity levels from the given days were interpreted as not established (category 0 and 1) or successfully established (category 2 and 3). The number of long-term establishments and the size of the transplantation area were used to calculate the density of established nests.

Master thesis by Jesper Stern Nielsen 30 Results

Difference in nest activity levels between dates indicated movements of ants in TS1 (figure 6-9). The indication of movement is supported by observations of pupae transportation. At first, ants were observed to have moved pupae and needles into the feeding boxes and stayed there the first weeks, until the feeding boxes were removed. Later, pupae were observed being transported from TS1-N1 to TS1-N2 the 13-05- 2015 and only low or none activity was observed in TS1-N1 afterwards, which could indicate a merger (figure 6). Ants were also seen moving pupae from TS1-N4 to TS1-N3 the 13-05-2015 (merge). A track between the nests was observed the 21-06-2015, and a shift in which nest that had activity of ants the 16- 07-2015 indicated that ants moved from TS1-N3 back to TS1-N4 (figure 7). Transport of pupae also occurred from TS1-N6 to TS1-N5 the 13-05-2015 (merge). A shift in which nest that had activity of ants was seen the 16-07-2015, indicating that the ants moved from TS1-N5 back to TS1-N6 (figure 8). No trails were observed between TS1-T7 and TS1-T8. The activity level of TS1-N8 was quite steady the whole season, while the activity level for TS1-N7 fluctuated (figure 9). Nests in TS1 went hibernating after the 15th of September.

Activity - TS1-N1 and TS1-N2 - "Æbletoften"

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Figure 6: Activity levels in TS1-N1 and TS1-N2 on 16 dates from 01-05-2015 to 11-10-2015. Ants were observed moving pupae from TS1-N1 to TS1-N2 the 13-05-2015, and only low or none activity was observed in TS1-N1 afterwards. Note that four levels of activity are used to visualize the dynamics.

Master thesis by Jesper Stern Nielsen 31 Activity - TS1-N3 and TS1-N4 - "Æbletoften"

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Figure 7: Activity levels in TS1-N3 and TS1-N4 on 16 dates from 01-05-2015 to 11-10-2015. Ants were observed moving pupae from TS1-N4 to TS1-N3 the 13-05-2015. A track between the nests was observed the 21-06-2015, and a shift in which nest that had activity of ants the 16-07-2015 indicates that ants moved from TS1-N3 to TS1-N4. Note that four levels of activity are used to visualize the dynamics.

Activity - TS1-N5 and TS1-N6 - "Æbletoften"

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Figure 8: Activity levels in TS1-N5 and TS1-N6 on 16 dates from 01-05-2015 to 11-10-2015. Ants were observed moving pupae from TS1-N6 to TS1-N5 the 13-05-2015. A shift in which nest that had activity of ants was seen the 16-07-2015, indicating that the ants moved from TS1-N5 to TS1-N6. Note that four levels of activity are used to visualize the dynamics.

Master thesis by Jesper Stern Nielsen 32 Activity - TS1-N7 and TS1-N8 - "Æbletoften"

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Figure 9: Activity levels in TS1-N7 and TS1-N8 on 16 dates from 01-05-2015 to 11-10-2015. No trail connection was observed between the two nests. The activity of TS1-N7 was fluctuating while TS1-N8 was quite steady in activity level. Note that four levels of activity are used to visualize the dynamics.

Only few observations on activity level were made in TS2 and TS3 and these showed only small fluctuations (Appendix 1: TS2: figure 12-15, TS3: figure 16-19). Ants in TS2 and TS3 were also observed to live in the feeding boxes the first weeks, but these were later abandoned. Most nests had an activity level of 2 or 3 on the three to four observation days, but nest TS3-N6 showed only an activity level of 1 the 12- 08-2015, possibly indicating a merger.

In August 2015, 20 nests out of the total 24 transplanted (83.3 %) nests showed a medium or high activity and were thus considered successfully short-term established. TS2 had the highest short-term establishment success rate of 100 % (8 out of 8 nests), while TS3 had a success rate of 87.5 % (7 out of 8 nests) and TS1 had a success rate of 62.5 % (5 out of 8 nests) (figure 10). The numbers of nest with medium and high activity was as follows: TS1: 0 and 5, respectively, TS2: 4 and 4, respectively, TS3: 2 and 5, respectively (figure 10). Activity by ants was in the end of the year only observed in the original artificial nests, at the exact transplantation position, though movement from and to the artificial nests was observed occasionally during the spring and summer.

Master thesis by Jesper Stern Nielsen 33 Division of successfully short-term established nests

8 N 100

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1 10 0 0 TS1 – Æbletoften TS2 – Lilleheden TS3 - Near Beder 23rd of August, 2015 12th of August, 2015 12th of August, 2015

Figure 10: The number and division of successfully short-term established nests observed in the three plantations at the 23rd of August and 12th of August, respectively. The nest IDs and which groups they belong to are indicated in the columns. Transplantation led to a relatively high number of successful short-term establishments in each of the transplantation sites. The total number of successful short-term established nests was 20 out of 24, corresponding to a total establishment success of 83.3 %.

In April 2016, 18 nests out of the total 24 transplanted nests (75 %) showed a medium or high activity and were thus considered successfully long-term established (figure 11). Long-term establishment was highest in TS2 with an establishment success rate of 100 % (8 out of 8 nests). TS1 and TS3 both had a success rate of 62.5 % (5 out of 8 nests). The division of nest with medium and high activity was as follows: TS1: 1 and 4, respectively, TS2: 2 and 6, respectively, TS3: 2 and 3, respectively (figure 11). The nests that were active in August 2015 were the same being active in April 2016, except for TS3-N2 and TS3-N8, which had no or low activity in April 2016. It was observed that nests in TS3 showed signs of predation by birds, deer, or badger in April 2016 as many of the tree stumps from the artificial nests were excavated and free of conifer needles. Nest TS3-N4 had increased its size and a high number of ants were seen on the nest, which could be a result of a merger.

The established nest density in TS1 was 5 on an area of 0.25 ha, which corresponds to approximately 20 nests/ha. In TS2 the number was 8 on an area of 1.1 ha corresponding to 7.3 nests/ha, and in TS3 the number was 5 on an area of 0.5 ha corresponding to 10 nests/ha.

Master thesis by Jesper Stern Nielsen 34 Division of successfully long-term established nests

8 N 100

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: 4, 5, 7 5, 4, : 20

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0 0 TS1 – Æbletoften TS2 – Lilleheden TS3 - Near Beder 8th of April, 2016 1st of April, 2016 1st of April, 2016

Figure 11: The number and division of successfully long-term established nests observed in the three plantations at the 8th of April and 1st of April, respectively. The nest IDs and which groups they belong to are indicated in the columns. The total number of successful long-term established nests was 18 out of 24, corresponding to a total establishment success of 75 %. The nests that were active in August 2015 were the same being active in April 2016, except for TS3-N2 and TS3-N8, which had no or low activity in April 2016.

Master thesis by Jesper Stern Nielsen 35

Discussion

In this experiment nests of F. polyctena were fragmented, transplanted and successfully established in three different types of plantations. Of the 24 transplanted fragments, 83.3 % (20 out of 24) were short- term established and 75 % (18 out of 24) were long-term established. Nest establishment was achieved at the exact position of transplantation and a nest density of 20, 7.3, and 10 nests/ha was achieved in TS1, TS2 and TS3, respectively. Some ants remained at their transplantation position, while others were observed to move between the artificial transplant nests, which indicated that fragments merged and that there was variation in which nests were used. In this experiment F. polyctena seems to do well in different environments, despite varying establishment successes in the three plantations, and thus indicate the possibility of using F. polyctena for biocontrol purposes in a wide array of plantation setups.

The seemingly low short-term and long-term establishment success of 62.5 % (5 out of 8) in TS1 does not speak the whole truth about the establishment success. Movement of ants was observed to cause merging of the artificial nests. Merging of nests lowered the measurable percentage of successful establishments, but did not necessarily mean an unsuccessful transplantation of individual nests. The merging could be a result of the short distance between the transplanted nests (10-20 m), a too low worker force in each fragment, or environmental conditions that made the transplantation position unsuitable (e.g. flooding). Others have had similar experiences with merging of transplanted nests originating from the same colony that were closely situated (Finnegan, 1975; Pisarski & Czechowski, 1990; Sorvari et al., 2014). The merging could increase the number of workers and thus the strength in the number of ants to collect food and protect the colony (Franks & Partridge, 1993). Though some of the ants have united in one nest they may later divide their worker force and expand their territory by budding (Adlung, 1966; Mabelis, 1979; Pisarski & Czechowski, 1990). This structure of group settlement with several related nests being connected by exchange trails is often found in natural colonies of F. polyctena (Bugrova & Reznikova, 1990) and could be a potential long-term outcome of the transplantation. The short-term establishment success of TS1 was the smallest of the three plantations, which coincide with it being the most alien habitat for the wood ants. However, when looking at the long-term establishment TS1 do as well as TS3, and thus questions whether the ants do better in the habitats of TS2 and TS3, which are more similar to their natural habitat, than in TS1.

In TS2, 100 % (8 out of 8) of the artificial transplant nests had successful short-term and long-term establishment of ants. Movement was not observed and the high establishment success could be due to no observed merging, because of the higher distance between nests (minimum 25 m) than in the other plantations (10-20 m). The ants were well-established even though they were disturbed by birds in the

Master thesis by Jesper Stern Nielsen 37 early season. The protection of the nests by chicken wire seemed to reduce the impact from birds. Despite a reduced impact and a high activity in nests, the nest sizes in TS2 were quite small, compared to natural nests and the artificial transplant nests in the apple orchard. The small nest sizes are not likely the result of a small fragment size, since this was more or less similar to the fragment sizes in the apple orchard. Thus the initial high disturbance of the nests by birds may be a reasonable explanation. Over time the sizes of the long-term established nests are expected to increase.

In TS3, 87.7 % (7 out of 8) of the artificial transplant nests had a successful short-term establishment of ants. The establishment success was high despite initial challenges in weather conditions (rain events and strong wind). Ants from many of the nests were observed exploiting the area, and probably met associates from neighboring artificial transplant nests, but only from TS3-N6, movement was observed, as pupae were transported to another nest. Merging was rare despite a distance of 15-20 m between nests which was more or less even with the distances in the apple orchard. In April 2016 only 62.5 % (5 out of 8) of the artificial transplant nests were found to be successfully long-term established. This difference in establishment from 2015 to 2016 could be the result of a merge in the early spring, indicated by the high number of ants on some of the nests, or of extirpation due to predation by birds, badger, or deer, indicated by the signs of excavation in April, 2016.

The total short-term and long-term establishment successes in this experiment were 83.3 % and 75 %, respectively, which are comparable to the results from transplantation by Gösswald (1984), Paulson & Akre (1992a), and Sorvari et al. (2014), who had a success rate of 88 % (22 out of 25 nests), approximately 80 % (22 out of 27 nests), and 77 % (20 out of 26 nests), respectively. Both Gösswald (1984), who transplanted F. lugubris to a mixed forest, and Paulson & Akre (1992a), who transplanted F. neoclara in wooden boxes to a pear orchard, experienced that ants vacated their transplant positions and established in new nests nearby. The same has been experienced by others (Wellenstein, 1973; Finnegan, 1975; Campbell et al., 1991). Pisarski & Czechowski (1990) concluded that the movement as well as the merging of transplanted nests is inevitable. In the current experiment ants were only observed to establish at the exact position of transplantation, which to my knowledge only have been achieved by Bradley (1972), but only for few non-fragmented nests and only in a jack pine plantation. In this experiment, movement was observed and merging occurred, but in the end, ants established in the original artificial fragment nests, at the exact position of transplantation in all three plantations. This can have different explanations. One explanation is that the construction of the artificial nests in this experiment followed the construction of natural found nests, with sandy soil in the bottom, a tree stump, and conifer needles on top. To my knowledge, sandy soil has only been applied in one other transplantation setup, the one by Bradley (1972),

Master thesis by Jesper Stern Nielsen 38 who in his successful establishment of nests at the exact transplant position, poured the sand around the artificial nests and did not use it as a part of the nest. Another explanation is that the conifer needles and twigs plus the sandy soil used for the artificial nest originated from the donor nest, while the tree stumps had been in contact with the nests for a period of 10 days before the transplantation. The materials were thus scent-wise familiar to the ants and could cause some kind of “homing effect”. A different explanation is that ants chose to establish in the artificial nests in lack of better building material or nest sites in the plantations. This is likely in TS1, where conifer needles were absent, but in TS2 these were present and the ants still did not move permanently away from the artificial nests. In TS3, the plantation consisted of young conifer trees, which caused a low amount of available needles as building material, because conifer trees do not naturally defoliate needles at a young age (Personal communication with one of the plantation owners). The larger conifer trees to the east and north, however, may have supplied some amount of conifer needles. Still, the ants did not end up establishing outside the artificial nests. A fourth explanation of why the ants ended up establishing in the original artificial fragment nests could be that abandonment of a nest and the building of a new one is likely to be costly in terms of energy (Ellis & Robinson, 2014), and would thus only be expected under unfavorable conditions.

