Zainab Ali Saad Abdelatti Bsc, Msc

Insect neurophysiology and botanical pest control

“Stochastic resonance in insect hearing and alternative locust control”

Doctoral thesis Presented to the Faculty of Natural Science of the University of Graz In Fulfilment of the Requirements for the Degree of Doctor rerum naturalium (Dr. rer. nat.)

submitted by Zainab Ali Saad Abdelatti BSc, MSc

at the Institute of Biology Neurobiology and Behaviour

Under the supervision of Assoz. Prof. Dr. Mag. rer. nat. Manfred Hartbauer

1. Reviewer: Assoz. Prof. Dr. Mag. rer. nat. Manfred Hartbauer

2. Reviewer: Associate Prof. Dr. Alberto Pozzebon

2021

For my mother’s soul

III

Zusammenfassung

Orthopteren-Insekten bilden die wichtigste Gruppe pflanzenfressender Insekten. Dieses Insektentaxon fasziniert Forscher seit Jahrzehnten aufgrund der stereotypen akustischen Kommunikation und des verheerenden Schwarmverhaltens mancher Arten. Der Zweck des ersten ist das Anlocken von potentiellen Partnern, während das Schwarmverhalten durch eine hohe Bevölkerungsdichte ausgelöst wird und als Gegenmaßnahme zur Vermeidung von Nahrungsmittelknappheit und Kannibalismus angesehen werden kann. In meiner Doktorarbeit habe ich beide Themen an Modellarten der Ordnung der Orthopteren untersucht. Zuerst untersuchte ich die Signaldetektion unter lärmhaften Bedingungen bei der tropischen Laubheuschreckenart Mecopoda elongata, wo Männer periodische Chirps erzeugen, um Weibchen akustisch anzulocken. Im Lebensraum dieser zirpenden Art erzeugt eine geschwisterliche Mecopoda-Art kontinuierliche Trillergesänge mit hoher Intensität und mit einer ähnlichen spektralen Zusammensetzung im Vergleich zum Lockgesang der zirpenden Art. Daher ist das Hintergrundrauschen im natürlichen Lebensraum hoch, und ich habe die Hypothese getestet, ob der heterospezifische Gesang die Erkennung von leisen konspezifischen Chirps, die unterhalb der Hörschwelle sind, aufgrund eines Phänomens verbessern kann, das als stochastische Resonanz (SR) bezeichnet wird. SR ist allgemein als paradoxes Phänomen bekannt, das die Erkennung schwacher Signale unter verrauschten Bedingungen verbessert. Zweitens entwickelte ich ein neuartiges botanisches Pestizid auf Basis von Leinöl, um die Wüstenheuschrecke Schistocerca gregaria und die Wanderheuschrecke Locusta migratoria zu bekämpfen. Beide Arten können zerstörerische Schwärme bilden und eine große Bedrohung für die Landwirtschaft darstellen, wo immer sie auftreten. In der Tat entdeckte ich eine neuartige botanische Ölmischung, die gegen alle Stadien dieser Heuschrecken hochwirksam ist. Ich bestimmte die letale Konzentration von 50% (LC50) für diese botanische Pestizidformulierung und untersuchte eine mögliche toxische Wirkung auf nützliche und alternative Nichtzielarten. Schließlich testete ich die Hypothese, ob in Leinöl enthaltene Fettsäuren als schwarmstörendes Mittel für Wüstenheuschrecken wirksam sind. Zumindest bei Männchen scheint dies der Fall zu sein. In weiteren Experimenten bestätigte ich, dass Wüstenheuschrecken ein nekrophobes Verhalten zeigen.

Abstract

Orthopteran insects constitute the most important group of plant-feeding insects. This insect taxon has fascinated researchers for decades because of its stereotyped acoustic communication and devastating swarming behaviour. The purpose of the first is mate attraction, whereas the latter is triggered by high population density and can be seen as a countermeasure to avoid food shortage and cannibalistic interactions. In my doctoral thesis, I studied both of these topics in model species belonging to the order of Orthoptera. First, I studied signal detection in noisy conditions in the tropical bushcricket Mecopoda elongata, where males produce periodic chirps to attract females acoustically. In the habitat of this chirping species, a sibling Mecopoda species produces continuous trills at high intensity with a similar broadband spectral composition compared to the conspecific song of the chirper. Therefore, background noise is high in the natural habitat and I tested the hypothesis whether or not the heterospecific calling song may improve the detection of subthreshold conspecific signals due to a phenomenon that is termed stochastic resonance (SR). SR is generally known as a paradox phenomenon enhancing the detection of weak signals in noisy conditions by stochastic noise. Second, I developed a novel botanical pesticide based on linseed oil to control the desert locust Schistocerca gregaria and the migratory locust Locusta migratoria. Both species can form destructive swarms in their gregarious phase and pose a great threat to agriculture wherever they appear. Indeed, I discovered a novel botanical oil mixture that is highly effective against all stages of gregarious locusts. I determined the lethal concentration 50% (LC50) for the botanical pesticide formulation and studied a possible toxic effect on beneficial and alternative non-target beetle species. Finally, I tested the hypothesis whether or not fatty acids contained in linseed oil act as a swarm disruptive agent for desert locusts. At least for males this seems to be the case. In additional experiments, I confirmed that males of desert locust show a necrophobic behaviour.

Content

Subject Pages

Zusammenfassung IV

Abstract V

List of Abbreviations VII

Chapter 1: Synopsis 1

Main goals of the present thesis 7

Summary Chapter 2 8

Summary Chapter 3 9

Summary Chapter 4 11

References 13

Chapter 2: The heterospecific calling song can improve 19 conspecific signal detection in a bushcricket species

Chapter 3: Plant oil mixtures as a novel botanical pesticide to 38 control gregarious locusts

Chapter 4: Linseed oil affects aggregation behaviour in the desert 59 locust Schistocerca gregaria—A potential swarm disruptive agent

Bibliography 79

List of Figures 92

List of Tables 94

Scientific contributions 95

Statutory Declaration 98

Acknowledgement 100

List of Abbreviations

SR Stochastic resonance

TN-1 T-shaped interneuron

SPL Sound pressure level

SNR Signal-to-noise ratio

ISI Interspike intervals

L. emulsion Linseed oil/bicarbonate emulsion

FAO Food and Agriculture Organization of the United Nations

WHO World Health Organization

MMM Male-male mounting

PAN Phenylacetonitrile

VII

Chapter 1: Synopsis

The order Orthoptera (Eng. "straight wings") is one of the oldest lineages of insect species (Grzimek et al., 2004). It includes various hemimetabolous insect species, many of them are economically important, such as swarming locusts. Orthopterans are also famous for the airborne sounds they produce for the purpose of acoustic communication, with most grasshoppers calling during the day and katydids and crickets singing at night (Gwynne et al., 1996). Sound production and swarm formation are common behaviours in many orthopteran species (Parsons, 2010), thus insects of this order were considered important subjects for numerous scientific studies: singing orthopterans serve as model organisms for questions addressing intraspecific communication and swarming locusts as model organisms for the phenotypic transition that may cause a destructive plague (Parsons, 2010). Orthopterans are the dominant terrestrial herbivorous insects with chewing mouthparts and hind legs modified for rapid escape jumps (Naskrecki, 2013). Based on the size of the antennae, orthopterans are divided into two suborders: Ensifera and Caelifera. Ensiferans are long-horned insects including crickets, katydids, weta and their relatives. Most ensiferans are nocturnal species and females possess a long ovipositor (Ax, 2000; Grzimek et al., 2004). The Tettigoniidae (katydids or bushcrickets) is the largest ensiferan family and includes many acoustically communicating insect species. Caeliferans are short-horned grasshoppers including true grasshoppers, locusts and their relatives. Most caeliferan species are diurnal and females possess a short ovipositor (Ax, 2000; Grzimek et al., 2004). The majority of caeliferan species belong to the family Acrididae (field grasshoppers and locusts). Some of locust species are known for their devastating agricultural potential once they form large swarms.

Singing katydids and crickets are fascinating and their songs have been appreciated in Japan and China for decades. In parts of China crickets are still sold as singing pets and their calling songs had a remarkable impact on art (Grzimek et al., 2004). The sound production of insects plays an important role in the attraction of mates, species recognition, defence of territory and intraspecific aggression (Gerhardt & Huber, 2002; Grzimek et al., 2004). Production of sound signals in orthopterans is commonly achieved by stridulation, which involves two parts of the body that are rubbed against each other. In acridids, stridulation organs are found on the inner edge of the femur of the hind legs and the outer surface of the forewings or the abdomen. Hearing organs of acridids are located in the first abdominal segment. In contrast, stereotyped singing of tettigoniids is achieved by rubbing both forewings against each 1 other and their ears are located in the tibia of forelegs (Ax, 2000; Grzimek et al., 2004). The majority of tettigoniid species live in the tropical regions (Naskrecki, 2013), where various organisms communicate acoustically at the same time which leads to a high level of background noise, especially after sunset. Acoustic communication in the context of mate attraction and mate choice leads to information exchange between a sender and indented (or unintended) receivers. In noisy situations, receivers face challenges to detect the signals of conspecific signallers. However, a positive effect of noise can be found in a paradox phenomenon named stochastic resonance (SR), which enhances the detection of subthreshold signals due to the resonance between stochastic noise and a periodic signal. One subject of my thesis addresses SR that may play a role in the acoustic communication of katydids (bushcrickets). In neurophysiological experiments, I was able to reveal the biological relevance of stochastic resonance in the auditory system of a tropical tettigoniid species, which is exposed to a high level of background noise that is generated by a sibling species.