The number of nests required per area unit to deliver a full protection against arthropod pests was evaluated by Gösswald (1951 in Travan, 1994) and by Scheblanov (1963), Dmitrienko & Petrenko (1976), Petrenko (1966), and Dmitrienko (1969) (in Rosengren et al., 1979). Gösswald (1951 in Travan, 1994) estimated that the requirement for conifer forest protection was approximately 4 wood ant nests/ha, while the others found that the required number of nests varied between 5-8 nests/ha (Scheblanov, 1963; Dmitrienko & Petrenko, 1976; in Rosengren et al., 1979) and 30-40 nests/ha (Petrenko, 1966; Dmitrienko, 1969 in Rosengren et al., 1979). The amount of nests per area in the current study was in 20, 7.3, and 10 nests/ha in TS1, TS2, and TS3, respectively, and are thus within the estimated requests by Gösswald (1951 in Travan, 1994) and Scheblanov (1963) and Dmitrienko & Petrenko (1976) (in Rosengren et al., 1979). Whether this number of nests can be maintained if ants were transplanted on a larger area is unknown. Another consideration is that the sizes of the nests in the current study still are small and that their contribution to biocontrol of pest may be limited. Experiments by Gösswald (in Travan, 1994) show that the number and sizes of nests increased during the first years after transplantation, but that after a following stabilization phase, which for some of the nests extended for more than 10 years, the nests gradually diminished in numbers and sizes, which in most cases led to extinction of the nests. From a biocontrol perspective the period of protection seems reasonable, and if nests go extinct new transplants can be made, either from nests in the plantation or from a source colony. Whether the source colony should be of same origin as the nest already present should be considered. Pisarski & Czechowski (1990) argued though

Master thesis by Jesper Stern Nielsen 39 that transplants do not merge with existing nests from same donor colony, and that they should be founded at an adequatly distance to the existing ones to reduces the risk of any conflicts.

Another consideration is the choice of area for transplantation, and especially that area’s presence of building materials and other essential materials. Conifer trees provides ants with both conifer needles, which are one of the preferred nest materials for wood ants (Douwes et al., 2012), and with resin that are used to reduce the density of many bacteria, fungi and other micro-organisms (Lenoir et al., 1999; Lenoir et al., 2003; Chapuisat et al., 2007; Castella et al., 2008; Brütsch & Chapuisat, 2014), which seems to be essential for the well-being of the ants. As mentioned earlier, no conifer trees were nearby the transplantation area in TS1 to provide the ants with needles and resin. Needles were thus a limited resource that needed to be provided artificially, which could be the case for other plantations as well. Whether the amount of resin on the provided needles is sufficient is unknown. In TS2 and TS3 needles and resin were present although needles were not in high numbers in the young conventional plantation (TS3). To increase the number of needles in such plantation, one could leave some of the branches or trees when harvesting or pruning. Whether this should be avoided due to the risk of spreading plant diseases is unknown. A way of designing plantations in the future that secures the necessary building materials and resin requirements could be to plant conifer trees among the crop to make the conifer needles and resin naturally available for the ants. Mixed plantations have been found to cause better nest development for the wood ants than pure spruce plantations (Wellenstein, 1973). Whether the crop or tree mixing will reduce the pest control effect and distribution of the ants in the plantation is unknown, but it is likely to ease short- and long-term establishment.

In conclusion, this experiment confirmed that F. polyctena can be fragmented and transplanted into different types of plantations and settle in these different environments successfully. The establishment success was comparable to the findings of other studies, and the achieved number of nests per area was 7.3, 10, and 20 nests/ha, which was within the limits of what have been found by others to be needed for effective pest control (Gösswald 1951 in Travan, 1994; Scheblanov, 1963; Dmitrienko & Petrenko, 1976; in Rosengren et al., 1979). Movement between nests were seen, but ants from fragments ended up to establish in the original artificial transplant nests, opposite to what has been achieved in most other studies, where transplanted nests vacated their transplant positions and established in new nests nearby. The achievement of established nests at the exact position of transplantation can be imputed to the fragmentation and transplantation method, the use of scent-familiar materials for nest construction that would cause a “homing effect”, the absence of appropriate nest building materials in the transplant area, or the likely cost of energy associated with abandonment and building of a new nests by the ants. When

Master thesis by Jesper Stern Nielsen 40 choosing a transplantation site the accessibility of building materials, like conifer needles, and other essential materials, like resin, should be considered, and if not present, an artificial provision is needed. The transplantation method used in this experiment has proven to be successful and the achievement of a relatively high establishment success indicates the possibility of using F. polyctena in a wide array of plantation setups for biocontrol purposes, if the effect of wood ants as biocontrol agents in plantation and orchards proves to be effective.

Master thesis by Jesper Stern Nielsen 41

References

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Master thesis by Jesper Stern Nielsen 43 Gösswald, K. 1990. Die Waldameise. Band 2. Die Waldameise in Ökosystem Wald, ihr Nutzen und ihre Hege (The Wood ant. Volume 2. The Wood Ant in the Forest Ecosystem, Its Uses and Protection). AULA- Verlag Wiesbaden. Hajek, A. 2004. Natural Enemies: An Introduction to Biological Control. Cambridge University Press. Karhu, K. J. 1998. Effects of Ant Exclusion During Outbreaks of a Defoliator and a Sap-sucker on Birch. Ecological Entomology 23: 185-194. Kilpeläinen, J., P. Punttila, L. Finer, P. Niemela, T. Domisch, M. F. Jurgensen, S. Neuvonen, M. Ohashi, A. C. Risch, & L. Sundstrom. 2008. Distribution of Ant Species and Mounds (Formica) in Different-aged Managed Spruce Stands in Eastern Finland. Journal of Applied Entomology 132: 315-325. Lafleur, L. J. 1941. The Founding of Ant Colonies. Biological Bulletin 81: 392-431. Lassen, J. W. 2015. Evaluation of Formica polyctena (Hymenoptera: Formicidae) as a Control Agent of Dreyfusia nordmannianae (Hemiptera: Adelgidae) in Danish Christmas Trees. Master Thesis, Unpublished. Lenoir, L., J. Bengtsson, & T. Persson. 1999. Effects of Coniferous Resin on Fungal Biomass and Mineralisation Processes in Wood Ant Nest Materials. Biology and Fertility of Soils 30: 251-257. Lenoir, L., J. Bengtsson, & T. Persson. 2003. Effects of Conifer Resin on Soil Fauna in Potential Wood-ant Nest Materials at Different Moisture Levels. Pedobiologia 47: 19-25. Lind, K., G. Lafer, & K. Schloffer. 2003. Organic Fruit Growing. CAB International, Wallingford, GB. Mabelis, A. A. 1979. Nest Splitting by the Red Wood Ant (Formica polyctena Foerster). Netherlands Journal of Zoology 29: 109-125. Mabelis, A. A. 1994. Flying as a Survival Strategy for Wood Ants in a Fragmented Landscape (Hymenoptera, Formicidae). Memorabilia Zoologica 48: 147-170. Müller, H. 1956. Können Honigtau liefernde Baumläuse (Lachnidae) ihre Wirtspflanzen schädigen? (Can Honeydew Producing Aphids Cause Damage to Their Host Trees?). Zeitschrift für Angewandte Entomologie 39: 168-177. Nielsen, J. S. 2015. Fragmentation and Transplantation of Red Wood Ant Colonies (Formica polyctena) for Biological Control Purposes. Students project, Unpublished. Offenberg, J. 2007. Myrer som Plantebeskyttere (Ants as Plant Protectors). moMentum 2007, nr. 1: 31-34. Offenberg, J. 2015. Ants as Tools in Sustainable Agriculture. Journal of Applied Ecology 52: 1197-1205. Oxforddictionaries.com. accessed 23-03-2016. Oxford Dictionaries. http://www.oxforddictionaries.com/definition/english/. Paulson, G. S., & R. D. Akre. 1992a. Introducing Ants (Hymenoptera: Formicidae) into Pear Orchards for the Control of Pear Psylla, Cacopsylla pyricola (Foerster) (Homoptera: Psyllidae). Journal of Agricultural Entomology 9: 37-39. Paulson, G. S., & R. D. Akre. 1992b. Evaluating the Effectiveness of Ants as Biological Control Agents of Pear Psylla (Homoptera: Psyllidae). Journal of Economic Entomology 85: 70-73. Peng, R. K., & K. Christian. 2008. Potential for Organic Mango Production in the Northern Territory of Australia Using Weaver Ants, Oecophylla smaragdina, (Hymenoptera: Formicidae). In: R. K. Prange and S. D. Bishop (eds.) Proceedings of the International Symposium on Sustainability through Integrated and Organic Horticulture. Acta Horticulturae: 81-88. Pinkalski, C., C. Damgaard, K.-M. V. Jensen, R. Peng, & J. Offenberg. 2015. Quantification of Ant Manure Deposition in a Tropical Agroecosystem: Implications for Host Plant Nitrogen Acquisition. Ecosystems 18: 1373-1382. Pisarski, B., & W. Czechowski. 1990. The Course of Artificial Colonization of Red Wood Ants in the Gorce National Park. Memorabilia Zoologica 44: 37-46. Risch, S. J., & C. R. Carroll. 1982. The Ecological Role of Ants in Two Mexican Agroecosystems. Oecologia 55: 114-119. Rosengren, R., K. Vepsäläinen, & H. Wuorenrinne. 1979. Distribution, Nest Densities, and Ecological Significance of Wood Ants (The Formica rufa Group) in Finland. Bulletin SROP 2: 181-213.

Master thesis by Jesper Stern Nielsen 44 Rosumek, F. B., F. A. O. Silveira, F. de S. Neves, N. P. de U. Barbosa, L. Diniz, Y. Oki, F. Pezzini, G. W. Fernandes, & T. Cornelissen. 2009. Ants on Plants: A Meta-analysis of the Role of Ants as Plant Biotic Defenses. Oecologia 160: 537-549. Savoläinen, R., & K. Vepsäläinen. 1988. A Competition Hierarchy Among Boreal Ants: Impact on Resource Partitioning and Community Structure. Oikos 51: 135-155. Skinner, G. J., & J. B. Whittaker. 1981. An Experimental Investigation of Inter-relationships Between the Wood-ant (Formica rufa) and Some Tree-canopy Herbivores. Journal of Animal Ecology 50: 313- 326. Sorvari, J., E. Huhta, & H. Hakkarainen. 2014. Survival of Transplanted Nests of the Red Wood Ant Formica aquilonia (Hymenoptera: Formicidae): The Effects of Intraspecific Competition and Forest Clear- cutting. Insect Science 21: 486-492. Storer, A. J., M. F. Jurgensen, A. C. Risch, J. Delisle, & M. D. Hyslop. 2008. The Fate of an Intentional Introduction of Formica lugubris to North America from Europe. Journal of Applied Entomology 132: 276-280. Tesanovic, D., & R. Spasic. 2013. Pests of Apple Leaf and Flower Buds in the Region of East Sarajevo. Fourth International Scientific Symposium "Agrosym 2013", Jahorina, Bosnia and Herzegovina, 3-6 October, 2013. Book of Proceedings: 546-552. Travan, J. 1994. Über Prof. Gösswalds vieljährige Versuche mit der künstlichen Ansiedlung und Vermehrung von Völkern der Roten Waldameisen (Hym., Formicidae) in Unterfranken (Professor Gösswald's Long-term Experiments on the Artificial Establishment and Propagation of Colonies of Red Wood Ants in Lower Franconia). Waldhygiene 20: 47-130. Tsuji, K., A. Hasyim, Harlion, & K. Nakamura. 2004. Asian Weaver Ants, Oecophylla smaragdina, and Their Repelling of Pollinators. Ecol Res 19: 669-673. Vidkjær, N. H., B. Wollenweber, R. Gislum, K.-M. V. Jensen, & I. S. Fomsgaard. 2015. Are Ant Feces Nutrients for Plants? A Metabolomics Approach to Elucidate the Nutritional Effects on Plants Hosting Weaver Ants. Metabolomics: 1-16. Warrington, S., & J. B. Whittaker. 1985. An Experimental Field-study of Different Levels of Insect Herbivory Induced by Formica rufa Predation on Sycamore (Acer pseudoplatanus) I. Lepidoptera Larvae. Journal of Applied Ecology 22: 775-785. Way, M. J., & K. C. Khoo. 1992. Role of Ants in Pest Management. Annual Review of Entomology 37: 479- 503. Wellenstein, G. 1973. The Development of Artificially Founded Colonies of Hill-building Red Wood Ants of the Formica rufa-Group in South-western Germany. EPPO Bulletin 2: 23-34. Wesolowski, T., & P. Rowinski. 2006. Tree Defoliation by Winter Moth Operophtera brumata L. During an Outbreak Affected by Structure of Forest Landscape. Forest Ecology and Management 221: 299- 305. Whittaker, J. B., & S. Warrington. 1985. An Experimental Field-study of Different Levels of Insect Herbivory Induced by Formica rufa Predation on Sycamore (Acer pseudoplatanus) III. Effects on Tree Growth. Journal of Applied Ecology 22: 797-811. Wilkinson, R. C., A. P. Bhatkar, W. H. Whitcomb, & W. J. Kloft. 1980. Formica integra (Hymenoptera, Formicidae) 3. Trial Introduction into Florida. Florida Entomologist 63: 142-146.

Master thesis by Jesper Stern Nielsen 45

Appendix 1

The appendix includes observations on activity level for TS2 and TS3 (See results).

Activity - TS2-N1 and TS2-N2 - "Lilleheden"

3

Aug

May

Jun

-

-

3

-

12

21

2

May

Aug

-

-

12

12

TS2-N1

Activity Activity level 1 TS2-N2

0 May-12 Jun-11 Jul-11 Aug-10 Date

Figure 12: Activity levels in TS2-N1 and TS2-N2 on the 12-05-2015, 21-05-2015, 03-06-2015, and 12-08-2015. Observations were only made on four dates.

Activity - TS2-N3 and TS2-N4 - "Lilleheden"

3

Jun

May

-

-

3

21

2

May

Aug

-

-

12

12

TS2-N3

Activity Activity level 1 TS2-N4

0 May-12 Jun-11 Jul-11 Aug-10 Date

Figure 13: Activity levels in TS2-N3 and TS2-N4 on the 12-05-2015, 21-05-2015, 03-06-2015, and 12-08-2015. Observations were only made on four dates.