Swarming constitutes a collective behaviour that is the outcome of an extreme example of “phenotypic plasticity”, which includes a transformation in morphology, physiology and behaviour of individuals in response to their environment. Most orthopteran species live solitary, i.e. individuals avoid each other, except when they are ready to mate. However, gregarious tendencies are commonly found among some tettigoniid species (e.g., Mormon crickets) and some acridid species (locusts). In species belonging to the latter, a serious agricultural problem arises when habitat conditions change (Grzimek et al., 2004). At high population densities and certain environmental conditions, locusts shift from the solitarious phase to a gregarious phase (Uvarov, 1966; Steedman, 1988). This phase transition is called polyphenism because the genotype of individuals stays the same. After phenotypic transition, locust individuals become social, aggregate and move collectively in marching bands before they form migratory swarms that can cover long distances (Steedman, 1988). A big swarm is rapidly growing and can devour entire fields within a very short period of time, thus, locusts constitute one of the most destructive plagues on earth. Currently, the control of locust upsurges or outbreaks is mainly based on chemical pesticides that are sprayed on large areas of land, which causes severe environmental problems. Chemical pesticides are also problematic in many countries because locusts are used in the animal food industry. Natural enemies, such as birds and lizards, and the use of bio-pesticides insufficiently control the rapidly expanding swarms. Therefore, it is highly important to develop alternative control measures with less side effects on the ecosystem. In my PhD thesis, I approached this aim by testing several natural oils for their insect toxicity. Moreover, a better understanding of intraspecific communication signals 2 of locusts can unravel novel effective control strategies. For example, results obtained in scientific studies of the acoustic communication of pest insects has inspired various pest management strategies: acoustic traps, manipulation of natural behaviours or interference of intraspecific communication (Mankin, 2012). Locusts, like other insects, can sense swarm members using olfactory, visual and tactile cues (Simpson et al., 2001; Pener & Simpson, 2009; Ma et al., 2014). In this context, a recent study on desert locusts has shown that repetitive tactile stimuli among individuals are the most potent cues for the induction of phase transition and swarm formation (Simpson et al., 2001). In the course of my PhD study, I revealed linseed oil to kill locusts and this oil contains fatty acids, some of which are known to exert a necrophobic (avoiding) behaviour in some insect species. Therefore, I performed behavioural experiments to test linseed oil as a novel swarm disruptive agent, which may reduce the aggregation behaviour of gregarious locusts. Therefore, results of my PhD study may inspire new measures for sustainable and integrated pest management.

In my thesis, I studied several members of the major families belonging to the order Orthoptera (table 1). The synchronizing bushcricket species Mecopoda elongata that generates periodic chirps to attract mates in their natural habitat, the tropical rainforest of Malaysia. There, biotic background noise is usually high because a trilling Mecopoda species produces ear deafening trills. I also studied the desert locust Schistocerca gregaria and the migratory locust Locusta migratoria. Both of these species can reversibly shift from a solitarious to a gregarious phase in response to population density and food availability.

Table 1: Taxonomy of orthopteran species that were studied in my thesis. Class Insecta Order Orthoptera Suborder Ensifera Caelifera Superfamily Tettigonioidea Acridoidea Family Tettigoniidae Acrididae Species Mecopoda elongata Schistocerca gregaria Locusta migratoria

1.1. Stochastic resonance in acoustically communicating bushcrickets

After sunset, biological noise is steadily increasing in the tropical rainforest of Malaysia. This chorus consists of many insect and anuran species that advertise themselves via airborne sound to attract potential mating partners. Such a multi-species chorus is challenging for receivers and, thus, various mechanisms evolved to enable receivers to detect and respond to

3 the signals of conspecifics. The genus Mecopoda includes several sibling species that are morphologically similar, but can be differentiated on the basis of their species-specific calling songs differing in the temporal and spectral composition, intensities and song durations (Nityananda & Balakrishnan, 2006; Siegert et al., 2011). In the Malayan tropical rainforest, two Mecopoda species live in sympatry: a chirping species and a trilling species. Males of both sibling species generate distinct calling songs to attract females from a distance. The “chirper males” produce periodic sound signals, called chirps, with frequencies that overlap with the songs of the “triller males” generating long lasting high intensity trills. Due to the high overlap of frequency compounds of these calling songs, one has to expect that the calling songs of the trilling species masks the calling song of the chirping species, when both species are simultaneously active. However, Siegert et al. (2013) demonstrated that chirper males are able to establish synchronous entrainment in playback experiments, despite the presence of high intensity “triller songs”. This ability strongly depends on a 2 kHz frequency component that is weak in the calling song of the trilling species and very low for acoustically communicating bushcrickets since even most frog calls use higher carrier frequencies. The authors of this study found a slight improvement in the detection of periodic 2 kHz signals in the response of an auditory interneuron (T-shaped neuron, TN-1) when the heterospecific trill was broadcast simultaneously. This enhancement of signal detection in the presence of “noise” may be attributed to a phenomenon known as stochastic resonance (SR), which describes accidental temporal overlap (resonance) of sub-threshold signals and noise causing an improvement of signal detection (Benzi et al., 1981; Benedix et al., 1994; Wiesenfeld & Moss, 1995).

SR improves the detection of weak periodic signals by adding rather low levels of random noise, whereas at high noise levels signal masking dominates (Collins et al., 1996; Gammaitoni et al., 1998; Henry, 1999). It is possible to study SR in behaviour or in neurophysiological experiments by testing the ability of receivers to detect subthreshold signals during steadily-increasing noise levels. The benefit of noise in the detection of weak signals has been showed in various organisms. For example, Douglass et al. (1993) found that noise of varying intensities improved the sensitivity of mechanoreceptor hair cells in the tailfin of crayfish in the context of predator avoidance. Levin and Miller (1996) found that adding broadband white noise to a weak periodic air flow improved the escape behaviour of crickets. SR was also found in the feeding behaviour of paddlefish, where the sensitivity to zooplankton prey was improved in the presence of low levels of electric noise (Russell et al., 1999). Additionally, the presence of broadband noise enhanced the response of auditory neurons in frogs (Ratnam & Feng, 1998; Bibikov, 2002). Moreover, Spezia et al. (2008) provided evidence 4 for SR in the context of mate attraction in the stink bug Nezara viridula, where males track vibrating females. In this experiment, white noise improved the detection of weak vibratory signals transmitted over the substrate.

In my study, I performed neurophysiological experiments to demonstrate whether or not SR plays a vital role in the acoustic communication of the chirping Mecopoda species in the natural habitat, where rather high levels of insect-generated background noise dominate the tropical rainforest night. For this purpose, I studied the response of an auditory neuron with T- shaped morphology (TN-1; Suga & Katsuki, 1961; McKay, 1969) in the chirping Mecopoda species to subthreshold 2, 8 and 20 kHz pure tone signals, while I steadily increased the amplitude of the trill of the heterospecific Mecopoda species. I selected 2 kHz because this frequency band of the chirping species is absent in the calling song of the trilling species (Siegert et al., 2013). In contrast, 8 kHz is the dominant frequency of the heterospecific trill and the TN-1 neuron is tuned to 20 kHz (highest sensitivity) (Siegert et al., 2013). Since many SR studies used white noise as artificial masker, I also broadcast increasing levels of white noise together with subthreshold pure tone signals to obtain results that can be compared with previous SR studies.

1.2. Alternative pest control for gregarious locusts

Gregarious locust outbreaks create agricultural problems at a global dimension (Wright, 1986; Brader et al., 2006; Millist & Abdalla, 2011; Latchininsky, 2013; Zhang & Hunter, 2017). Big swarms were already found in Africa, the Middle East, Asia, South and Central America, Australia and Southern Europe. The desert locusts Schistocerca gregaria and the migratory locust Locusta migratoria are considered to be among the most serious agricultural pest species. They display density-dependent phase polyphenism when solitarious individuals face certain environmental conditions. For example: heavy rainfalls that end long periods of drought can boost the increase in population density. Behavioural transition of locusts is evoked in a short period of time and leads to the formation of swarms that are very destructive (Pener & Simpson, 2009; Ma et al., 2014). Currently, locust control is mainly based on organophosphorus pesticides that are sprayed over large areas, which can lead to severe environmental problems because some of these pesticides are known for their harmful side effects on the ecosystem (WHO, 1990, 2012; Alavanja, 2009; Alavanja & Bonner, 2012; Köhler & Triebskorn, 2013; Carvalho, 2017). To counteract this problem, a bio-pesticide was developed under the auspices of the Food and Agriculture Organization of the United Nations (FAO). It is based on the fungus Metarhizium acridum and infests locusts and grasshoppers depending on environmental 5 temperature and humidity (Lecoq, 2010). Because of several shortcomings of this bio-pesticide, such as its long incubation period and strong dependence on ambient temperature, there is a strong interest in the development of effective agents for locust control with only moderate side effects on the ecosystem. Since a large locust swarm can eat up tons of crops each day, safe alternative agents or novel alternative control technologies should have maximum priority (Nicolopoulou-Stamati et al., 2016; Tlak Gajger & Dar, 2021).