Master thesis by Jesper Stern Nielsen 47 Activity - TS2-N5 and TS2-N6 - "Lilleheden"

3

Jun Aug

May

Jun

Aug

May

-

-

-

-

-

-

3

3

12

12

21

12

2

TS2-N5

Activity Activity level 1 TS2-N6

0 May-12 Jun-11 Jul-11 Aug-10 Date

Figure 14: Activity levels in TS2-N5 and TS2-N6 on the 12-05-2015, 21-05-2015, 03-06-2015, and 12-08-2015. Observations were only made on four dates.

Activity - TS2-N7 and TS2-N8 - "Lilleheden"

3

Aug

May

Jun

May

-

-

3

12

-

-

21

12

2

Aug

May

-

12

- 12

TS2-N7

Activity Activity level 1 TS2-N8

0 May-12 Jun-11 Jul-11 Aug-10 Date

Figure 15: Activity levels in TS2-N7 and TS2-N8 on the 12-05-2015, 21-05-2015, 03-06-2015, and 12-08-2015. Observations were only made on four dates.

Master thesis by Jesper Stern Nielsen 48 Activity - TS3-N1 and TS3-N2 - Near Beder

3

May

May

Aug

-

-

-

12

2 19

2

Aug

- 12

TS3-N1

Activity Activity level 1 TS3-N2

0 May-2 Jun-2 Jul-3 Aug-3 Date

Figure 16: Activity levels in TS3-N1 and TS3-N2 on the 02-05-2015, 19-05-2015 and 12-08-2015. Observations were only made on three dates.

Activity - TS3-N3 and TS3-N4 - Near Beder

3

Aug

May

May

-

-

-

12

19

2

2

TS3-N3

Activity Activity level 1 TS3-N4

0 May-2 Jun-2 Jul-3 Aug-3 Date

Figure 17: Activity levels in TS3-N3 and TS3-N4 on the 02-05-2015, 19-05-2015 and 12-08-2015. Observations were only made on three dates.

Master thesis by Jesper Stern Nielsen 49 Activity - TS3-N5 and TS3-N6 - Near Beder

3

Aug

May

May

-

-

-

12

19

2

2

TS3-N5

Activity Activity level 1 TS3-N6

Aug

-

12

0 May-2 Jun-2 Jul-3 Aug-3 Date

Figure 18: Activity levels in TS3-N5 and TS3-N6 on the 02-05-2015, 19-05-2015 and 12-08-2015. Observations were only made on three dates.

Activity - TS3-N7 and TS3-N8 - Near Beder

3

May

Aug

May

-

-

-

12

19

2

2

Aug -

12 TS3-N7

Activity Activity level 1 TS3-N8

0 May-2 Jun-2 Jul-3 Aug-3 Date

Figure 19: Activity levels in TS3-N7 and TS3-N8 on the 02-05-2015, 19-05-2015 and 12-08-2015. Observations were only made on three dates.

Master thesis by Jesper Stern Nielsen 50

Manuscript 2:

Contribution from Wood Ants to Fruit Production and Control of Winter Moths in an Organic Apple Orchard

Jesper Stern Nielsen

Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark

Photo by Anne Aagaard Lauridsen

Contribution from Wood Ants to Fruit Production and Control of Winter Moths in an Organic Apple Orchard

Jesper Stern Nielsen

Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark

Abstract

8. Pests like winter moths can reduce monetary returns in organic fruit production in form of reduced yield. Predaceous wood ants of the Formica rufa group may contribute to controlling winter moths Operophtera brumata, and may have positive effects in fruit production. 9. In an organic apple orchard transplanted nests of the species Formica polyctena and their effect on apple yield, winter moth larvae damage and number was examined. 10. Apple trees with high ant activity were found to have an average apple yield of 5.1 apples per tree, which was more than twice as much as trees with no or low ant activity. The distance from trees to the nearest active nest was significantly negatively correlated with the apple yield. No clear pattern of significance was seen from ant activity on winter moth larvae, apple bud loss, or leaf damage, despite the fact that ants were observed to predate and deter winter moth larvae. 11. The presence of ants caused an increase in yield, despite no measurable effect was seen on the underlying levels: leaf damage and number of winter moth larvae.

Key words: Biological control, Wood ants, Formica polyctena, Winter moth, Operophtera brumata, Fruit yield.

Master thesis by Jesper Stern Nielsen 53 Resumé

1. Frostmålere og andre skadedyr kan reducere det økonomiske udbytte i form af et reduceret frugtudbytte. Skovmyrer fra Formica rufa-gruppen kan bidrage til kontrol af frostmålere Operophtera brumata og kan have andre positive effekter i frugt produktion. 2. Effekten af transplanterede tuer af arten Formica polyctena i en økologisk æbleplantage blev undersøgt på æbleudbytte, antallet af frostmålerlarver samt skade herfra. 3. Æbletræer med høj myreaktivitet havde et gennemsnitligt æbleudbytte på 5.1 æbler pr. træ, hvilket var mere end det dobbelte af hvad der blev fundet på træer med lav eller ingen myreaktivitet. Afstanden fra træer til nærmeste aktive tue var signifikant negativt korreleret med æbleudbyttet. Intet klart signifikansmønster var set af myreaktivitet på antallet af frostmålerlarver, æbleknoptab eller bladskade, selvom myrer blev set prædere på og afskrække frostmålerlarver. 4. Tilstedeværelsen af myrer medførte et højere æbleudbytte, selvom målbare effekter af myrer ikke blev fundet på de underliggende niveauer: bladskade og antallet af frostmålerlarver.

Dictionary Due to the structural complexity of Formica polyctena colonies, some terms used in this paper are here defined:

Nest: Referring to a single dome within a colony.

Colony: A community of ants that are socially connected and live close together in one or several nests. Resources, individuals and brood are exchanged via connecting trails (Definition inspired by Bugrova & Reznikova, 1990; Oxforddictionaries.com, accessed 23-03-2016).

Polydome colony: Ant colonies that inhabit several spatially separated, but socially connected nests (Ellis & Robinson, 2015).

Polygyne: The condition of having more than one egg-laying queen in a colony (Oxforddictionaries.com, accessed 23-03-2016).

Budding: Formation of new nests by colony splitting (Mabelis, 1994).

Master thesis by Jesper Stern Nielsen 54 Introduction

Pests in organic fruit production can represent a substantial challenge. Not only can pests increase the workload for plantation owners, but they can also lead to economic loss due to reduced yield, reduced quality of the fruit, or increased expenses for control agents to suppress the pests (De Ros et al., 2015; MacPhee et al., 1988; Tesanovic & Spasic, 2013). The choice of pesticides is very restricted in organic production, and the few that are available are often less effective than in integrated production (Lind et al., 2003; Sigsgaard et al., 2013). Alternatives to pesticides include the use of biological control (biocontrol) agents. The idea is to use nature’s own agents to control the pests. An organism that shows promising results in biocontrol is ants.

Predaceous ants have in many cases been used for biocontrol purposes due to their many beneficial abilities. The first indication of the use of ants for biocontrol comes from records that describe habitat manipulation in 324 B.C. to favor the population of weaver ants Oecophylla smaragdina in citrus trees to control large boring beetles and caterpillars (Hajek, 2004; Way & Khoo, 1992). Many experiments have since elucidated the effects of ants, which involves both their direct predatory effects on other arthropods, but also their indirect effects on their surroundings. Some effects from the presence of ants that could be beneficial in fruit production are: higher yield (Peng & Christian, 2008), better fruit quality (Peng & Christian, 2013; Peng & Christian, 2008), a lower number of Lepidoptera larvae (Skinner & Whittaker, 1981), better tree growth (Whittaker & Warrington, 1985), less leaf damage (Karhu, 1998; Rosumek et al., 2009; Skinner & Whittaker, 1981; Warrington & Whittaker, 1985), nutrient deposition from ant manure on host trees (Pinkalski et al., 2015; Vidkjær et al., 2015), soil improvement and nutrient cycling (Way & Khoo, 1992), and antibacterial and antifungal effects (Chapuisat et al., 2007; Lenoir et al., 2003). The presence of ants unfortunately also has some effects that could be negative in fruit production, for example: removal or deterrence of beneficial insects like pollinators or other predators (Assunção et al., 2014; Tsuji et al., 2004), an increased number of damaging aphids (Hemiptera sp.) (Karhu, 1998), and nuisance for farm workers (Offenberg, 2015; Peng & Christian, 2008). However, solutions are available for elimination or reduction of many of these negative effects (Offenberg, 2015), thus the potential to use ants as biocontrol agents is encouraging.

The relevance of ants as biocontrol agents can be attributed to their social way of life and their aggressive behavior against many other organisms (Gridina, 1990; Way & Khoo, 1992). Their social way of life provides them with features like: being in great numbers, being able to recruit colony mates, share resources, and have a division of labor (Gösswald, 1989; Offenberg, 2015). Their aggressiveness interacts well with their generalistic feeding behavior, which enables them to deter or gather and remove high

Master thesis by Jesper Stern Nielsen 55 numbers of other arthropods from their territory (Gridina, 1990; Way & Khoo, 1992). The collected arthropods serve as a protein source, and the large requirement of proteins for brood and queens makes the ants hard to satiate (Offenberg, 2007; Paulson & Akre, 1992b). Also, the ants can store gathered food within their nests for later use, which means they do not respond on satiation but on prey density (Gridina, 1990; Way & Khoo, 1992), and if prey density is too low and they are short of food they can cannibalize their own brood (Lafleur, 1941; Paulson & Akre, 1992b; Way & Khoo, 1992). Ant colonies remain more or less stationary, because their workers lack wings, but they are able to spread into other areas through winged mated queens (Mabelis, 1994). Furthermore, the adaptiveness of ants towards their environment makes the ants able to survive in many types of habitats (Paulson & Akre, 1992b; Pisarski & Czechowski, 1990; Way & Khoo, 1992). Many of the abovementioned properties are useful in the control of pests.

In fruit production many different arthropod pests can occur. The pests are found within different groups, and examples include mites (Acari), aphids (Hemiptera), sawflies (Hymenoptera), borers and weevils (Coleoptera), and moths (Lepidoptera) (Lind et al., 2003). Especially the winter moth Operophtera brumata L. (Geometridae, Lepidoptera), have been subject to many studies due to its appearance and impact on different tree crops and on fruit production (MacPhee et al., 1988; Sigsgaard et al., 2013; Tesanovic & Spasic, 2013). It is found on both fruit trees and deciduous hardwood trees like oak and birch, but can also occur on Sitka spruce, where it can cause great damage (Stoakley, 1985). The damage from the winter moth is often seen as dry buds, disturbed development of leaves and flowers, reduction in yield and fruit quality from damaged or deformed fruit, and tree defoliation which can have great impact on tree fertility the following years (Graf et al., 1995; Holliday, 1977; Lind et al., 2003; MacPhee et al., 1988; Tesanovic & Spasic, 2013; Wesolowski & Rowinski, 2006). Even at a low population level winter moth can reduce monetary returns to the grower (MacPhee et al., 1988). The major damages are caused in the larval stage and the larvae are thus often the main targets of control, but more of the winter moth stages can be the target of biocontrol (Frank, 1967a, 1967b; Hand et al., 1987; MacPhee et al., 1988). The stages and the active period of the winter moth are seen in figure 1. The winter moth larvae hatches in the early spring, at time of budburst, from eggs hidden in crevices in the tree bark or under lichens (Holliday, 1977; Lind et al., 2003; Peterson & Nilssen, 1998). Hereafter the larvae feed on leaves and flower buds before pupating in the soil (Lind et al., 2003; MacPhee et al., 1988). The adult winter moths typically emerge in the late fall or early winter season, before snowfall. At this time they avoid most predatory insects and the most specialized insect predators among birds (Peterson & Nilssen, 1998). The merged male moths have functional wings and fly out to find females to mate, while the females are not able to fly due to reduced wings (Tenow, 1972). Females attract males with a pheromone, they copulate on the ground or on the tree trunk, and the female climb the tree and find a place to lay her eggs (Bestmann et al., 1982; Holliday, 1977).

Master thesis by Jesper Stern Nielsen 56 The approved organic control methods that have been used to control the winter moths depend on the developmental stage of the moths. Method previously used to control the larvae include: parasitoids, (Embree, 1991; MacPhee et al., 1988; Roland, 1994; Roland & Embree, 1995), application of oil or soap (Hardman & Gaul, 1990; Sigsgaard et al., 2013), promoting conditions for larvae-eating birds (Lind et al., 2003; Roland et al., 1986), application of Bacillus thuringiensis (Embree, 1991; Hardman & Gaul, 1990; Sigsgaard et al., 2013), and application of viruses or microsporidian diseases (Roland & Embree, 1995). In the pupation state control attempts include the use of rove beetles (Staphylinidae, Coleoptera) and ground beetles (Carabidae, Coleoptera) as pupal predators (Frank, 1967a, 1967b; Kowalski, 1976). Control of the adults has been done by aid of sticky traps preventing the females from crawling up the trees to oviposit, and sticky traps have also been used to monitor female winter moth activity. (Graf et al., 1995; Hand et al., 1987; Lind et al., 2003; Petersen, 2015). However, despite a wide array of biocontrol methods to minimize damage from winter moths many of the methods seems to be labor-intensive (i.e. sticky bands, Sigsgaard et al., 2013), monitoring dependent (i.e. timing of spraying with Bacillus thuringiensis, Sigsgaard et al., 2013), weather dependent (i.e. B. thuringiensis requires dryness, while frost and low temperature cause low predation by soil predators, Frank, 1967a; Jaastad et al., 2001; Lind et al., 2003), expensive (i.e. cost of spraying, Hardman & Gaul, 1990), or dependent on a high winter moth density to be effective (i.e. parasitoids, Horgan et al., 1999; Varley, 1971). Ants have the potential to overcome many of these challenges by being somewhat self-caring, relatively cheap to apply, generalist feeders that are not dependent on only winter moth as a food source, and by potentially being able to control the winter moth in more of its stages (Finnegan, 1975; Paulson & Akre, 1992b; Way & Khoo, 1992). Despite wood ants being associated with woods they may be suitable as control agents in orchards and plantations as well.

Figure 1: Life cycle of the winter moth (Operophtera brumata) (from Lind et al., 2003). Roman numerals refer to months.

Master thesis by Jesper Stern Nielsen 57 Wood ants of the Formica rufa group show many qualities that make them suitable as biocontrol agents against pests in orchards and plantations. In northern Eurasia they have no significant ant competitors and they are thus a dominant invertebrate predator in woodland (Savoläinen & Vepsäläinen, 1988; Way & Khoo, 1992). The activity of wood ants is typically seen from early spring, approximately March, when the ants awake from hibernation until mid-autumn, approximately October, where they reenter hibernation (Douwes et al., 2012; Rosengren et al., 1979). Foraging activity has been recorded down to 6°C, but nest activity has been observed at even lower temperatures (Rosengren et al., 1979). Their activity period is consistent with the activity period of most other arthropods which can be predated by the ants, but an exception includes winter moth adults which typically are active from October to December and thus avoid most predators (Lind et al., 2003). Within an activity season a wood ant nest can collect large amounts of food items. Estimates indicate that a medium sized nest of approximately 100,000 forager ants can collect between 64,000 and 8,000,000 pieces of food items per year (Adlung, 1966). Even though these numbers include items that from a biocontrol perspective are of no interest, like chitin, carrions, beneficial arthropods, plus a number of insects not killed by the ants, a great number of pest species have been observed to be included as well (Rosengren et al., 1979). Rosengren et al. (1979) estimated that 3200 larvae of Lepidoptera and sawfly were collected by their wood ant study nests each day in late June and early July, which is a promising result for biocontrol. Another observation that is encouraging from a biocontrol point of view was the appearance of “green islands” of birch around wood ant nests in otherwise defoliated areas in Finish Lapland, due to the hunt of Oporinia autumnata larvae (Lepidoptera) by wood ants (Rosengren et al., 1979).

A wood ant species of the F. rufa group that possess promising qualities for biocontrol purposes in orchards and plantations is Formica polyctena (Förster). F. polyctena is polygynous which enables their colonies to have a high reproductive output of workers, and makes the colony less vulnerable to the loss of a single queen (Way & Khoo, 1992). In addition F. polyctena often has a polydomous colony structure where the grouped nests are connected by trails and can exchange resources (Bugrova & Reznikova, 1990; Finnegan, 1975; Mabelis, 1994). This quality is useful from a biocontrol point of view because it allows a high nest density and thus the coverage of a large forage area without intraspecific competition between nests (Finnegan, 1975; Lafleur, 1941). Spreading of F. polyctena happens mainly by budding, and several new nests can thus come into existence nearby (Mabelis, 1994). Allthough the main habitat for F. polyctena is conifer woods the species has been found to have a high ecological flexibility (Pisarski & Czechowski, 1990). A recent study has utilized this ecological flexibility and developed a new method for successful transplantation and establishment of the wood ant F. polyctena in highly different types of plantations: an organic Christmas tree plantation, a conventional Christmas tree plantation, and an organic apple orchard

Master thesis by Jesper Stern Nielsen 58 (Nielsen, 2016, unpublished). The successful transplantation of ants into an orchard led to the current study of the beneficial effects of F. polyctena in apple production.

The aim of this study was to test whether transplanted nests of F. polyctena contributed to increased fruit production in an apple orchard. To answer this, the following sub-hypotheses were tested:

1) Leaf damage is higher on trees with a high number of winter moth larvae. 2) Apple yield is lower on trees with a high number of winter moth larvae. 3) The number of winter moth larvae is lower in trees with high ant activity. 4) Leaf damage is lower on trees with high ant activity. 5) Apple yield is higher on trees with high ant activity. 6) Apple yield is higher on trees close to the nearest active nest.

Furthermore, observations were made on the behavior during encounters of ants and winter moth larvae.

Master thesis by Jesper Stern Nielsen 59 Methods

Study site

The experiment was conducted in an organic apple orchard located near Tirstrup, East Jutland, Denmark (56° 18' 36.9"N, 10° 41' 28.1"E) in 2015. The total size of the orchard was approximately 1.3 ha and included about 3000 row-arranged trees of 11 varieties. The experimental area covered about 0.25 ha with apple trees that were approximately seven years old. Trees that were severely damaged or recently re- grafted were excluded from the study, and thus the number of trees included in the study was 425. The 425 trees were distributed in 9 rows of 5 different varieties (figure 2), plus 14 random positions of crab apple varieties (Malus sp.) that was used for pollinators. A two meter wide vegetation buffer strip, consisting of non-hoed herbs and grasses, and two machinery tracks divided the 9 rows in two, with 4 rows to the west and 5 rows to the east. Two vegetation buffer strips framed the study area to the west and the east, respectively (figure 2). The varieties in the experimental area were Holsteiner-Cox (HOL, row 1 and 6), Alkmene (ALK, row 2, 7, and 9), Collina (COL, row 3 and 8), Angold (ANG, row 4), and Resista (RES, row 5). The number of trees in each row was: row 1: 55, row 2: 33, row 3: 53, row 4: 48, row 5: 55, row 6: 53, row 7: 25, row 8: 50, and row 9: 53. In row 7 many trees were excluded because the trees had been re-grafted and were not developed. There was a distance of 3.5 m between rows to allow machines to maintain the area. Concrete poles were positioned for approximately every 7.5 m in each row, and wires were secured to them at approximately 60 cm and 200 cm height (see Petersen, 2015). Between each concrete pole were 6 wooden poles, positioned with approximately 1 m distance, which connected the wires and acted as support for the trees. Trees were positioned at and fastened to each wooden pole and each concrete pole, though some trees were missing. The lower wire had a garden hose attached for drip irrigation of the apple trees in the summer. A low dike (10 – 30 cm height) beneath the apple rows sometimes caused a lower distance between the wires and the ground. No herbicides or insecticides were used in the orchard. The area between the trees and between vegetation strips and trees was hoed mechanically every, or every second week. The vegetation in the orchard was heterogenic due to the variation in plant height and species composition in vegetation strips, machinery tracks, and apple rows. The main herbs and grasses were meadow-grass (Poa sp.), dandelion (Taraxacum sp.), orchard grass (Dactylis glomerata), couch grass (Elytrigia repens), common tansy (Tanacetum vulgare), cow parsley (Anthriscus sylvestris) and creeping thistle (Cirsium arvense). This difference in vegetation structure allowed both shaded paths and paths with plenty of sun. Honey bee (Apis sp.) hives were present next to the plantation and the bees functioned as primary pollinators. The area was drained by drainpipes but standing water was found sporadically in more rainy months (September-November).

Master thesis by Jesper Stern Nielsen 60

Veg. Strip

N3

N7

N4 N2

N8

N5

N1

N6

Veg. Strip

Veg. Strip

Figure 2: The experimental area. 425 apple trees were included (Red dots). The trees were distributed in 9 rows of 5 apple varieties, plus 14 random positions of crab apple. 8 nests (N1-N8) were transplanted into vegetation strips (Veg. Strip) in the area. Asterisks indicate nest positions.

Master thesis by Jesper Stern Nielsen 61 Transplantation and maintenance of ants

One donor nest of the wood ant Formica polyctena was dug up and transplanted as eight fragments into the study area the 27th of April, 2015 (For more information on the transplantation see Nielsen, 2016). In the first week the ants were fed with sugar dough (Ambrosia®, 85 % sucrose, Nordzucker) in perforated cash boxes and water in a plastic container next to the nest to facilitate establishment. After the first week the cash boxes were removed and sugar dough was provided in chosen trees (described in the following part). On the 16th of June and the 11th of October, each nest was provided one sack of extra conifer needles from dried cut-off branches of Christmas trees, next to the nests.

Treatments

A total of 34 trees were chosen as controls and 34 trees were chosen as sugar treatment trees (figure 3). Control trees were chosen by dividing each row into four groups. One tree in a group was then randomly chosen to be the control tree of that group. Treatment trees were chosen to be positioned randomly one or two positions away from the control tree, to minimize local differences in soil conditions, and the trees thus constituted a test pair. All randomization was done by using a random number generator command in Microsoft Excel. Row no. 7 was only divided into two groups and thus only had two control trees and two treatment trees. Control trees were isolated with sticky barriers on the tree stem, on the wooden poles if they were in possible contact with the ground, and on the upper wire that otherwise connected trees. The lower wire was only isolated if the stem and wooden stick isolation was positioned below the wire. Sticky barriers were made by a piece of duct tape glued with insect glue (OecoTak A5, Oecos). In two cases one ant was found on an isolated tree, probably due to wind transportation from the adjacent tree, but otherwise were ants observed to avoid the barriers. Treatment trees were provided a 50 ml centrifuge plastic tube with 60-80 g sugar dough, fixed horizontally to the trees at approximately a height of 120 cm above ground. Tubes were exchanged when empty or when they were expected to be emptied before next inspection.

Master thesis by Jesper Stern Nielsen 62

Figure 3: Distribution of isolated (red) and treatment trees (green). A total of 34 pairs of control and treatment trees were randomly positioned, with 4 pairs in each row, except row 7 where only 2 pairs were present. Asterisks indicate nest positions.

Data collection and arrangement

Tree and nest positions and distance

Tree and nest positions were captured with a high precision RTK-GPS (Sokkia GRX1 and Sokkia SHC250, accuracy: 1-10cm) and mapped in the ArcGIS® program ArcMap (version 10.3, ESRI®) (figure 2). The distances from each nest to each tree were calculated with the ArcMap analysis tool: Point distance. In the statistical tests, the distance from the nearest active ant nest to a tree is referred to as: Distance ActNest.

Master thesis by Jesper Stern Nielsen 63 Ant activity

The activity of the ants in the 425 apple trees was assessed on 6 dates during their active season (13-05, 22- 05, 04-06, 11-06, 21-06, and 23-07). Activity was assessed by counting the ants found on the tree in a period of 15-20 seconds. The number of ants was categorized as; 0: No ants, 1: 1-10 ants, 2: 11-50 ants, and 3: More than 50 ants. Ant activity category 2 and 3 were in the analyses grouped as one category in order to increase the sample size, and is referred to as high activity. Category 1 is referred to as low activity, and category 0 is referred to as no activity (none). In the statistical tests these activity levels are referred to as: Ant activity. Due to challenges in calculating an average of the ant activity levels from the observation dates, the highest observed ant activity level for each tree was used to represent the overall effect of ants. This will be referred to as: Maximum ant activity. Maps of the spatial distribution of ant activity for the six observation dates were made by interpolation using the Inverse Distance Weighted tool in ArcMap (IDW).

Damage assessment and number of larvae

16 test tree pairs were chosen for assessment of winter moth damage on the trees. Only pairs in the rows closest to the ant nests were chosen for this investigation to examine the maximum and minimum effect of ants on winter moth damage. On each of the trees a branch from the lower part of the tree and a branch from the upper part of the tree were chosen randomly. Randomness was assured by numbering the branches and let a 20-sidet dice select the number. The chosen branches were marked with a string for continuous recognition and they were examined the 10-05 (24 trees), 27-05 (32 trees), and 05-06 (32 trees). At each branch the following was counted: the number of winter moth larvae, the number of leaves with damage from gnawing larvae, the total number of leaves on the branch, and the total number of apple buds. The ant activity level for the tree was also noted. On the 27-05 counting of the number of leaves was deliberately omitted, due to high fragility of the buds. For the statistical tests, parameters were set up as: No. of WM larvae: The total number of winter moth larvae on the two branches on a given date, Leaf damage: the fraction of leaves with gnaws for the two examined branches, Apple bud loss: The number of lost apple buds on the two branches from one date to another. Trees with a gain of buds were excluded from apple bud loss analyses.

Master thesis by Jesper Stern Nielsen 64 Apple yield

The number of apples on each tree was counted on 411 trees the 3rd of September 2015. The 14 crab apple trees were not included. The parameter for the statistical tests was set up as: Apple yield: the number of apples larger than 2 cm in diameter that did not have the pathogen Monilia fructigena.

Observations on behavior

The behavior of ants in apple trees was observed regularly during the study (approximately 15-30 minutes on 16 data collection days). Encounters between wood ants and different pests were observed, including the encounter with winter moth larvae, garden chafer Phyllopertha horticola and other beetles (Coleoptera spp.), flies (Diptera spp.), Heteroptera species, apple ermine Yponomeuta malinellus, aphid species (Aphididae spp.), pollinators (primarily honeybees Apis spp.), harvestmen (Opiliones spp.), and spiders (Aranea spp.). The behavior of winter moths was also observed when encountering ants.

Tests

The effect of the No. of WM larvae was tested on: Leaf damage, Apple bud loss, and Apple yield. The effect of Ant activity was tested on: No. of WM larvae, Leaf damage, Apple bud loss, and Apple yield. The effect of Distance ActNest was tested on: Apple yield. This was done for each date, as the activity of nests changed during the study due to ant movement. The difference in pairs of control and treatment trees was tested on: No. of WM larvae, Leaf damage, Apple bud loss, and Apple yield.

Statistics

All analyses were made in the statistical program JMP® 12.1.0 (SAS, 2015). A Goodness-of-fit test (Shapiro- Wilk W test) was used to test whether numerical data followed a normal distribution. This included: No. of WM larvae, Leaf damage, Apple bud loss, and Apple yield. As all but one test did not follow a normal distribution, and it was not possible to transform data to obtain normality and variance homogeneity, non- parametric tests were used for the analyses. Correlations were tested using the Kendall’s tau (τ). Differences in matched tree-pairs were tested using the Wilcoxon Signed Rank test (JMP), and p-values were chosen according to the directional expectation of the results (one-tailed test). The non-parametric Wilcoxon Each Pair (JMP) was used to test differences in any groups. Analyses are tested at the significance level of 5 % (p < 0.05). * will be used for indicating significance.