Recently, plant oils have received great attention as promising alternatives for an environmental-friendly control of important plant pests and pathogens (Isman, 2004; Tlak Gajger & Dar, 2021). Plant oils have been used for centuries in alternative medicine and aromatherapy. They have also been used as food flavourings, perfumes, preservatives and biological agents (Tak, 2015). Natural plant oils are suggested for organic agriculture because they are biodegradable, moderate toxic to humans and vertebrate animals, easy to prepare and apply (El Rasheed & El Rasheed, 2017). Plant oils are divided into two groups: vegetable oils and essential oils. Vegetable oils (e.g., sesame oil, sunflower oil, olive oil, linseed oil) are usually extracted from the seeds by mechanical pressing and are often used as carrier oil for essential oils. Essential oils (e.g., orange peel oil, peppermint oil, clove oil, lavender oil) consist of volatile, concentrated compounds that are extracted by distillation. Essential oils were suggested as effective compounds against locusts and grasshoppers in recent studies (For example, Sharaby et al., 2012; Abdellah et al., 2013; Halawa & Hustert, 2014; Lahsen et al., 2015). In these studies, essential oils were found to exert insect toxicity in different ways.

In my PhD thesis, I developed a novel botanical mixture against two target species of gregarious locusts: the desert locust and the migratory locust. Linseed oil is a drying oil that becomes very viscous over time. In order to speed up its drying process, linseed oil can be mixed with a saturated solution of sodium-hydrogencarbonate (Sodium bicarbonate). I used this linseed/bicarbonate emulsion in order to coat the locusts with a thin layer that is difficult to remove and becomes hard over time. Sodium bicarbonate may also have an additional beneficial effect as it is commonly used for the control of some fungal plant diseases (Horst et al., 1992; Kuepper et al., 2001). In order to increase the toxicity of this linseed emulsion, I added low concentrations of different essential oils that were sprayed on desert locusts in a screening study. Essential oil emulsions that were found to be very toxic for desert locusts were tested either individually or in combination to reveal a possible synergistic toxic effect as this would allow to lower the concentration of essential oils. To test the toxic effect of the botanical emulsion on a beneficial and an alternative non-target species, ladybirds and mealworm beetles

6 were treated with this novel botanical pesticide as well. I also studied a possible phytotoxic effect of this oil formulation on the growth of wheat seedlings.

1.3. Botanical oil for the disruption of locust aggregation

My botanical pesticide formulation contains 53% linseed oil that is rich in unsaturated fatty acids (50-55% linolenic acid, 15-20% oleic acid and 11-20% linoleic acid; Bayrak et al., 2010). Interestingly, the fatty acids of linseed oil were described as necromones in the scientific literature (Yao et al., 2009; Sun & Zhou, 2013). Therefore, these chemical substances are associated with the injury and death of insects (Yao et al., 2009). Necromones evoke different behaviours in other individuals such as: 1) necrophoric behaviour which is the removal, burial and cannibalism of dead bodies and, 2) necrophobic behaviour which is the avoidance of dead or injured individuals (Yao et al., 2009; Sun & Zhou, 2013). Since linseed oil “necromones” may act as an agent that disrupts the formation of locust swarms, I performed behavioural experiments to test whether linseed oil affects the aggregation behaviour of small groups of gregarious desert locusts after the wings of one individual (the target individual) has been brushed with this oil. For this purpose, I evaluated the time the target individuals spent in groups with other individuals within a time span of 30 minutes before and after brushing the wings of the target locusts with linseed oil. In additional behavioural experiments, I also tested the response of groups of five desert locusts to a stationary linseed oil target as well as intact and crushed dead locust bodies that were placed in one corner of the experimental arena. I evaluated the time locust individuals spent in the target corner within a 30 minutes time span before and after the addition of the stationary linseed oil, dead or crushed locust bodies.

1.4. Objectives of my PhD thesis:

My thesis was dedicated to the study of the acoustic communication in sibling Mecopoda species and the development of a novel botanical pesticide that is effective against different locust species. My research addressed the following hypotheses:

• Stochastic resonance (SR) is present in the auditory system of acoustically communicating bushcricket species. ▪ Heterospecific calling song improves the detection of a subthreshold conspecific signal component in a chirping Mecopoda species from Malaysia. ▪ The range of distances in which SR occurs between a sender and receiver is rather high in the presence of trilling songs.

• Certain botanical oils are effective against two gregarious locust species. ▪ The lethal concentration 50% (LC50) for locusts is rather low after treatment with my novel botanical pesticide formulation. ▪ The toxic effect of this botanical pesticide is less severe in beneficial and alternative non-target species. ▪ My botanical pesticide stops the feeding activity of desert locusts. ▪ My botanical pesticide formulation does not affect the growth of wheat seedlings. ▪ Fatty acids contained in linseed oil exerts a swarm disruptive effect on the aggregation behaviour of desert locust. ▪ Gregarious individuals of the desert locust avoid dead or injured conspecifics (necrophobic avoidance behaviour).

1.5. Summary of Chapter 2: The heterospeciﬁc calling song can improve conspeciﬁc signal detection in a bushcricket species

This chapter addresses a paradox phenomenon, named stochastic resonance (SR) in the acoustically communicating bushcricket species Mecopoda elongata. Contrary to what one would expect, SR improves the detection of subthreshold signals when noise is added to the system. I performed neurophysiological experiments to investigate the existence of SR in the auditory system of a chirping bushcricket from the genus Mecopoda. For this purpose, I prepared insects as shown in figure 1 and recorded from an auditory interneuron (TN1) that forwards the information about external acoustic signals from the thorax to the brain. In order to broadcast subthreshold signals, the hearing threshold for the periodic pure tone signal was determined by increasing stimulus intensity until 50% of presented signals elicited a TN1 response. Then, the signal intensity was reduced by 1 dB and either white noise or a pre- recorded trill of the sympatric bushcricket species was presented at various intensities. To demonstrate the existence of SR, I evaluated the proportion of TN1 responses to subthreshold triple-pulsed signals with carrier frequencies of 2, 8 or 20 kHz at various signal to noise ratios (SNR). Results demonstrate the existence of SR when 20 kHz or 2 kHz signals were presented simultaneously with moderate levels of white noise. This was evident from the percentage of TN1 response that exceeded the detection threshold of 50% at various signal-to-noise ratios. On the contrary, the detectability of these pure-tone signals decreased due to masking effects at high noise levels. However, between-individual variability of TN1 response was high when periodic 2 kHz or 8 kHz signals were broadcast simultaneously with the heterospecific trill.

Nevertheless, further analysis of this variability of TN1 revealed a significant correlation of TN1 response with the phase lag between the signal pulses and the syllable pattern of the trill. The highest TN1 response occurred when there was an overlap of 2 kHz pulses with loud syllables of the trill. In contrast, TN1 response was lower when a smaller number of loud syllables overlapped with the triple pulse. I also simulated the propagation of sound signals in a Netlogo model. I used this multi-agent simulation environment to estimate the distances between the signal source and the trill in which SR may occur. This computer model suggests a wide range of sender-receiver distances in which the calling song of the trilling species may improve the detection of subthreshold signals of the chirping species. In conclusion, my results demonstrate the existence of SR in acoustically communicating bushcricket species and suggest that the calling song of a heterospecific species may facilitate the detection of subthreshold conspecific signals under certain SNRs and phase lags.

Fig. 1: Preparation of the bushcricket in the neurophysiological experiments.

1.6. Summary of Chapter 3: Plant oil mixtures as a novel botanical pesticide to control gregarious locusts

The work described in Chapter 3 of my PhD thesis is related to the development of a novel botanical oil mixture that kills two species of swarming locusts; the desert locust Schistocerca gregaria and the migratory locust Locusta migratoria. Linseed oil is a vegetable 9 oil that becomes very viscous over time and forms a thin film that may lead to suffocation of insects after single spray treatment. To speed up this mechanical hardening process, I mixed linseed oil with a saturated water solution of Sodium-hydrogen carbonate. To increase the toxicity of this linseed oil/bicarbonate emulsion, different essential oils were added at low concentrations. To rule out the most toxic essential oils, I performed screening studies in which different oil emulsions were sprayed on adult locusts and the number of dead individuals were counted 24 hours after each single spray treatment. In this screening experiment, garlic, orange peel, caraway and wintergreen oil emulsions were found to cause high insect mortality. Therefore, these essential oils (excluding garlic oil) were identified as potential candidates for a novel formulation of a botanical pesticide. I combined these oils and was able to show that a certain combination of these essential oils exerts a synergistic toxic effect that significantly increased the mortality rate of desert and migratory locusts. In an additional experiment, I was able to demonstrate that this botanical pesticide formulation had an antifeedant effect because locust individuals stopped feeding after single spray treatment. Furthermore, this novel botanical pesticide formulation was tested on a beneficial non-target species (adults and larvae of the mealybug ladybird Cryptolaemus montrouzieri) and an alternative non-target species (mealworm beetles Tenebrio molitor). To my surprise, mealworm beetles behaved normally within a time span of 8 days after spray treatment. On the contrary, a significant toxic effect was observed in ladybird adults 8 days after spray treatment and larvae of this bug species was even more vulnerable and showed toxic effects already one day after treatment. Therefore, we cannot exclude an unwanted side-effect on non-target insect species that are relevant for the ecosystem. However, this botanical pesticide may have only minor negative effects on sun exposed vegetation, since the growth of wheat seedlings on a balcony was not significantly affected. The insecticidal effects on locusts exerted by linseed, caraway and wintergreen oil is novel and have not been described elsewhere before. The components of my botanical pesticide formulation can be obtained easily, and no laboratory equipment is needed to mix the ingredients before spray treatment. In conclusion, my results, demonstrate that my novel botanical pesticide formulation is an effective agent with a great potential to combat locust outbreaks (fig. 2). The aim of future research should be to replace chemical pesticides that are harmful for man and nature. In further research projects, performed under field conditions, the effects of novel botanical formulations on locust swarms and the ecosystem have to be examined.

Fig. 2: Caricature for an effective treatment of locusts using a novel botanical pesticide.

1.7. Summary of Chapter 4: Linseed oil affects aggregation behaviour in the desert locust Schistocerca gregaria

Based on the results obtained in Chapter 3, I performed behavioural experiments to test a potential swarm disruptive effect of linseed oil in the gregarious desert locust Schistocerca gregaria. As described in chapter 3, I developed a highly effective botanical pesticide formulation against two species of locusts that is mainly composed of linseed oil that was used as a carrier for essential oils in a pesticidal emulsion. Linseed oil exerts a lethal effect, but may also have an additional adverse effect on the aggregation behaviour of swarming locusts mediated by its unsaturated fatty acids. Similar, fatty acid necromones were found to elicit necrophobic behaviour in other insect species (Yao et al., 2009; Sun & Zhou, 2013). Necrophobic behaviour may disrupt swarm formation when some locust individuals come into contact with linseed oil in the course of a botanical pesticide spray treatment. Therefore, I performed a series of aggregation experiments with groups of six mature locust individuals of the same sex and brushed the wings of one individual (target individual) with linseed oil. Experiments were performed inside an illuminated anechoic chamber with a heater, to maintain 11 a constant temperature of 30°C. The movement of locusts in each trial was recorded for 30 minutes using a top-view camera (see Fig. 2 for the experimental condition). Results showed that brushing the wings of the target locusts with linseed oil affected the amount of time target male individuals spent in groups with other males. This was obvious in a significant decrease in the group formation time in male groups after the linseed oil application, compared to the time before treatment. This result may indicate necrophobic avoidance behaviour in locust males mediated through fatty acids contained in linseed oil. However, this effect was absent in female groups because of the attraction between female individuals mediated by the release of oviposition pheromone. Aggregation of ovipositing females supports the gregarious cohesion among members of the next generation (Ferenz & Seidelmann, 2003). In contrast, males of desert locusts are responsible for the release of aggregation pheromone and thus for swarm cohesion. Since the reduction in the amount of aggregation time among males leads to a reduction in tactile stimuli, linseed oil seems to be a promising agent for disrupting swarm formation in gregarious desert locusts. This has to be expected because mechanical stimuli in crowded swarms are required to keep individuals in the gregarious phase. To provide evidence that male locusts respond to necromones, I performed additional experiments on mature desert locusts of both sexes to study the attraction/avoidance behaviour after exposing locust individuals to a stationary linseed oil-soaked paper. I also studied locust movement in an arena experiment after positioning dead or crushed locust bodies in one corner. I was able to show that the average amount of time males spent in the target corner after adding crushed male bodies was significantly decreased. This result clearly demonstrates that locust males in my experiments showed a necrophobic avoidance behaviour. It is known from previous studies that the intact dead bodies of some insect and isopod species can be an indicator of contagion, but crushed bodies or body extracts of conspecifics can be a sign of an injury caused by a predator and swarm mates should avoid it (Yao et al., 2009). On the contrary, other results showed that the average amount of time spent in the target corner before and after adding the linseed oil paper or intact dead bodies did not change significantly within a 30 minutes time span. This may be explained by the fact that the necrophobic response is usually weak to fresh fatty acids and dead bodies, but gets stronger over time (for example: Aksenov & David Rollo, 2017). In conclusion, linseed oil not just has a high potential as novel insecticide but can also become a promising candidate for the control of aggregation behaviour in gregarious desert locusts. Therefore, fatty acids contained in linseed oil needs to be considered as necromones with important consequences for pest management. Further experiments are necessary to show which concentrations and types of fatty acid necromones are more effective.

Fig. 3: Setup and locust arena used in behavioural experiments. (A) Arena, camera and lamps inside the anechoic chamber. (B) Top view of the locust arena.

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Chapter 2

The heterospeciﬁc calling song can improve conspeciﬁc signal

detection in a bushcricket species

2.1. Summary

In forest clearings of the Malaysian rainforest, chirping and trilling Mecopoda species often live in sympatry. We investigated whether a phenomenon known as stochastic resonance (SR) improved the ability of individuals to detect a low-frequent signal component typical of chirps when members of the heterospecific trilling species were simultaneously active. This phenomenon may explain the fact that the chirping species upholds entrainment to the conspecific song in the presence of the trill. Therefore, we evaluated the response probability of an ascending auditory neuron (TN-1) in individuals of the chirping Mecopoda species to triple-pulsed 2, 8 and 20 kHz signals that were broadcast 1 dB below the hearing threshold while increasing the intensity of either white noise or a typical triller song. Our results demonstrate the existence of SR over a rather broad range of signal-to-noise ratios (SNRs) of input signals when periodic 2 kHz and 20 kHz signals were presented at the same time as white noise. Using the chirp-specific 2 kHz signal as a stimulus, the maximum TN-1 response probability frequently exceeded the 50% threshold if the trill was broadcast simultaneously. Playback of an 8 kHz signal, a common frequency band component of the trill, yielded a similar result. Nevertheless, using the trill as a masker, the signal-related TN-1 spiking probability was rather variable. The variability on an individual level resulted from correlations between the phase relationship of the signal and syllables of the trill. For the first time, these results demonstrate the existence of SR in acoustically-communicating insects and suggest that the calling song of heterospecifics may facilitate the detection of a subthreshold signal component in certain situations. The results of the simulation of sound propagation in a computer model 19 suggest a wide range of sender-receiver distances in which the triller can help to improve the detection of subthreshold signals in the chirping species.

Highlights

• Stochastic resonance (SR) improves acoustic signal detection in a chirping insect. • White noise or a heterospecific trill leads to SR in a broad range of SNRs. • The trill improves the detection of a conspecific frequency band. • The response variability is related to the timing of the signal and the trill. • Sound simulation suggests a wide range of distances in which SR occurs.

2.2. Introduction

In the nocturnal tropical rainforest, many insect and anuran species communicate via airborne sound simultaneously. In this situation, which is somewhat like that of a cocktail party, the background noise level is usually high (Riede, 1997; Ellinger & Hödl, 2003; Lang et al., 2005; Hartbauer et al., 2010; Römer, 2013), and receivers must face challenges while listening and responding to the signals of conspecifics due to potential masking interference (Bee & Micheyl, 2008). Signallers usually evaluate the temporal signal pattern to discriminate conspecific from heterospecific signals, but this task is difficult to perform when a multitude of heterospecific signals degrade the amplitude modulations of calling songs that contain information about species identity. Some animals have found a solution to this problem in that the frequency of their calling songs matches the sensitivity of the receiver (Schmidt & Römer, 2011; Schmidt et al., 2012). However, this type of frequency tuning in receivers is less effective in communication systems whereby the signaller generates songs with more broadband frequencies. This is exactly the case in many bushcricket species in which the males attract phonotactically-responding females from a distance.

In the genus Mecopoda, several sibling species are morphologically similar, but these can easily be distinguished by differences in their species-specific calling song patterns (Nityananda & Balakrishnan, 2006; Siegert et al., 2011). Two Mecopoda species live sympatrically in forest clearings of the Malayan tropical rainforest: a trilling species and a chirping species, the males of which advertise themselves by producing periodic signals with frequency compositions that strongly overlap those of the songs of the trilling species (Fig. 1). Since both species are active at the same time (i.e., after sunset), and the calling songs of the trilling species are of high intensity, it was expected that the trilling species' song would mask the calling song of the chirping species. On the contrary, Siegert et al. (2013) demonstrated that 20 a rather low frequency component at 2 kHz allowed males to establish synchronous entrainment even when the trill of the sympatric Mecopoda species was broadcast 8 dB louder than the conspecific calling song. This frequency component is weak in the calling song of the trilling species but rather high in the chirping one. Surprisingly, Siegert et al. (2013) found an improvement in the detection of periodic 2 kHz signals in the response of an auditory neuron (TN-1) when the heterospecific masking trill was broadcast simultaneously. This unexpected result may be attributed to a phenomenon known as stochastic resonance (SR), which explains noise-enhanced signal detection due to the resonance of random and uncorrelated noise with the signal (Benzi et al., 1981; Benedix et al., 1994; Wiesenfeld & Moss, 1995).