Master thesis by Jesper Stern Nielsen 65

Results

Effect of winter moth larvae on leaf damage

Winter moth larvae were observed on the trees from the beginning of the experiment, the 27th of April, until the 24th of June. The major part was gone by the 10th of June.

For the 10-05 there was a significant positive correlation between No. of WM larvae and leaf damage (τ = 0.42, p = 0.0065*, N = 24, figure 4 A)) indicating a higher leaf damage at higher numbers of winter moth larvae. For the 05-06 there was no significant correlation (τ = 0.24, p = 0.083, N = 32), although the low p-value and the steep tendency line showed a tendency towards a correlation (figure 4 B)).

Figure 4: Fraction of leaf damage as a function of the number of winter moth larvae counted on two branches per tree for: A) 10- 05, and B) 05-06. The red line represents a tendency line. For A) there was a significant positive correlation between the parameters, while for B) there was no significant correlation. However, a tendency towards a positive correlation was seen both from the low p-value and the steep tendency line.

Master thesis by Jesper Stern Nielsen 67 Effect of winter moth larvae on bud loss and apple yield

No significant correlation was seen between the average No. of WM larvae and the Apple bud loss for the period 10-05 to 27-05 (τ = 0.095, p = 0.56, N = 21), and for the period 27-05 to 05-06 (τ = - 0.14, p = 0.32, N = 30) (figure 5).

Figure 5: Apple bud loss in one period as a function of the average number of winter moth larvae for: A) The 10-05 to 27-05, and for B) 27-05 to 05-06. No significant correlations were found.

Similarly, there were no significant correlations between No. of WM larvae and apple yield on any of the three dates or on average (figure 6).

Master thesis by Jesper Stern Nielsen 68

Figure 6: Apple yield as a function of the number of winter moth larvae for: A) 10-05, B) 27-05, C) 05-06, and D) the average from 10-05 to 05-06. No significant correlations were found.

Effect of ant activity on the number of winter moth larvae

A significant effect of ant activity on the No. of WM larvae was seen the 27-05 (p = 0.036*, N= 32), but not on any of the other dates or on average (figure 7). The No. of WM larvae was significantly lower in trees with low ant activity compared to trees with no ant activity (p = 0.011*), but there was no significant difference between high and low ant activity (p = 0.060) and high and none ant activity (p = 0.63). The sample size for low ant activity was quite low with only five trees in this category. This may explain the rather curious significance pattern.

Master thesis by Jesper Stern Nielsen 69

Figure 7: The number of winter moth larvae at different ant activity levels for: A) 10-05, B) 27-05, C) 05-06, D) the average number of winter moth larvae and the maximum ant activity. Groups that are not connected by same letter are significantly different. A significant effect of ant activity on the number of winter moth larvae was seen the 27-05, but not on any of the other dates or on average. The number of winter moth larvae was significantly lower in trees with low ant activity compared to trees with no ant activity (p = 0.011*), but there was no significant difference between high and low ant activity (p = 0.060) and high and none ant activity (p = 0.63). Values are overall means (+SE).

The Wilcoxon Signed Rank test showed no significant difference in the No. of WM larvae between control and treatment trees for any of the dates (10-05: S = - 1.00, p < S = 0.50, N = 12 pairs, 27-05: S = 22.0, p > S = 0.14, N = 16 pairs, 05-06: S = 21.0, p > S = 0.17, N = 16 pairs) or the average No. of WM larvae of the period (S = 26.5, p > S = 0.084, N = 16 pairs), though there was a tendency towards a higher No. of WM larvae on control trees for the average of the period.

Master thesis by Jesper Stern Nielsen 70 Effect of ant activity on leaf damage

A significant effect of the maximum ant activity was seen on leaf damage 10-05 (p = 0.049*, N = 24) (figure 8 B)), where trees with low ant activity had significantly less leaf damage than trees with high ant activity (p = 0.025*), but not significantly less than trees with no ant activity (p = 0.39). Only 3 trees had low ant activity, and the low sample size may explain the rather curious significance pattern. No significant effect were seen when analyzing the effect of ant activity on leaf damage on any of the other dates, but a tendency for higher leaf damage on trees with a low ant activity compared to no ant activity was seen for the 10-05. This pattern may also be explained by the low sample size (N = 3) (figure 8 A)).

Figure 8: Leaf damage at different ant activity levels for: A) 10-05, B) Maximum ant activity on leaf damage 10-05, C) 05-06, D) Maximum ant activity on leaf damage 05-06. Groups that are not connected by same letter are significantly different. A significant effect of the maximum ant activity was seen on leaf damage 10-05. No significance was seen on the other dates, though a low p-value indicated a tendency in A), but at a low sample size. Values are overall means (+SE).

Master thesis by Jesper Stern Nielsen 71 The Wilcoxon Signed Rank test showed no significant difference in the leaf damage between control and treatment trees for any of the dates (10-05: S = - 11.0, p < S = 0.21, N = 12 pairs, 05-06: S = 15.0, p > S = 0.23, N = 16 pairs).

Effect of ant activity on apple buds and apple yield

No significant effect of the maximum ant activity was seen on the total apple bud loss (figure 9).

p = 0.88 N = 24

A A A

N = 12 N = 3 N = 9

Figure 9: Total apple bud loss at different maximum ant activity levels. Groups that are not connected by same letter are significantly different. No significant difference is seen in apple bud loss between the three maximum ant activity levels. Values are overall means (+SE).

However, a significant effect was seen by maximum ant activity on the apple yield (figure 10). The mean values of apples on trees in each group of maximum ant activity were: High: 5.1 apples/tree, low: 2.1 apples/tree, and none: 2.2 apples/tree, suggesting that the presence of more than 10 ants per tree may lead to increased yields. When looking at the effect of maximum ant activity on apple yield by varieties, a significant effect was seen in Alkmene and Holsteiner-Cox, while a tendency of effect was seen in the other varieties (figure 11). The yield of Resista was generally higher for all three ant activity levels, which is in line with it being better at holding on to its apple buds (personal communication with the owner).

Master thesis by Jesper Stern Nielsen 72

p < 0.0001* A N = 411

B B

N = 91 N = 179 N = 141

Figure 10: Effect of maximum ant activity on apple yield. Groups that are not connected by same letter are significantly different. A significant difference is seen between high ant activity and low and no ant activity. Values are overall means (+SE).

p < 0.0001* p = 0.67 p = 0.57 p = 0.043* p = 0.38 N = 105 N = 45 N = 102 N = 108 A N = 51 A

N= 3

A

A

A A

A

N=18

N= 30

N= 14

N= 23

N= 8

N= 55 N= 70

A

N= 20

A

N= 20 N= 55

N= 16 N= 7

N= 40

N= 32 A A

A

B

AB B

Figure 11: Average yield (+SE) for the five varieties within each maximum ant activity group: = High ant activity, = Low ant activity = No ant activity. Groups that are not connected by same letter are significantly different. A large variation in the number of apples is seen at each ant activity levels.

Master thesis by Jesper Stern Nielsen 73 The Wilcoxon Signed Rank test showed no significant difference in apple bud loss between control and treatment trees (S = 8.0, p > S = 0.28, N = 12 pairs). Neither was a significant difference seen between control and treatment trees in apple yield (S = - 85.0, p < S = 0.065, N = 34 pairs), but a tendency towards more apples on treatment trees was seen.

Effect of distance from nearest active nest on apple yield

A significant negative correlation between the distance ActNest and the apple yield was seen on all dates (figure 12), suggesting that the low distances to ant nests lead to an increased yield. The correlation was higher for dates late in the season compared to dates early in the season.

A) B) C) p = 0.0059* p = 0.0083* p = 0.033* τ = - 0.10 τ = - 0.097 τ = - 0.079

D) E) p < 0.0001* p < 0.0001* τ = - 0.15 τ = - 0.14

Figure 12: Effect of distance to nearest active nest on apple yield for the 411 apple trees the: A) 13-05, B) 22-05 and 04-06, C) 11-06, D) 21-06, E) 23-07. A significant negative correlation is seen for each date.

Master thesis by Jesper Stern Nielsen 74 Ant distribution

The interpolated spatial distribution of ant activity for the six observation dates are illustrated in figure 13. The observations showed that ants at first (13-05) visited trees near the nests and in the nearest apple rows, and that high ant activity primarily was found closest to the nests. From the 13-05 to the 04-06 ants expanded their range of trees that they visited, but after the 04-06 this range was reduced. This change was seen approximately at the same time as apple tree flowering ended, as many aphids were seen, and as the main part of the winter moths pupated in the soil. On the 23-07 ants were found to be concentrated around trees with sugar feeding (green dots) or presence of aphids, and mostly in trees near nests with activity on that date (nest 2, 4, 6, 7 and 8).

The percentwise distribution of ant activity categories is seen in table 1. The lowest percentage of trees with at least one ant (low + high ant activity) was 9 % and was found the 23-07. The highest percentage of trees with at least one ant (low + high ant activity) was 53 % and was found the 04-06. Looking only at trees with at least 10 ants (high ant activity) the lowest percentage of trees was 4 %, which was found the 23-07, while the highest percentage of trees was 11 %, and was found the 21-06. The average percentage of trees with at least one ant was 34 %, and the average percentage of trees with at least 10 ants was 8 %.

Master thesis by Jesper Stern Nielsen 75 Ant activity 13-05 Ant activity 22-05 Ant activity 04-06

Ant activity 11-06 Ant activity 21-06 Ant activity 23-07

Figure 13: Interpolated ant activity at the study site on six dates of observation ( = No ant activity, = Low ant activity, = High ant activity). Dots indicate tree positions, green dots indicate trees with sugar feeding, and asterisks indicate nest positions. Tree positions have been made smaller the 23-07, to make areas with high ant activity more visible. From the 13-05 to the 04-06 ants expanded their range of trees that they visited, but after the 04-06 this range was reduced, and ants were on the 23-07 primarily found in trees with sugar feeding or presence of aphids.

Master thesis by Jesper Stern Nielsen 76 Table 1: Number and percentwise division of ant activities in the 411 trees. Percentage is rounded up or down, and may not sum to 100 %. The lowest percentage of trees with at least one ant (low + high ant activity) was 9 % (23-07), and the highest percentage was 53 % (04-06). The lowest percentage of trees with at least 10 ants (low + high ant activity) was 4 % (23-07), and the highest percentage was 11 % (21-06). The average percentage of trees with at least one ant was 34 %, while the average percentage of trees with at least 10 ants was 8 %.

No. of trees with no No. of trees with No. of trees with Date ant activity low ant activity high ant activity # % # % # % 13- 05 316 74 67 16 42 10 22-05 228 54 161 38 36 8 04-06 200 47 194 46 31 7 11-06 286 67 112 26 27 6 21-06 277 65 101 24 47 11 23-07 385 91 22 5 18 4 Average 282 66 110 26 34 8

Observations on behavior

Observations on ants were mainly made on their foraging behavior. Ants were observed to forage in the apple trees, when their original sugar source in the cash box was taken away from their eight nests. The workers from nest 2, 3, 4, 5, 7, and 8 quickly found a tube with sugar dough near their nest (figure 13, 13- 05). An ant track was observed from nest 1 to nest 2, and from nest 6 to nest 5 (For further information on the movement and difference in nest activity see Nielsen, 2016). Ants foraging in the trees were observed to spend a lot of time on and by the flower buds, probably to collect nectar from within the flower buds. Ants were not seen destroying the flower buds, though damage may have occurred. Ants were also observed in the flowers when the flowers bloomed (approximately 22-05). The foraging of ants was observed to happen mostly within apple rows compared to between rows. Ants were seen using paths both at the ground and at the lower wire and water hose that connected most of the apple trees in each row.

Master thesis by Jesper Stern Nielsen 77

Figure 14: A wood ant has deterred a winter moth larva. The larva escaped by letting itself drop from the leaf in a silk thread.

Many encounters were observed between ants and different arthropods. For example, winter moth larvae were observed to flea when ants were marching on the same branch or leaf as the larva (approximately 10 observations). If ants managed to come close, two outcomes were possible. The first outcome was that the larva let itself drop from the leaf or branch in a silk thread, and thus escaped the ants (figure 14). After some time the larvae would either crawl back up or slack itself to a lower position. The other outcome was that the ants caught the larva and brought it back to the nest (figure 15). A wood ant was also observed to throw itself on a P. horticola that was gnawing in an apple bud. As a counterattack the P. horticola started twisting in a bronc riding manner to buck off the ant. This “bronc riding” went on for 15 minutes before the observation was terminated. Other ants were nearby but did not assist the “rider”. The wood ants were also observed bringing other arthropods back to their nests. This included different beetles, flies, and Heteroptera species like the Italian striped-bug Graphosoma lineatum. The ants also tried to enter the webs of the Y. malinellus, but despite persistence from the ants no encounter was observed. The apple orchard had many harvestmen and spiders, but encounters were never observed between those and the ants, probably due to the rapidness of the spiders and the deterring scent from scent glands of the harvestmen (Hara & Gnaspini, 2003). One encounter was observed between a pollinator (honey bee) and ants. The bee was observed to fly into a flower where ants were present, and did not fly out instantly. Whether the bee managed to pollinate the flower is unknown, but the bee was not captured by the ants. Ants were also

Master thesis by Jesper Stern Nielsen 78 observed to engage in a mutualistic relationship with aphids on the apple trees, where they protected and attended the aphids in exchange for the sugary honey dew (Gösswald, 1989). Many colonies of aphids were observed to attract the ants. Even though ants were seen to nurse aphids they also collected sugar dough from the tubes. Ants were also observed nursing aphids on other plants than apple trees, for example on A. sylvestris and on Rumex species.

Figure 15: A wood ant has caught a winter moth larva and take a walk in the tree in triumph.