Figure 1. Oscillograms of the calling song of the two Mecopoda species (reprinted with permission from Siegert et al., 2013). (A) Calling song of the chirping species and the temporal pattern of a single chirp (below). (B) Calling song of the trilling species and the train of syllables magnified below. A two-second part of this trill was used for playback in this study. SR refers to a paradox phenomenon that improves the sensitivity of a system to external stimuli at rather weak levels of noise (e.g., Collins et al., 1995, 1996; Gluckman et al., 1996; Gammaitoni et al., 1998; Russell et al., 1999; Henry, 1999; Tougaard, 2000; Ward et al., 2002; Lyttle, 2008). Noise is usually considered to be something detrimental that should be minimized whenever possible; however, noise is enhancing the detection of weak periodic signals in certain cases. Therefore, McDonnell and Abbott (2009) described SR as a ‘‘noise benefit in a signal-processing system’’, or ‘‘noise-enhanced signal processing’’. Low levels of stochastic noise usually improve the detection of subthreshold signals, while higher noise levels adversely affect signal detection because signals are masked by noise (e.g., Collins et al., 1996; Gammaitoni et al., 1998; Henry, 1999).

To date, SR has been found in many different receiver systems, either of biological or technical origin. SR has generally been found in a nonlinear input-output system (for an 21 exception, see Fuliński & Góra, 2000) when signals are broadcast at subthreshold levels (but see Collins et al., 1995) and moderate levels of noise are added. The detection of subthreshold, periodic signals is improved, which reduces the probability of missing signals by increasing the hit rate at the same time according to signal detection theory (Tougaard, 2002). SR can be investigated by studying the receiver's ability to detect signals during steadily-increasing noise levels.

Several studies have shown that SR can improve signal detection in various organisms. For example, SR seems to improve the sensitivity of mechanoreceptor hair cells in the crayfish (Douglass et al., 1993). SR was also found in a study by Russell et al. (1999), who investigated the feeding behaviour of paddlefish. Their results demonstrated that the success of capture rate was increased in the presence of low electrical background noise levels. Moreover, Levin and Miller (1996) conducted SR experiments to examine the cercal system of crickets. They believe that adding noise to a weak periodic air flow is improving the detection of predatory wasps. SR was additionally found to play a role in the mating system of the stink bug Nezara viridula, in which it improved the detection of weak vibratory signals in a noisy environment (Spezia et al., 2008). As another positive effect, SR was found to improve the nervous processing of auditory information in the brains of frogs. In this case, the response of midbrain auditory neurons to a weak periodic input signal was enhanced in the presence of broadband noise (Ratnam & Feng, 1998; Bibikov, 2002).

Currently, it is unclear whether SR plays a vital role in acoustically-communicating organisms that live in habitats that have rather high levels of background noise such as the nocturnal tropical rainforest. One reason for this lack of knowledge is that many SR studies use white noise as a masker because the characteristics of this artificial noise strongly deviates from natural signals that are characterized by a species-specific spectral content as well as temporal structure. Furthermore, the noise intensity must be moderate to favour SR, which is not necessarily the case when heterospecific signallers are in close proximity to one another or when their signals are of high intensity. In order to study whether SR improves the acoustic communication in Mecopoda, we studied the response to subthreshold, species-specific 2 kHz signals in the nervous system of individuals of the chirping Mecopoda species while increasing the amplitude of the song of the trilling heterospecific Mecopoda species. To compare our results with those of previous SR studies, we also broadcast increasing levels of white noise together with subthreshold signals. We recorded signals from an afferent auditory neuron with T-shaped morphology (TN-1; Suga & Katsuki, 1961; McKay, 1969) because it has been shown

22 to reliably encode conspecific chirps even under conditions of natural background noise (Siegert et al., 2011). This allowed us to study SR with only little influence of internal neuronal noise, which can be crucial for the effectiveness of SR (Aihara et al., 2008, 2010; Krauss et al., 2016).

2.3. Material and methods

2.3.1. Insects

We included males and females of a chirping song variant of the genus Mecopoda elongata (Orthoptera, Ensifera, Tettigoniidae, Mecopodinae) in our study, which had originally been described as species S by Sismondo (1990). Insects were taken from a laboratory breed, founders of which had been originally collected in Malaysia in the years 2010 and 2011 (Ulu Gombak, Selangor, Kuala Lumpur). Males of this species produce calling songs that consist of chirps repeated at a period of 2 s (27°C ambient temperature). In a chorus, males of this species tend to produces chirps in synchrony. In the breeding room, insects were exposed to a light:dark cycle of 12:12 h and maintained at a constant temperature of 27°C, 70% relative humidity. Their diet consisted of fresh lettuce, apples, fish food and oatmeal. There is no publication so far that describes the trilling Mecopoda species morphologically, but Korsunovskaya (2008) described this species acoustically as “Mecopoda sp. 4”. One male and female voucher specimen of the triller were deposited at the National History Museum in Vienna (NOaS-11/2013).

2.3.2. Neurophysiology

SR was studied at the level of a first-order auditory interneuron ascending to the brain. Because this neuron has a T-shaped morphology, it is described as TN-1 neuron (Suga & Katsuki, 1961). Before dissection, insects were anesthetized with Chloroethylene gas. The legs (except the forelegs), antennae and wings were removed, and individuals were mounted ventral side up to a platform using dental wax. The metathoracic ganglion was also removed to avoid the neuronal activity that is normally generated by the flight oscillator and input from the cercal organ. The connectives between the metathorax and the first abdominal segment were cut, and a small piece of paper was inserted into the abdomen to relieve haemolymph pressure. The leg opposite the auditory stimulus was removed to avoid any contralateral inhibition of the TN-1 response. Subsequently, the cervical connectives were exposed, and the right connective was lifted using a tungsten-wire hook electrode. After removing the haemolymph, both connectives were cut between the sub-oesophageal ganglion and prothoracic ganglion. The neck was covered with petroleum jelly (Vaseline) to prevent the desiccation of the connectives. Signals 23 recorded with the electrode were amplified against an indifferent silver electrode that had been inserted into the abdomen (Suga & Katsuki, 1961; McKay, 1969). The experiments were performed in an anechoic chamber in which two loudspeakers were positioned adjacent to one another, 24 cm away from the insect preparation. The insect holder was placed on top of a heating platform (G. Maier, Electrotechnik GmbH) which was maintained a constant temperature of about 27 °C at the position of the insect.

The neuronal response was amplified via a custom-made amplifier that was fabricated as described by Land et al. (2001). Analog to digital conversion was made using a Powerlab/4s (AD Instruments), and converted data were saved in Chart (Version 5.5.6, AD Instruments, Spechbach, Germany).

2.3.3. Acoustic stimulation

Acoustic stimuli consisted of triple-pulsed pure tones (further referred to as signal) and were presented in several signal and noise combinations: 1) a triple-pulsed 20 kHz signal was presented together with white noise to test for the existence of SR at the carrier frequency at which the TN-1 neurons were tuned (Siegert et al., 2013), 2) a triple-pulsed 2 kHz signal and white noise, 3) a triple-pulsed 2 kHz signal and a trill of a heterospecific Mecopoda species that lacked this frequency band and 4) a triple-pulsed 8 kHz signal and a Mecopoda trill with high energy at this frequency band (see Fig. 2). The signal period was limited to 2 s in order to mimic the natural chirp period observed at an ambient temperature of 27 °C (Sismondo, 1990; Siegert et al., 2013). Noise was broadcast in loop mode, and care was taken that the phase relationship between the signal and noise was changed in a random manner. With the exception of the trill, all signals were generated in Cool Edit Pro 2.0 (Syntrillium Software Corporation, Phoenix, AZ, USA).

2.3.4. Playback

For acoustic playback, Cool Edit Pro was used to control an external audio interface for D/A conversion (RME Fireface UC, Haimhausen Bavaria, Germany). Analogue output signals were attenuated via a two-channel programmable attenuator (PA5, Tucker Davis Technologies, Alachua, FL, USA) and amplified using a NAD stereo power amplifier (C275BEE, NAD Electronics International, Canada) with a flat frequency response up to 100 kHz. Acoustic signals were broadcast via two leaf tweeters that exhibited a rather flat frequency response between 1 kHz and 45 kHz (EAS- 10TH400A, Technics, Kadoma, Osaka, Japan).

Figure 2. Oscillograms of the acoustic stimuli used in SR experiments. (A) Triple-pulsed 20 kHz signal: 25 ms pulse duration and 5 ms pause separating pulses. (B) The white noise signal broadcast in loop mode. (C) Triple-pulsed 2 kHz signal: 30 ms pulse duration and 5 ms pause separating pulses. (D) Triple-pulsed 8 kHz signal: 30 ms pulse duration and 5 ms pause separating pulses.

2.3.5. Sound calibration

The SPL of playback signals were calibrated at the position of the insect preparation with a calibrated microphone (model 2450, Larson Davis Laboratories, USA) that was connected to a sound level meter (CEL 414, CEL Instruments Ltd. Hitchin, Herts, England, attached to a filter unit CEL 296). Sound calibration of pulsed signals was carried out in the fast reading mode by broadcasting sound signals in the loop mode. The sound level meter operated with a flat frequency response in a range between 100 Hz and 45 kHz. Although we also recorded the peak values of pulsed signals, the signal to noise ratios given in the results refer to averaged values obtained in the fast reading mode. Sound signal amplitudes were calibrated relative to 20 µ Pa.

2.3.6. Stochastic resonance experiments

The thresholds of the pure tone signals were determined by systematically changing the signal intensity until 50% of the signal presentations elicited a TN-1 response of at least one spike per signal presentation. The SPL of the signal was then decreased by 1 dB (1 dB subthreshold). If the SR increased the detection of the subthreshold signal, noise added to the signal would increase the TN-1 response probability to a value higher than 50%. To quantify the effect of noise on the spiking response of TN-1 to pure tone signals, the noise was steadily 25 increased in steps of 2 or 3 dB after the presentation of a sequence of 35 identical pure tone signals (2 s signal period). SNR values refer to the dB difference between the signal and noise amplitude. To exclude a possible effect caused by neuronal adaptation processes, the responses to five signal presentations at the beginning of a playback sequence were excluded from data evaluation. Results represent the proportion of signal presentations that elicited at least one spike during a sequence of 30 stimulus presentations. Since the phase relationship between the pulses of the signal and the trill differed between stimulus sequences, we assumed that the signal detection rate not only depended on the SNR, but also on the phase lag between the signal and the syllables comprising the trill. Therefore, we also quantified the variability of the TN-1 response on an individual level at several SNRs by repeating the sequence of the pure tone signals 10 times at a given SNR. In this experiment, the intensity of the noise was increased in steps of 10 dB.

2.3.7. Data evaluation

TN-1 recordings were exported to Spike2 (V5.21, Cambridge Electronic Design) to run a custom-written evaluation script which counted the instances of presence or absence of TN-1 related spikes in a time window of 100 ms during the signal presentation, taking a response latency of 10 ms into account. The extracellular potentials of TN-1 neurons are of high amplitude so TN-1 activity can be easily discriminated from other neuronal activity by setting a user-defined amplitude threshold. With the exception of signal onset, white noise rarely elicited a spiking response, whereas the presentation of the trill at low SNRs frequently elicited spikes. In order to quantify the trill-related TN-1 response, we also counted the number of spikes that occurred in a time window of 1 s before the signal onset. To convert this noise- related spike count for a given SNR into a spiking probability, the average number of noise- related spikes was divided by 10 to obtain the same time basis as the stimulus (100 ms), and the result was multiplied by 100. Individuals with a trill-related spiking probability that exceeded 30% were excluded from the SR analysis. Since spike intervals shorter than 100 ms may lead to confusion between noise-related spikes and signal-related spikes, we also evaluated the probability of trill-related, interspike intervals (ISIs). Generally, noise-related ISIs shorter than 100 ms rarely occurred and, therefore, we used the first method to quantify the trill-related spiking response. In additional experiments, we studied the influence of the phase relationship between the pure tone pulses and the first soft syllable of the trill on TN-1 response (Fig. 3). The correlation between the phase lag and TN-1 response obtained from single individuals was tested for significance in Sigma Plot (v. 13.0, Systat Software, Inc.).

Figure 3. Example showing the phase relationship between the 2 kHz signal and the syllables of the trill.

2.3.8. Modelling sound propagation

The sound propagation of pure-tone signals was simulated in NetLogo (v. 5.3.1) to estimate the spacing between conspecific and heterospecific signallers in which SR was likely to occur. Simulations of the active space were based on realistic sound propagation properties observed in the field (Römer & Lewald, 1992) and the hearing threshold of the receivers was taken into account. Signal attenuation over a distance was simulated using equation (1) (see below) for a pure tone of carrier frequency of 5 kHz to simulate the relatively low frequency bands that are contained in the chirp. The signal attenuation of the trill was simulated by modelling a pure tone with a carrier frequency of 10 kHz using equation (2) (see below) because this frequency is similar to a prominent frequency band in the trill. The SPL of the receiver was set to 81 dB at a distance of 1 m, a value that corresponds to the average peak SPL of M. elongata males measured at a distance of 1 m (Hartbauer et al., 2014). The amplitude of the 10 kHz signal was simulated assuming an amplitude of 85 dB SPL and measured at a distance of 1.2 m (corresponds to 103 dB measured at 15 cm; see Krobath, 2013). A hearing threshold of 52 dB SPL (-1 dB subthreshold) was simulated for the 5 kHz signal which corresponds to the hearing threshold for 2 kHz signals. A hearing threshold of 40 dB SPL was defined for the 10 kHz signal which corresponds to the TN-1 threshold of the 8 kHz signal.

I = 81 dB – [10.05 * ln(x) - 0.865] (Eq. 1)

I = 85 dB – [14.18 * ln(x) - 0.891] (Eq. 2) 27

2.3.9. Statistics

If SR improves signal detection, we should expect a higher number of relative TN-1 responses exceeding the 50% threshold at moderate SNRs compared to very high SNRs (noise amplitude is low compared to the amplitude of the subthreshold signal). This was tested by performing z-tests with Yates correction, which tests the proportions for statistically significant differences taking the sample number into account. Data used to perform the z-test are indicated by dashed boxes in Figs. 4 and 6. All statistical tests were performed in Sigma Plot version 13 (Systat Software Inc.).

2.4. Results

2.4.1. White noise

The average threshold required to elicit a TN-1 response to 50% of the 20 kHz signals was 35 ± 4.4 dB SPL (mean ± SD; N = 10). The percentage of TN-1 responses to 30 repetitions of this signal, presented 1 dB subthreshold, frequently exceeded the 50% value when white noise was broadcast at SNRs between +16 and -7 dB (Fig. 4A). Exceeding the threshold occurred significantly more often at moderate SNRs compared to high SNRs (see dashed boxes in Fig. 4A; p < 0.05, N = 10, z-test). However, at SNRs lower than -6 dB, the percentage of TN1 responses decreased remarkably. Since the TN-1 neuron is tuned to frequencies higher than 10 kHz (Siegert et al., 2013), the average hearing threshold for triple-pulsed 2 kHz signals was much higher (52.6 ± 3.2 dB SPL). The percentage of TN-1 responses to 2 kHz signals broadcast at 1 dB subthreshold exceeded the 50% threshold significantly more often at moderate amplitudes of white noise (indicated by dashed boxes in Fig. 4B; p < 0.05, N = 10, z- test) and strongly declined at SNRs less than +5 dB (Fig. 4B). The between-individual variability of TN-1 responses was higher during presentations of the 2 kHz signal as compared to the 20 kHz signal. These results indicate that white noise improved signal detection more reliably. To compare the TN-1 responses of a single individual to repeated presentations of the same stimulus sequence, we broadcast either 2 kHz or 20 kHz signals 10 times at certain SNRs. The results obtained from two individuals are shown in Fig. 5, the detection rate of 20 kHz signals increased significantly at various levels of white noise (p < 0.05, Mann-Whitney U test followed by a Tukey post hoc test), whereas the detection ability for 2 kHz signals improved only marginally at all tested SNRs in one individual and was significantly higher at a SNR of 22.8 dB in another individual.

Figure 4. Percentage of TN-1 responses to pure tone signals broadcast at a 1 dB subthreshold together with different amplitudes of white noise. (A) 20 kHz signal. (B) 2 kHz signal. Colours in A and B refer to the percentages of TN-1 responses of different individuals. Data in A were obtained from 5 females (orange colour) and 5 males (blue or violet colours). Data in B were obtained from 7 males and 3 females (orange colour). Dashed boxes refer to the data that were used for comparing the proportions of suprathreshold responses using a z-test. (For interpretation of the references to colour in this figure legend, the reader should refer to the web version of this article.) 29

Figure 5. The mean percentage of the TN-1 responses of two individuals to repeated presentations of subthreshold pure tone signals at various amplitudes of white noise. Results are based on 10 repetitions of the stimulus sequence at each SNR. (A) 20 kHz signal. (B) 2 kHz signal. Error bars indicate standard deviation. A significant increase in the percentage of TN-1 response is indicated by asterisks (p < 0.05, Mann Whitney U test followed by a Tukey post hoc test).