Master thesis by Jesper Stern Nielsen 79

Discussion

Summary of results Despite no clear pattern of significance from ant activity on winter moth larvae, apple bud loss, or leaf damage, apple yield was found to be significantly higher on trees with high ant activity compared to trees with no or low ant activity. A positive correlation was seen between the numbers of winter moth larvae and amount of leaf damage on one observation date, but no correlation was seen on the effect of winter moth larvae on the loss of apple buds or the apple yield. The distance from trees to the nearest active nest was significantly negatively correlated with the apple yield on each of the observation dates and the distribution of ants expanded until the 04-06, after which the ants reduced their expansion and concentrated around trees with sugar feeding and aphids. Ants were observed to: 1) cause escape behavior of winter moths, 2) predate on winter moth larvae and other arthropods except spiders and harvestmen, and 3) attend aphid populations both on apple trees and other plant species.

Effect of ants on yield

The significantly higher apple yield on trees with high ant activity compared to trees with low or none activity indicate an effect of wood ants on the fruit production in the apple orchard. Despite large variations in the number of apples within ant activity categories (figure 11) and a generally low apple yield in the study area, the yield was 5.1 apples/tree on trees with high ant activity, which is more than twice the yield on trees with low or no ant activity. This is supported by the significant negative correlation between distance from nearest active nest and apple yield and the tendency of a higher yield on treatment trees than on control trees. By looking at the effect of ants on yield by apple varieties, a significant effect was seen in the varieties Alkmene and Holsteiner-Cox, which were the best represented varieties (Alk: N = 105, Hol: N = 108), though Collina also was present in similar numbers (Col: N = 102), but did not show any significant difference. The less represented Resista showed no significant effect of ants on apple yield, which indicates that the overall association between ant activity and apple yield is unlikely to be caused by Resista being close to the ant nests (figure 2) and being better at holding on to its apple buds, but is likely to be an effect of the observed difference in ant activity. Regarding apple yield it was found to increase when an active ant nest was nearby, since a significant negative correlation was seen on all observation dates between the distance from apple trees to the nearest active nests and apple yield. This correlation is likely to be the outcome of a higher number of foraging ants near the nest, which decrease with increased distance to the nest (Adlung, 1966; Way & Khoo, 1992).

Master thesis by Jesper Stern Nielsen 81 Others have also looked at the effect of ants in fruit production. In a mango orchard in Australia Peng & Christian (2008) experienced that trees with weaver ants produced either similar amounts or more fruit per tree per year than treatments without weaver ants. Even though the climatic conditions, the mode of life, and the distance between nests and trees are different between weaver ants in Australia and wood ants in Denmark, they both have caused a higher yield of fruit by their presence. A quality assessment of the fruit was not conducted in the current experiment, but the findings by others show varying results. Stewart-Jones et al. (2008) found that apple trees that had been accessed by natural occurring ants had a greater proportion of apples damaged by the rosy apple aphid Drysaphis plantaginea, than trees where ants were excluded by a band of cloth gaffer tape coated with insect glue (OecoTak A5). Sugar attraction was, however, not used by Stewart-Jones et al. (2008) to tempt the ants to collect sugar instead of attending aphids (as was seen with Lasius niger by Offenberg, 2001). Peng & Christian (2008) found, in contrast to Stewart-Jones et al. (2008), that mango trees with weaver ants produced more first class fruit than treatments without ants. However, very few studies address this issue in fruit orchards and plantations, and the effect of ants on fruit quality should be addressed in further studies, as it is of high importance that fruit is saleable.

Both the density of nests and the distribution of ants in the area are of importance in relation to biocontrol in orchards and plantations. The density of nests is important, since a too low density could result in insufficient protection from ants on the crop (Rosengren et al., 1979). The nest density in the apple orchard was found to be 20 nests/ha (Nielsen, 2016, unpublished), which is above the number that according to Gösswald (1951 in Travan, 1994) is required. However, nest size is also important to ensure a high enough number of ants. In this experiment the nest sizes were only 90 L and the number of ants thus not as high as in most natural nests. The foraging efficiency is thus expected to be low the first year, but may increase if and as the number of ants increase. The number and activity of ants seemingly contributed to apple production, but a higher number of ants may contribute even more. If more ants were transplanted into the area their foraging range and activity on each tree may have been increased, which potentially could have led to an increased yield on more trees. Limitations of the nest density would primarily be sugar availability, but artificial sugar feeding can be adjusted to the need. However, it is unknown whether ants would concentrate on individual trees, as were seen the 23-07, and thus not forage in more trees than were seen this year. As this experiment only look at first year effects of ants, the number of ants and thus their contribution to yield may increase the following years, though an upper limit of increased yield is expected.

Master thesis by Jesper Stern Nielsen 82 The yield in the study area was very low compared to trees in other areas in the plantation, which on average have produced approximately 3.5 kg/tree (2004 to 2008), corresponding to approximately 10- 30 apples/tree (Æbletoften.dk, accessed 24-04-2016). The area of lower yield expands to an area beyond the study site (visual assessment and communication with the owner) and is thus unlikely to be caused by the ants. The presence of winter moths may explain the low yields in this part of the plantation, but as they were not found on all low-yielding trees this explanation is questionable. Instead, the low yield can be imputed to mineral shortage (personal communication with the orchard owner), other undetected pests, or too wet soil. Low temperatures and loss from wind exposure are not considered causing effects, because of the seemingly unaffected production in the other parts of the orchard. The effect of pests like winter moths could be a reason, as the results show a significant correlation between winter moth larvae and leaf damage the 10-05-2015, but this explanation seems unlikely, as no significant correlation was seen between the number winter moth larvae and apple bud loss or apple yield, and the low production seemed to expand beyond areas where winter moths or damage on leaves has been observed.

Ant distribution

The observations on ant distribution in the apple trees showed that ants at first expanded their range of trees that they visited, but after the 04-06 reduced their expansion and concentrated around trees with sugar feeding or presence of aphids. Different explanations are possible for this pattern. The first explanation is that ants explore the surrounding area to locate resources, and when persistent resources such as aphids were found, the exploration was lowered and more efforts were given to exploitation. Another explanation is that ants utilized nectar from flower buds in the apple trees, and when flowers faded and floral nectaries disappeared, ant movement was driven by presence of aphids and sugar feeding. The concentration of ants on trees with sugar or aphids can also be because no other sugar sources were available. The most widespread distribution was seen in late May and early June, which overlaps with the observed and expected activity of the winter moths (Lind et al., 2003, and own observations). If this distribution follows the same pattern the following years, the ants may control winter moths and it can be evaluated whether the moth damage was the cause of low production or if there are other issues that cannot be solved by ant presence.

Effect of ants on winter moth larvae and leaf damage

In the current experiment a measureable effect by ants on the fruit yield was seen, but only low or no effect of ants were found on the underlying factors; the number of winter moth larvae and leaf damage.

Master thesis by Jesper Stern Nielsen 83 The tests, regarding the effect of ant activity on larvae, did for one date show a significantly lower number of winter moth larvae in trees with low ant activity compared to no ant activity, but no significant difference was seen between high ant activity and the two other activity levels. This effect can be the result of the low sample size of trees in category low (Low: N = 5) and may therefore be misleading. A similar situation was seen for the effect of maximum ant activity on leaf damage 10-05, where trees with high ant activity had a significantly higher amount of leaves with gnaw than trees with low ant activity, but no significant difference was seen between trees with no ant activity and trees with higher activity. The results of this experiment therefore indicate that the predatory effect of ants is not the driving force, as it was not possible to measure a significant reduction in the number of winter moths and amount of leaf damage. However, the effect on other arthropod species was not measured, and therefore predatory behavior may still be a very important factor.

The reason that higher yield cannot be explained by ants removing winter moth larvae may have a number of different explanations. One explanation is that the sample size of damage assessment and winter moth larvae count was too small (N = 24 and 32). Another explanation is that ants deterred the larvae and therefore lowered herbivory from winter moth on apple buds and leaves, though no significant effect of ants was measurable. Other explanations could be that ants predated on other damaging arthropods not measured in the current study or that the presence of ants had other positive effects, like nutrient deposition from ant manure on host trees, or antibacterial or antifungal effects, which has been found by Pinkalski et al. (2015), Vidkjær et al. (2015), Chapuisat et al. (2007), and Lenoir et al. (2003). The possibility that another herbivor has caused some of the leaf damage, and thus may have blurred the effect of ants on the winter moth larvae may be an explanation, but it would be expected that ants would deter or predate on such a herbivor as well. A quite different explanation is that the higher number of apples on trees with high ant activity is a coincidence, but this seems unlikely due to the large sample size and the highly significant result (N = 411, P < 0001). A similar explanation is that because the study setup is descriptive, the significant association might be an artifact of the choice of ants to forage on trees with a high number of apples. However, even though no significant predatory effect of ants on winter moths could be measured, predation by ants on the winter moths was observed.

An effect of ants on the winter moths may be measureable after ants being present in the orchard for more than one season, when the ants are better established. Paulson & Akre (1992a) found that transplanted wood ants, Formica neoclara, began to contribute to pear psylla Cacopsylla pyricola (Hemiptera) control two years after introduction to a pear orchard, and that ant-excluded trees contained four to five times as many pear psylla as trees with ants (Paulson & Akre, 1992b). In the current experiment a contribution from the ants was already seen within the first year after transplantation, at least on the overall apple production. The longer presence of ants in the orchard also gives the ants the opportunity to

Master thesis by Jesper Stern Nielsen 84 control the winter moths in more of its life stages. Also, disturbance by ants probably caused a lower feeding rate by the winter moth, and as starved winter moth larvae produce adults that lay fewer eggs (MacPhee et al., 1988), the deterrence effect by the ants may eventually result in a lower number of winter moth larvae, despite no direct predatory effect of the ants were found. Thus the deterring and predatory effect of ants on winter moths may still become a measurable factor in the coming years.

Effect of ant presence in the apple orchard

The observed deterrence effect of the ants can be both positive and negative in a fruit orchard or plantation. The positive effects include, in addition to deterrence of winter moth, also deterrence of other damaging arthropods, for example P. horticola, different fly species, or maybe Y. malinellus in a stage before they spin their protective web. Similar effect was found by Olofsson (1992) who found that the survival of the European pine sawfly larvae Neodiprion sertifer increased sharply beyond 40 meters from a F. polyctena nest. Unfortunately there is a possibility that ants deter or predate on beneficial arthropods like spiders, harvestmen, soil-living predators like rove beetles and ground beetles, and pollinators like bees. In the study area a bee was observed to fly into a flower where ants were present, but whether the bee managed to pollinate the flower is unknown. Tsuji et al. (2004) found that the presence of weaver ants significantly lowered the flower-visiting rate of flying insects in a fruit orchard. The findings by Assunção et al. (2014) indicate that pollinators identified ants as a danger, which caused a lower fruit production by flowers of the Brazilian shrub Heteropterys pteropetala. Also, they found that only the constant presence of ants deterred pollinators. The presence of ants could be constant during flowering, but some flowers may occasionally be left unguarded by ants and allow access of pollinators. Ballantyne & Willmer (2012) concluded that bumblebees are not inherently repelled by ants, but that they by experience can learn to ignore flowers with ant-deposited scent marks, meaning that they can avoid flowers were ants has been present recently. Despite a potential deterrence of pollinators, Peng & Christian (2008) concluded that trees with weaver ants produced either similar amounts or more fruit than trees without ants, as was also seen in the current experiment, and whether pollinator deterrence is an issue of high importance is thus questionable. The deterrence effect of ants on other beneficials was investigated by Gridina (1990), who found a negative correlation between the number of ants present in an area and the number of individuals within the groups of spiders, harvestmen, rove beetles, and ground beetles in a spruce-leaved forest and a spruce-pine forest, though this varied over the season. Also, refuges were found where the activity of ants was low. The effects of ants on beneficial arthropods are well-summarized in Adlung (1966). Wellenstein (1959 in Adlung, 1966) found that the population of spiders and mites near an ant nest was 83 % of the population 80 meters away from the nest. He also found that large parasitic wasps and small parasitic wasps (Ichneumonidae sp.) was 42 % and 83 % of the population 80 meters away from the nest, Master thesis by Jesper Stern Nielsen 85 respectively. Even though soil predators and parasitoids have been proved to have a high influence on for example winter moth mortality (MacPhee et al., 1988), and the negative effect of ants on other beneficials seems unavoidable, the ants have the potential to control many different arthropods which may compensate for the lesser control from other beneficials.

Problems with aphids

In the current experiment the wood ants were observed to engage in a mutualistic relationship with aphids. Aphids provide ants with honeydew in exchange for protection and tending (Gösswald, 1989). However, this mutualistic relationship can cause troubles in orchards and plantations, since aphids may affect trees negatively by loss of growth, shoot deformation, rolling of leaves, increased risk of fungal infections, increased susceptibility to secondary invasion by other pests, or reduced yield (Adlung, 1966; Lind et al., 2003; Müller, 1956). Artificial sugar feeding (with honey solution) has successfully been used to interrupt the mutualism between Lasius ants and aphids and led to an increased predation on the aphids (Offenberg, 2001). In another experiment on Camponotus ants no effect was seen on the tending of aphids when the ants were presented an alternative sugar source (Del-Claro & Oliveira, 1993). Another test on sugar acceptance has shown that a sugar solution can limit the attendance of the wood ant Formica lugubris on aphids in the spring and early summer (Sudd & Sudd, 1985). Wood ants have been found to bypass sugar baits (Ambrosia®, sugar dough) if honeydew producing aphids were in the neighborhood (Lassen, 2015). It is thus important to develop or find sugar sources that can compete with the honeydew to interrupt ant- aphids mutualism, limit the negative effects of aphids on trees, and optimize management of ants in pest control situations.