2.4.2. Triller song

When the heterospecific trill was broadcast at the same time as the 2 kHz signal, the TN-1 responses were variable between different individuals. Therefore, the average percentage of TN-1 responses obtained from 10 individuals remained below 50% at various SNRs when the 2 kHz signal was broadcast at 1 dB subthreshold (Fig. 6A). At SNRs between +15 dB and 0 dB (right dashed box in Fig. 6A), the percentage of the TN-1 responses was high in some individuals and low in others (indicated by the large error bars and the dashed lines in Fig. 6A). Nevertheless, the proportion of TN-1 responses exceeding the 50% threshold was significantly higher in this range of SNRs compared to high SNRs at which the trill amplitude was very low (left dashed box in Fig. 6A; p < 0.05, N = 10, z-test). The trill exceeded the average hearing threshold of receivers at a SNR of 12 dB (vertical line above the graph in Fig. 6A). At SNRs lower than -5 dB, however, the average percentage of TN-1 responses decreased monotonically. The average hearing threshold for the 8 kHz triple-pulsed signal was 40 ± 5.7 dB SPL. Presenting this signal one dB subthreshold together with the trill resulted in a significant increase of relative TN-1 responses exceeding the 50% threshold at SNRs between 0.8 and -5 dB compared to high SNRs (Fig. 6B; p < 0.05, N = 10, z-test). This range of SNRs is close to

30 the hearing threshold of the trill (-3 dB, vertical line above the graph in Fig. 6B). At SNRs less than -8 dB, the average proportion of the TN-1 responses decreased gradually.

Figure 6. The mean percentage of TN-1 responses to the pure tone signals broadcast at a 1 dB subthreshold together with different amplitudes of the trill. (A) 2 kHz signal, mean ± SD of 10 individuals. (B) 8 kHz signal, mean ± SD of 10 individuals. Dashed lines refer to the maximum and minimum values. The vertical tick on top of the figures indicates the mean threshold required to elicit a spiking response to the trill. Dashed boxes refer to the data that were used for comparing the proportions of suprathreshold responses using a z-test.

2.4.2.1. Phase relationship between signals and the trill

We examined the TN-1 response variability on the level of a single individual because the TN-1 response to the 2 kHz triple-pulsed signal showed rather high levels of variability at SNRs at which the heterospecific trill exceeded the hearing threshold (see Fig. 6A). The TN-1 response to the 2 kHz and 8 kHz signal obtained from a single individual was variable as indicated by the error bars shown in Figure 7A and C. A significant increase in TN-1 response was only found when 2 kHz signals were presented at a SNR of 14.8 dB (p < 0.05, Mann Whitney U test followed by a Tukey post hoc test). By plotting the TN-1 responses against the phase lags and referring to the time separating the onset of the signal and the next soft syllable of the trill, we could reveal a significant negative correlation between both parameters (2 kHz: p < 0.05, Spearman rank order correlation, cc = -0.510; 8 kHz: p < 0.05, Spearman rank order correlation, cc = -0.556; Fig. 7B and D). We observed the highest TN-1 response (the red dot in Fig. 7B) where a time lag of 11.8 ms separated the onset of signal and the next soft syllable of the trill (Fig. 7E). On the contrary, a time lag of 21.3 ms (Fig. 7F) resulted in the lowest percentage of TN-1 response (blue dot in Fig. 7B). Time lags that were nearly the same resulted in the highest and lowest percentages of TN-1 response when the 8 kHz signal was broadcast together with the trill as shown in Fig. 7D. These results demonstrate that a temporal overlap between a higher number of loud syllables and the triple-pulsed signal favored a TN-1 response, whereas the overlap of soft syllables diminished it (Fig. 7E and F).

2.4.3. Occurrence of stochastic resonance

The presence or absence of SR is shown in Fig. S1. In a large proportion of individuals, SR improved the TN-1 response probability at moderate amplitudes of white noise (Fig. S1, blue bars). In some individuals, the percentage of TN-1 response remained below 50% at all tested SNRs, which demonstrates the absence of SR (Fig. S1, red bars). We also evaluated the proportion of individuals for which the trill-related spiking activity exceeding 30% (see materials and method section for the calculation of the trill-related spiking response, Fig. S1, green bars). In contrast to the trill, white noise rarely elicited spiking activity in the interval between subsequent signal presentations.

Figure 7. Within-individual TN-1 response variability and phase relationships. Mean TN-1 response probability to subthreshold pure-tone signals broadcast at various SNRs together with the trill (A = 2 kHz, C = 8 kHz). Correlation between the spiking probability and stimulus phase lag. (B = 2 kHz; corresponds to +25 and +15 SNRs, D = 8 kHz; corresponds to +14 and +4 SNRs). (E) The phase relationship that led to the highest TN-1 response (F) The phase relationship that led to the lowest TN1 response. A significant increase in the percentage of TN-1 response is indicated by an asterisk (p < 0.05, Mann Whitney U test followed by a Tukey post hoc test).

2.5. Discussion

In the current study, the presence of stochastic resonance (SR) could be clearly demonstrated in the auditory system of a chirping Mecopoda species, as evinced by the percentage of the TN-1 response that exceeded the critical detection threshold of 50% at moderate levels of white noise. However, broadcasting high noise amplitudes resulted in a strong decrease in signal detection due to masking interference with the signal (Hänggi, 2002). In its natural habitat, the chirping species is confronted with noise from a multitude of sources. The song of the heterospecific trilling Mecopoda species has especially important implications for intraspecific acoustic communication among chirping species due to similarities in the frequency content of its calling songs. Siegert et al. (2013) showed that the 2 kHz band in the conspecific signal is essential for acoustic communication when a heterospecific trilling species is active at the same time. As shown in Figures 4 and 6, broadcasting white noise and the heterospecific trill improved the detection of subthreshold signals up to a critical SNR at which the high noise levels masked the signal. This optimum level of white noise and its effect on the detection of signals has been described in many studies and is known as the stochastic resonance peak (see review by McDonnell & Abbott, 2009).

Simultaneously broadcasting white noise clearly improved the detection of the 20 kHz signal, but also resulted in high levels of variability in TN-1 responses to the 2 kHz signal presented at moderate amplitudes of white noise (Fig. 4). Differences between the results obtained with white noise and the trill may be due to the tuning characteristics of the TN-1 neuron (Zhantiev & Korsunovskaja, 1983; Hartbauer et al., 2010) which favour the detection of the 20 kHz signal but not the 2 kHz signal. The average hearing threshold of individuals included in our study was 35 ± 4.4 dB SPL for the 20 kHz signal, 52.6 ± 3.2 dB SPL for the 2 kHz signal and 40 ± 5.7 dB SPL for the 8 kHz signal. Siegert et al. (2013) showed that TN-1 neurons are less sensitive to frequencies lower than 10 kHz, even though the 2 kHz band in the conspecific signal is essential to maintain acoustic communication when the heterospecific trilling species is simultaneously active. As a result of this frequency tuning, 2 kHz signals suffer from masking interference at much higher SNRs (<+5 dB), whereas masking of the 20 kHz signal was observed at SNRs less than -6 dB (Fig. 4). These TN-1 tuning characteristics may explain the stronger increase in signal detection rate observed when 8 kHz rather than 2 kHz signals were broadcast together with the trill at SNRs between 0 dB and -5 dB (Fig. 6B). Furthermore, the low sensitivity of TN-1 neurons to the 2 kHz signal may cause a temporal threshold shift in the TN-1 neuron when white noise is broadcast at higher amplitudes. Due to

34 this adaptation process, we rarely observed TN-1 responses to white noise except at noise onset. We observed relatively weak improvements in the ability of the individuals to detect 2 kHz signals when broadcast together with white noise (compare Fig. 4A with 4B and also see Fig. 5), and such a noise-dependent threshold shift may be responsible for this. This may be due to the fact that the level of noise was higher at SNRs in which we found an increase of signal detection in the 2 kHz playback experiment.

Simultaneously broadcasting pure-tone signals and the trill resulted in high levels of variability in TN-1 responses, which is clearly revealed by the high standard deviations shown in Figure 6. We propose two explanations for this result: First, the trill consists of a stereotyped sequence of syllables with a frequency composition that is broadband, but far from random with respect to its frequency content, the cross-correlations of the frequencies and the temporal signal pattern. This difference between the trill and white noise may limit the possibilities for SR. Second, the TN-1 response strongly depended on the time lag between the pulse onset of the signal and the next soft syllable of the trill (Fig. 7B and D). The highest TN-1 response occurred when the pulses of pure tone signals overlapped with a higher number of loud syllables of the trill (Fig. 7E), indicating that any improvement in signal detection strongly depended on the precision of the pulse and syllable timing.

It is still unclear whether the moderate SR detected is a consequence of central nervous mechanisms or the non-linear properties of the tympanal membrane, because auditory function can be enhanced on the periphery in the presence of low or moderate noise levels under certain circumstances (Henry, 1999; Indresano et al., 2003; Nadrowski et al., 2004). The dependence on the exact timing of signal pulses and syllables of the trill suggests that a peripheral mechanism could account for the moderate increase in the signal detection rate obtained with the 2 kHz signal, particularly because the ears of insects (energy detectors) have short integration time constants between 4 and 25 ms (Tougaard, 1998) that increase with the bandwidth of the noise (see Tougaard, 2000). In contrast, central nervous processing has longer integration time constants (>200 ms; Ronacher et al., 2000).