Other remarks

An issue that is of great concern by orchard and plantation owners is the annoyance of ants to plantation workers, especially in the form of biting at harvest (Peng & Christian, 2008). Peng & Christian (2008) suggested to spray mango trees with water to limit the ant activity in the trees for some time, or to put the harvested fruit shortly into water. The same could be applied in other orchard and plantation types. However, this issue has not caused problems in this experiment so far, since the density of ants in the trees was not that high. Another concern could be the possible damage from ants gnawing in flower buds to collect nectar, but in this setup the number of apples was higher on trees with high ant activity, which indicates that there was little or no damaging effect of ant activity on the flower buds.

Master thesis by Jesper Stern Nielsen 86 Conclusion

This experiment shows a clear positive effect of ants on apple yield, with an apple average of 5.1 apples/tree on trees with high ant activity, which was more than the double compared to trees with low or no ant activity. This effect was seen despite no or only low effect of ants could be measured on the underlying levels: leaf damage and number of winter moth larvae. A significant effect of winter moths on leaf damage was found, but no effect of winter moth on apple yield was seen, suggesting that there may be other pests or problems influencing fruit production in that part of the plantation. The correlation between the distance from a tree to the nearest active nest and apple yield was significant, indicating that ants are most effective closest to the nest. Ants were first observed to expand their distribution and reducing it later on and concentrating on or near trees with sugar feeding or presence of aphids. The timing of the largest distribution overlapped with parts of the winter moths active season in late May to mid-June, and if this is seen in the coming seasons the effect of ants may be measureable on winter moth larvae. Furthermore, ants were observed to predate and deter winter moths, predate on other arthropods, and engage in a mutualistic relationship with aphids, which may cause reduced yield. Future studies should include both fruit quality assessments plus comparison of yield in form of weight and numbers. Also, the effect of ants on winter moth numbers, weight, and size should be addressed more thoroughly to test the predatory and deterrence effect by ants. Furthermore, more investigations on the distribution and number of ants in trees, from transplanted nests, are of importance.

Master thesis by Jesper Stern Nielsen 87

References

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Master thesis by Jesper Stern Nielsen 89 Hardman, J. M., & S. O. Gaul. 1990. Mixtures of Bacillus thuringiensis and Pyrethroids Control Winter Moth (Lepidoptera, Geometridae) in Orchards Without Causing Outbreaks of Mites. Journal of Economic Entomology 83: 920-936. Holliday, N. J. 1977. Population Ecology of Winter Moth (Operophtera brumata) on Apple in Relation to Larval Dispersal and Time of Bud Burst. Journal of Applied Ecology 14: 803-813. Horgan, F. G., J. H. Myers, & R. Van Meel. 1999. Cyzenis albicans (Diptera : Tachinidae) Does Not Prevent the Outbreak of Winter Moth (Lepidoptera : Geometridae) In Birch Stands and Blueberry Plots on the Lower Mainland of British Columbia. Environmental Entomology 28: 96-107. Jaastad, G., D. Roen, & L. Nornes. 2001. Field Evaluation of Bacillus thuringiensis Against Lepidopterans in Norwegian Apple Orchards. Entomol Exp Appl 100: 347-353. Karhu, K. J. 1998. Effects of Ant Exclusion During Outbreaks of a Defoliator and a Sap-sucker on Birch. Ecological Entomology 23: 185-194. Kowalski, R. 1976. Biology of Philonthus decorus (Coleoptera, Staphylinidae) in Relation to Its Role as a Predator of Winter Moth Pupae Operophtera brumata (Lepidoptera, Geometridae). Pedobiologia 16: 233-242. Lafleur, L. J. 1941. The Founding of Ant Colonies. Biological Bulletin 81: 392-431. Lassen, J. W. 2015. Evaluation of Formica polyctena (Hymenoptera: Formicidae) as a Control Agent of Dreyfusia nordmannianae (Hemiptera: Adelgidae) in Danish Christmas Trees. Master Thesis, Unpublished. Lenoir, L., J. Bengtsson, & T. Persson. 2003. Effects of Conifer Resin on Soil Fauna in Potential Wood-ant Nest Materials at Different Moisture Levels. Pedobiologia 47: 19-25. Lind, K., G. Lafer, & K. Schloffer. 2003. Organic Fruit Growing. CAB International, Wallingford, GB. Mabelis, A. A. 1994. Flying as a Survival Strategy for Wood Ants in a Fragmented Landscape (Hymenoptera, Formicidae). Memorabilia Zoologica 48: 147-170. MacPhee, A., A. Newton, & K. B. McRae. 1988. Population Studies on the Winter Moth Operophtera brumata (L) (Lepidoptera, Geometridae) in Apple Orchards in Nova Scotia. Canadian Entomologist 120: 73-83. Müller, H. 1956. Können Honigtau liefernde Baumläuse (Lachnidae) ihre Wirtspflanzen schädigen? (Can Honeydew Producing Aphids Cause Damage to Their Host Trees?). Zeitschrift für Angewandte Entomologie 39: 168-177. Nielsen, J. S. 2016. Transplantation of Formica polyctena Förster (Hym., Formicidae) into Plantation Crops for Biological Control. Master Thesis, Manuscript 1, Unpublished. Offenberg, J. 2001. Balancing Between Mutualism and Exploitation: The Symbiotic Interaction Between Lasius Ants and Aphids. Behavioral Ecology and Sociobiology 49: 304-310. Offenberg, J. 2007. Myrer som Plantebeskyttere (Ants as Plant Protectors). moMentum 2007, nr. 1: 31-34. Offenberg, J. 2015. Ants as Tools in Sustainable Agriculture. Journal of Applied Ecology 52: 1197-1205. Olofsson, E. 1992. Predation by Formica polyctena Förster (Hym., Formicidae) on Newly Emerged Larvae of Neodiprion sertifer (Geoffroy) (Hym., Diprionidae). Journal of Applied Entomology 114: 315-319. Oxforddictionaries.com. accessed 23-03-2016. Oxford Dictionaries. http://www.oxforddictionaries.com/definition/english/ Accessed 23-03 2016. Paulson, G. S., & R. D. Akre. 1992a. Introducing Ants (Hymenoptera: Formicidae) into Pear Orchards for the Control of Pear Psylla, Cacopsylla pyricola (Foerster) (Homoptera: Psyllidae). Journal of Agricultural Entomology 9: 37-39. Paulson, G. S., & R. D. Akre. 1992b. Evaluating the Effectiveness of Ants as Biological Control Agents of Pear Psylla (Homoptera: Psyllidae). Journal of Economic Entomology 85: 70-73. Peng, R., & K. Christian. 2013. Do Weaver Ants Affect Arthropod Diversity and the Natural-Enemy-to-Pest Ratio in Horticultural Systems? Journal of Applied Entomology 137: 711-720. Peng, R. K., & K. Christian. 2008. Potential for Organic Mango Production in the Northern Territory of Australia Using Weaver Ants, Oecophylla smaragdina, (Hymenoptera: Formicidae). In: R. K. Prange and S. D. Bishop (eds.) Proceedings of the International Symposium on Sustainability through Integrated and Organic Horticulture. Acta Horticulturae. 81-88.

Master thesis by Jesper Stern Nielsen 90 Petersen, J. H. 2015. Drømmen om en Lille Økologisk Æbleplantage (The Dream of a Small Organic Apple Orchard). Gyldendal A/S. Peterson, N. A., & A. C. Nilssen. 1998. Late Autumn Eclosion in the Winter Moth Operophtera brumata: Compromise of Selective Forces in Life-cycle Timing. Ecological Entomology 23: 417-426. Pinkalski, C., C. Damgaard, K.-M. V. Jensen, R. Peng, & J. Offenberg. 2015. Quantification of Ant Manure Deposition in a Tropical Agroecosystem: Implications for Host Plant Nitrogen Acquisition. Ecosystems 18: 1373-1382. Pisarski, B., & W. Czechowski. 1990. The Course of Artificial Colonization of Red Wood Ants in the Gorce National Park. Memorabilia Zoologica 44: 37-46. Roland, J. 1994. After the Decline - What Maintains Low Winter Moth Density after Successful Biological Control. Journal of Animal Ecology 63: 392-398. Roland, J., & D. G. Embree. 1995. Biological Control of the Winter Moth. Annual Review of Entomology 40: 475-492. Roland, J., S. J. Hannon, & M. A. Smith. 1986. Foraging Pattern of Pine Siskins and Its Influence on Winter Moth Survival in an Apple Orchard. Oecologia 69: 47-52. Rosengren, R., K. Vepsäläinen, & H. Wuorenrinne. 1979. Distribution, Nest Densities, and Ecological Significance of Wood Ants (The Formica rufa Group) in Finland. Bulletin SROP 2: 181-213. Rosumek, F. B., F. A. O. Silveira, F. de S. Neves, N. P. de U. Barbosa, L. Diniz, Y. Oki, F. Pezzini, G. W. Fernandes, & T. Cornelissen. 2009. Ants on Plants: A Meta-analysis of the Role of Ants as Plant Biotic Defenses. Oecologia 160: 537-549. Savoläinen, R., & K. Vepsäläinen. 1988. A Competition Hierarchy among Boreal Ants: Impact on Resource Partitioning and Community Structure. Oikos 51: 135-155. Sigsgaard, L., H. Philipsen, H. B. Jakobsen, & M. Korsgaard. 2013. Mekanisk og Biologisk Bekæmpelse af Frostmålere, Viklere og Ribsbredvingemøl i Økologisk Solbær og Ribs (Mechanical and Biological Control of Winter Moths, Leafroller Moths and The Currant Shoot Borer Moth in Organic Blackcurrant and Redcurrant). Det Naturvidenskabelige Fakultet - Københavns Universitet. Skinner, G. J., & J. B. Whittaker. 1981. An Experimental Investigation of Inter-relationships Between the Wood-ant (Formica rufa) and Some Tree-canopy Herbivores. Journal of Animal Ecology 50: 313- 326. Stewart-Jones, A., T. W. Pope, J. D. Fitzgerald, & G. M. Poppy. 2008. The Effect of Ant Attendance on the Success of Rosy Apple Aphid Populations, Natural Enemy Abundance and Apple Damage in Orchards. Agricultural and Forest Entomology 10: 37-43. Stoakley, J. T. 1985. Outbreaks of Winter Moth, Operophthera brumata L. (Lep., Geometridae) in Young Plantations of Sitka Spruce in Scotland - Insecticidal Control and Population Assessment Using the Sex Attractant Pheromone. Zeitschrift Fur Angewandte Entomologie - Journal of Applied Entomology 99: 153-160. Sudd, J. H., & M. E. Sudd. 1985. Seasonal Changes in the Response of Wood-ants (Formica lugubris) to Sucrose Baits. Ecological Entomology 10: 89-97. Tenow, O. 1972. The Outbreaks of Oporinia autumnata Bkh. and Operophthera spp. (Lep., Geometridae) in the Scandinavian Mountain Chain and Northern Finland 1862-1968. Zoologiska Bidrag från Uppsala: 1-107. Tesanovic, D., & R. Spasic. 2013. Pests of Apple Leaf and Flower Buds in the Region of East Sarajevo. Fourth International Scientific Symposium "Agrosym 2013", Jahorina, Bosnia and Herzegovina, 3-6 October, 2013. Book of Proceedings: 546-552. Travan, J. 1994. Über Prof. Gösswalds vieljährige Versuche mit der künstlichen Ansiedlung und Vermehrung von Völkern der Roten Waldameisen (Hym., Formicidae) in Unterfranken (Professor Gösswald's Long-term Experiments on the Artificial Establishment and Propagation of Colonies of Red Wood Ants in Lower Franconia). Waldhygiene 20: 47-130. Tsuji, K., A. Hasyim, Harlion, & K. Nakamura. 2004. Asian Weaver Ants, Oecophylla smaragdina, and Their Repelling of Pollinators. Ecol Res 19: 669-673.

Master thesis by Jesper Stern Nielsen 91 Varley, G. C. 1971. The Effects of Natural Predators and Parasites on Winter Moth Populations in England. Proceedings of the Tall Timbers Conference on Ecological Animal Control by Habitat Management, No. 2: 103-116. Vidkjær, N. H., B. Wollenweber, R. Gislum, K.-M. V. Jensen, & I. S. Fomsgaard. 2015. Are Ant Feces Nutrients for Plants? A Metabolomics Approach to Elucidate the Nutritional Effects on Plants Hosting Weaver Ants. Metabolomics: 1-16. Warrington, S., & J. B. Whittaker. 1985. An Experimental Field-study of Different Levels of Insect Herbivory Induced by Formica rufa Predation on Sycamore (Acer pseudoplatanus) I. Lepidoptera Larvae. Journal of Applied Ecology 22: 775-785. Way, M. J., & K. C. Khoo. 1992. Role of Ants in Pest Management. Annual Review of Entomology 37: 479- 503. Wesolowski, T., & P. Rowinski. 2006. Tree Defoliation by Winter Moth Operophtera brumata L. During an Outbreak Affected by Structure of Forest Landscape. Forest Ecology and Management 221: 299- 305. Whittaker, J. B., & S. Warrington. 1985. An Experimental Field-study of Different Levels of Insect Herbivory Induced by Formica rufa Predation on Sycamore (Acer pseudoplatanus) III. Effects on Tree Growth. Journal of Applied Ecology 22: 797-811. Æbletoften.dk. accessed 24-04-2016. http://www.aebletoften.dk/projekt/hoestudbytter.htm.

Master thesis by Jesper Stern Nielsen 92 Additional thoughts, methods and tests

During the study, many considerations have been made and methods have been tested. This section is made to let the reader into some of the considerations, methods, and results that was not included in the final articles.

Estimation of nest activity level

Instead of estimating the activity of ants on a nest by the somewhat arbitrary visual examination, other approaches were considered. One of the contemplated methods was to count the number of ants walking to and from their nest on one of their tracks within a certain time interval. Unfortunately, the vegetation made it difficult to observe their tracks relatively close to their nests. Cutting some of the vegetation was attempted, but the counting was still impractical and therefore discarded. Another, but similar method that was contemplated was inspired by Turaki et al. (2012), who estimated activity of the harvester ant Messor galla by looking at seed collection. The amount of seeds removed from a position near a nest within a certain time interval could indicate the activity, and seeds could also make it easier to spot ants in the vegetation. This method was not used due to the expected time consummation compared to the visual estimation, which took about 15 min in total. The Lincoln-index method or mark-release-recapture method (Chew, 1959) was also dropped due to the expected time consummation and challenges regarding observing ant tracks in the vegetation. Another attempt was to evaluate activity of nests by looking at the removal of sugar dough (Ambrosia®, 85 % sucrose, Nordzucker) from the feeding tubes. Problems with the weighing machines occurred repeatedly during field collection and the sugar removal was only recorded on an overall basis. The overall removed amount of sugar dough is showed in figure M1. The sugar consumption by a nest was originally thought to equal the amount of sugar dough removed from tubes closest to the nest. The data included only sugar dough removal from the 27th of April to the 23rd of July, because after that date large numbers of wasps were observed in the feeding tubes. Another issue was that sugar dough was also removed from feeding tubes near visually abandoned nests, which put the reliability of the method in question. The problem with the weighing machine and the removal of sugar dough by other arthropods than ants were the main reasons for excluding these data and findings in the articles. Sugar removal could otherwise have been a proxy for or a supplement on assessing ant or nest activity.

Master thesis by Jesper Stern Nielsen 93

Figure M16: Sugar dough consumption in the feeding tubes in the apple orchard from the 27th April to the 23rd of July. Green colors indicate a low consumption and red colors indicate a higher consumption (see Legend in figure). The numbers in the legend are in grams of sugar dough (g).

Evaluating the effect of ants on winter moth larvae by differences in number, larvae weight and size

Originally the effect of ants was assumed to influence the number of winter moth larvae in the apple orchard, but as observations on the interaction between ants and larvae mostly showed a flea response by the winter moth larvae, the effect of ants were assumed to lower the feeding rate of the larvae. As starved winter moth larvae produce adults that lay fewer eggs (MacPhee et al., 1988), the deterrence effect by the ants might cause a lower number of winter moths in the long run. If this effect could be estimated, the deterrence effect of ants could be confirmed or disproved. The date for the counting and measurement was chosen half a week before carrying it out (Mid-June) to make sure that the larvae were well-developed.

Master thesis by Jesper Stern Nielsen 94 Unfortunately the larvae managed to enter the soil and pupate before the day of examination, thus rendering count, weight and size measurements impossible.

Evaluating effect of ants by looking at winter moth pupae

As the winter moth larvae managed to pupate before examination of their numbers, weights and sizes, it was attempted to find their pupae in the soil. A comparison could thus be made between pupae number, size, and weight, in areas with many ants compared to areas with few ants, or number of pupae, size, and weight underneath trees that was sugar feed compared to trees that had been isolated from ants. To test whether pupae could be found in soil samples, two trees, where many winter moths had been present, was chosen. From beneath each of those trees, two soil samples of 40 cm * 30 cm * 10 cm positioned approximately 15 cm from the stem were dug up, as the winter moth pupae typically is found within the upper 5 cm of the soil (Holliday, 1977). The soil samples were brought home and searched thoroughly by washing and sieving the soil, to remove all but material larger than 1 mm. Despite efforts, no pupae were found, and the methods was concluded too time and labor consuming.

Figure M18: Plastic pots are placed around the sticky ring barrier trap to protect birds from taking trapped Figure M17: An adult male (left) and an adult female (right) winter winter moths and to shield against rain. moth are trapped on the sticky ring barrier trap. The lightly green dots behind the female are eggs. Notice that the male is winged, while the female wings are reduced.

Master thesis by Jesper Stern Nielsen 95 Evaluating effect of ants by looking at the number of adult winter moths

As no pupae were found in the soil samples, it was attempted to quantify the effect of ants by comparing the number of adult winter moths on trees with high and low ant activity, respectively. Glue traps were used to catch the adult winter moths and hereby assess the number of adult female winter moths, since they are known to crawl up the tree and lack the ability to fly. Two types of traps were used: a sticky ring barrier (Neudorff®) on the stem of the apple trees, and an emerge-trap on the ground. Sticky ring barriers were made by winding the Neudorff® glued band around the stem of the tree and secure it with duct tape. A round plastic pot was placed over the sticky barrier round the stem to protect the trapped winter moths from birds, and to shield against rain (figure M2). Bot males and females were trapped on Figure M19: Emerge-trap. Sticky barriers were positioned on the inside of the box. A total of three winter moths were captured in the sticky ring barrier (figure M3). The emerge-trap the 32 emerge-traps. was made from a 34 * 24 * 16 cm plastic box (SmartStoretm) where sticky barriers were placed along the middle of the sides inside the box. The box was then placed with the opening towards the ground, so that emerging winter moth could be trapped on the inside (figure M4). The 14th-16th of October 32 emerge-traps and 42 sticky barriers were put up in the study area (figure M5). The emerge-traps thus covered a total area of approximately 2.6 m2. It was attempted to find two groups of trees: those where nearby ant activity had been high during the season, and those were ant activity had been low. The pairs of treatment and isolated trees were not used to avoid an effect of the sugar in the treatment trees, which attracted ants to the nearby ground area and therefore could have had an effect on the number of moth larvae that were pupating. Traps were checked for winter moths the 10- 11-2015, 20-11-2015, 01-12-2015, and the 08-12-2015, and trapped individuals were removed from the traps and put in separate plastic bags for later examination and species confirmation. Many harvestmen and small flies (Diptera sp.) were caught on the sticky ring barrier traps. Harvestmen were removed, but sticky barriers with too many flies were changed. A total of three winter moths were caught in the emerge- traps from the 16th of October to the 8th of December. This low number made a comparison impossible, and the emerge-trap design was thus concluded impractical or ineffective. On the sticky ring barrier traps the total number of trapped adult female winter moths was 45 and the total number of adult winter moth was 121 from the 16th of October to the 8th of December. As the males can fly, their number would not give a Master thesis by Jesper Stern Nielsen 96 good estimate of the number of winter moths from an area with or without ants, as they could have travelled from other trees. It was thus most obvious to use the females for analyses, but out of curiosity both an analysis including only females and an analysis including both were carried out. The analyses were done by testing for differences in adult winter moth numbers between areas with high ant activity and areas with low or no ant activity, using a Wilcoxon test. The result for differences in adult females was not significant (Prob>ChiSq =0.92) and neither was it for both adult male and female winter moths (Prob>ChiSq =0.17), which lead to the exclusion of the examination in article 2.

Figure M20: Locations of emerge-traps (blue dots) and sticky ring barrier traps (Orange dots). Nest positions are indicated with asterisks.

Evaluating the effect of ants on Monilia fructigena

To test whether ants had a positive, negative, or no effect on the plant pathogen Monilia fructigena (figure M6), the number of apples with M. fructigena was counted the 03-09-2015. The effect of maximum ant activity on the number of apples with M. fructigena was tested with a Wilcoxon test. Also, the effect of maximum ant activity on the presence of apples with M. fructigena (Presence/absence) was tested with a Contingency Analysis. No significant effect was seen for the ants on the number of apples with M. fructigena (Prob>ChiSq = 0.89) or on the presence of it (Prob>ChiSq = 0.94).

Other observations that was not used

During the study other observations were made in the field. This included presence/absence counts of aphids in trees, but because aphids were more easily seen when ants were present, the data was considered useless. Apple ermine Yponomeuta malinellus was also observed, and the number of webs in each tree counted. The effect of ants on the apple ermine was tested with a Wilcoxon test for four dates (04-06-2015, 11-06-2015, 21-06-2015, 23-07-2015), but no effect was found (Prob>ChiSq = 0.22, Master thesis by Jesper Stern Nielsen 97 Prob>ChiSq = 0.051, Prob>ChiSq = 0.51, Prob>ChiSq = 0.74, respectively). This was to be expected, as apple ermine creates a dense web around the colony, and ants were not observed to enter the web though they tried. Furthermore, the presence of other ant species occurred and was noted, but the occurrence where low and data thus limited. The species found included Formica fusca, black garden ant Lasius niger, and a Myrmica species. No analyses were made on those, due to the limited data.

To conclude, several methods were tested for activity assessments of the ants, but most were discarded. Different traps were tested for the collection of winter moths in various developmental stages, but no effect from ants was seen. Observations were also made on the presence of other arthropods in the apple trees, as well as the presence of the pathogen M. fructigena. All this was disregarded from the paper-manuscripts for various reasons.

Figure M21: Apple with Monilia fructigena.

References

Chew, R. M. 1959. Estimation of Ant Colony Size by the Lincoln Index Method. Journal of the New York Entomological Society 67: 157-161. Holliday, N. J. 1977. Population Ecology of Winter Moth (Operophtera brumata) on Apple in Relation to Larval Dispersal and Time of Bud Burst. Journal of Applied Ecology 14: 803-813. MacPhee, A., A. Newton, and K. B. McRae. 1988. Population Studies on the Winter Moth Operophtera brumata (L) (Lepidoptera, Geometridae) in Apple Orchards in Nova Scotia. Canadian Entomologist 120: 73-83. Turaki, Z. G. S., J. Z. Turaki, J. D. Zira, and A. B. Abba-Masta. 2012. Estimation of Haulage Capacity and Nest Activity of Different Sizes of Harvester Ant (Messor galla Forel) for Major Small Grain Cereals in Nigeria. Journal of Stored Products and Postharvest Research 3: 83-86.

Master thesis by Jesper Stern Nielsen 98 Picture Gallery

The Fragmentation and Transplantation Process

P 1 P 2

P 4 P 3

P 5 P 1: A donor nest, with tree stumps for the artificial nests, placed some time before to let the ants become accustomed to them.

P 2 and P 4: The chosen nest is dug up in horizontal layers, and the upper nest material is divided between eight 90 L tubs – each comprises material for one fragment. The fragmentation is done with shovels and spades.

P 3: The search for ovipositing queens is done by searching the lower part of the nest thoroughly with tweezers.

P 5: A look into the lower part of the nest. This nest has a large complex of galleries throughout the surrounding sandy soil. Nest material is carefully removed by scraping the material onto shovels. P 6: A tub with a part of a fragment, consisting of P 7 upper nest material and ants.

P 7: The tubs are covered with tulle and transported by trailer.

P 8: An ovipositing Formica polyctena queen. Found ovipositing queens were put in plastic containers with moist moss. The picture is kindly lent out by Jens H. Petersen.

P 9: The artificial transplanted nest is constructed. In the bottom is sandy soil from the donor nest, then a tree stump (see P 1), and then nest material from the tubs are poured carefully over, so that the ants can settle the needles and nest themselves. The picture is kindly lent out by Jens H. Petersen.

P 8

P 6

P 9

P 10 P 11

P 12

P 10: Artificial transplanted nest. A fine thatched surface structure was seen after 3 hours, and two to four ovipositing queens were added.

P 11: Sugar feeding box, made from a cash box. Two holes drilled in the lower left side allow entrance by ants to sugar dough.

P 12: The ovipositing queen is added to a nest. Workers examined her, before she disappeared into an entrance of the nest.

P 13: Artificial nest after approximately one month (04-06-2015) in the organic apple orchard (TS1).

P 14: Artificial nest in the conventional Christmas tree plantation (TS3) 19-05-2015.

P 15: Artificial nest in the organic Christmas tree P 13 plantation (TS2) (12-08-2015). The nest is protected with chicken wire.

P 15

P 14

Damage to Apple Trees

P 16 P 17

P 18

P 16: Leaf damage by winter moths on an apple tree.

P 17: Damage on an apple bud from a garden chafer Phyllopertha horticola.

P 18: Damaged dried out apple buds.

P 19: Larvae of Apple ermine Yponomeuta malinellus, sitting protected in their web gnawing on apple leaves.

P 19

Winter Moths

P 20 P 20: Winter moth larvae 14-05-2015 P 21: Winter moth larvae 14-05-2015

P 22: Winter moth larvae 21-06-2015

P 23: Winter moth larvae 14-05-2015

P 24: Adult winter moths trapped on a sticky ring barrier. The winged male is seen to the left and the female with reduced wings is seen to the right (20-11-2015).

P 21 P 22

P 24 P 23

Ants and Aphids

P 25 P 26

P 27

P 25: The wood ants attend aphids in the apple trees (11-06-2015).

P 26: Aphids on an apple leaf (04-06-2015).

P 27: Wood ants attend aphids on a Rumex species (04-07-2015).

P 28: Some leaves were full of attended aphids (23-08-2015).

P 28

Ant Deterrence and Predation

P 30 P 29 P 29: A wood ant has caught a Diptera species P 30: A wood ant has deterred a winter moth larvae that now hang in a silk thread (14-05- 2015).

P 31: Two wood ants has caught a winter moth larvae (14-05-2015)

P 32: A wood ant has jumped onto a garden chafer, and is given a “ride” – The garden chafer twists its body from side to side, quickly and repeatedly, but the ant is holding on.

P 33: A wood ant has caught a winter moth, and is making a victory parade (14-05-2015).

P 34: Wood ants have caught a beetle.

P 31 P 32

P 33 P 34

Apple Trees

P 35: Apple tree from the P 35 P 36 other end of the plantation. Many and fine apple are on the trees (23- 08-2015).

P 36: Apple tree from the study area. Few apples are available on each tree (23- 08-2015)

P 37: A damaged, underdeveloped apple (23-07-2015).

P 38: Apple trees from the other end of the plantation (23-08-2015).

P 37

P 38

The Other Stuff

P 39

P 39: The conventional Christmas tree P 40 plantation (TS3).

P 40: Feeding tube in the apple orchard. Sugar dough is available for the ants inside the tube.

P 41: Sugar dough is provided for one of the donor nests – and they like it.

P 42: Wood ants are presumably looking for nectaries in the flower buds (14-05- 2015).

P 41 P 42