2.6. Biological relevance of SR

In a theoretical receiver model, Tougaard (2000) came to the conclusion that the range of usable noise levels that lead to SR are limited, which leads researchers to question the biological relevance of SR (but see Hänggi, 2002). Nevertheless, several studies have provided evidence for this in the detection of prey (Russell et al., 1999), hydrodynamic turbulence

(Douglass et al., 1993), wind generated by a predatory wasp (Levin and Miller, 1996), weak vibratory signals (Spezia et al., 2008) and the reduction in the absolute threshold for the detection of pure tone signals in humans with normal hearing (Zeng et al., 2000). We simulated the active space of pure tone signals in a 2D computer model in order to study the biological relevance of the improved detection of the 2 kHz signal when the trilling species is active at the same time, taking realistic hearing thresholds into account. These simulation results suggest that, under ideal conditions (i.e., sound propagates uniformly in all directions in the absence of background noise), a chirper male that generates a 5 kHz signal and sings at a distance of 19 m remains 1 dB below the hearing threshold of a conspecific receiver (52 dB SPL). According to the data shown in Figure 6A, SR is likely to improve signal detection at SNRs between +15 dB and -3 dB, which correspond to simulated triller-receiver distances of 37 m and 10 m, respectively (see Fig. 8). This modelling result suggests a wide range of sender-receiver distances over which the triller male calling song may help to improve the ability of the chirping species to detect subthreshold signals. However, the SR mainly depends on the temporal overlap of signals and loud syllables of the triller song, which is more likely occur in the field where triller males form choruses in which their songs, but not their syllables, overlap in time (Krobath et al., 2017). Nevertheless, the high level of background noise in the habitat may restrict the area within which receivers may benefit from SR. Kostarakos and Römer (2015) made intracellular recordings of several prothoracic interneurons and revealed that their response to a 2 kHz signal was only marginally affected by the high intensities of the heterospecific trill. This result is either the outcome of a tuning to low-frequency sound or the result of strong stimulus-dependent adaptation processes that have taken place in auditory neurons. These neurons are believed to play an important role in the detection of conspecific signals in natural habitats in which the trilling species is also active. In this case, SR may improve the abilities of these neurons to detect signals when the conspecific signaller is singing at a greater distance.

Figure 8. Spatial arrangement of signallers and a receiver between which SR is possible. According to the simulation results, the conspecific receiver remained 1 dB subthreshold at a distance of 19 m. When a triller male sings 10 – 37 m away from the receiver, it can more easily detect the conspecific signaller according to results shown in Figure 6A. Note that these results neglect the possible influence of other signallers and complex acoustic properties of the natural habitat. Insect photos were taken by Isabel Krobath and Manuela Kainz. Supplementary materials

Figure S1. SR statistics. Proportion of individuals for which white noise or the trill improved signal detection (blue bars). Red bars represent the proportion of individuals for which an improvement of signal detection was absent. Green bars represent the proportion of individuals for which increasing amplitudes of the trill resulted in a significant increase in the TN-1 response (> 30% trill-related TN-1 response). N refers to the number of individuals 37

Chapter 3

Plant oil mixtures as a novel botanical pesticide to control gregarious locusts

3.1. Summary

For thousands of years, large locust swarms have been responsible for causing severe problems in agriculture. People fight against current outbreaks by using chemical pesticides or an insect fungus known as Green MuscleTM. While chemical pesticides may be harmful for humans and non-target species, the sporulation of the fungus takes a long period of time and requires conditions of high humidity that are not always found in the field. In this study, we tested the toxicity of a linseed oil/bicarbonate emulsion against gregarious desert locusts and screened for plant essential oils that enhance its toxicity. Finally, we combined three essential oils to develop a novel formulation that is effective against the locust species Schistocerca gregaria and Locusta migratoria after single spray treatment. Within 24 hours, this formulation caused a mean mortality rate of 80 ± 7.1% and 100% of desert and migratory locusts, respectively. Its toxicity is based on a synergistic effect resulting from the combination of caraway, orange-peel and wintergreen oils. In addition, we tested this botanical pesticide on two beetle species regarded either as beneficial or alternative non-target species. The first species, mealworm beetles, did not suffer from the spray treatment and behaved normally after eight days. In contrast, 67.7 ± 8.9% of ladybird adults died in the same time span. Interestingly, the growth of wheat seedlings was almost unaffected by spraying this botanical pesticide. These results suggest this botanical pesticide can be used as a strong agent against desert and migratory locusts, but needs to be used with care to minimize unwanted side-effects on the ecosystem.

Key message

• In many countries, locust outbreaks still constitute a big problem for farmers. Current pest management is potentially harmful for humans.

• We developed a novel botanical pesticide against locusts that consisted of natural plant oils. • We identified orange-peel, caraway and wintergreen oils to be effective against locusts when added to a linseed oil/bicarbonate emulsion. • Our novel botanical pesticide had a synergistic toxic effect. • This formulation is highly effective against two gregarious locust species and is considered harmless for humans.

3.2. Introduction

Locusts constitute a major problem for agricultural crops (Wright, 1986; Brader et al., 2006; Millist & Abdalla, 2011; Latchininsky, 2013; Zhang & Hunter, 2017). They can transform reversibly between two extreme phases, solitary and gregarious. The locusts differ in both of these phases in terms of their morphology, physiology and behaviour (Uvarov, 1966; Steedman, 1988). The distribution of gregarious locusts extends from Africa through the Middle East to Asia, South and Central America, Australia and Southern Europe. When these species transform into the gregarious phase, they form locust swarms that can devour entire fields and cause extensive crop damage over a very short period of time. A small swarm of locusts contains thousands of individuals that spread over several hundred square meters, but large swarms contain up to 80 million individuals per km2 and can infest more than 1000 km2. Since a swarm can cover a distance of 100 km per day (Steedman, 1988), farmers regard gregarious locusts as one of the most destructive plagues on earth.

Before 1980, persistent organochlorine compounds like dieldrin were used extensively to combat locust swarms (Steedman, 1988). Because organochlorine compounds have been banned, locust control is currently mainly based on organophosphorus pesticides. The reduced persistence of these compounds makes them less effective than dieldrin (Arthurs, 2008). From 2003-2005, authorities applied thirteen million litres of chemical pesticides to thirteen million hectares of land to control desert locust outbreaks (Lecoq, 2010). This intensive use of chemical pesticides is probably underestimated and has led to severe environmental problems, which have had harmful side effects on the overall ecosystem (WHO, 1990, 2012; Alavanja, 2009; Alavanja & Bonner, 2012; Köhler & Triebskorn, 2013; Carvalho, 2017). Therefore, there is an interest in developing potentially safe alternatives or additional control technologies (Nicolopoulou-Stamati et al., 2016). Natural enemies cannot be used because of the rapid development and migration of locusts (Steedman, 1988). Over the past twenty years, the Food

39 and Agriculture Organization of the United Nations (FAO) has been working with several countries to develop a bio-pesticide that is based on the fungus Metarhizium acridum, which infests locusts and grasshoppers (Lecoq, 2010). The commercial name of this bio-pesticide is Green MuscleTM. In 2009, the desert Locust Control Committee located in Rome recommended that properly trained professional teams use Green Muscle™ operationally for the preventive control of the desert locust when the temperature is suitable (FAO, 2009). Because Green Muscle™ has several shortcomings, such its long incubation period, scientists urgently need to carry out more studies to develop effective agents that control locusts and do not have severe side effects on the ecosystem.

In recent years, plant oils have received more and more attention as alternative, potentially valuable compounds that can be used against specific pests and some important plant pathogens (Isman, 2004). Several studies have confirmed that plant oils could be used as natural pesticides to control agricultural pests (e.g., Tripathi et al., 2009; Aly et al., 2012; Tak, 2015) and plant diseases (e.g., Arshad et al., 2014; Sturchio et al., 2014; El Rasheed & El Rasheed, 2017). Most natural plant oils are suitable for organic agriculture because they are non-residual, non-toxic to humans and vertebrate animals, easy to prepare and apply, cheap, effective and natural (El Rasheed & El Rasheed, 2017). Plant oils are available as vegetable oils and essential oils. The first type is produced by pressing the oils from the seeds and parts of the plant (e.g., sunflower oil, olive oil, linseed oil). The second type is extracted from the plants by means of distillation (e.g., orange oil, peppermint oil, garlic oil, lavender oil). Essential oils are volatile compounds and must be diluted in carrier oils before their use. For centuries, plant oils or extracts have been used in alternative medicine, aromatherapy, as food flavourings, perfumes, preservatives and biological agents (Tak, 2015). Recent studies have suggested that essential oils are effective against locusts and grasshoppers. For example, Sharaby et al. (2012) suggested that the essential oils of garlic, eucalyptus and mint could be used in the integrated pest management (IPM) against the grasshopper Heteracris littoralis. Abdellah et al. (2013) tested the effect of the crude leaf essential oil obtained from Peganum harmala L. (wild rue) on larvae and adult individuals of the desert locust. They reported that the desert locusts displayed toxic effects, imbalance problems and convulsive movements. Halawa and Hustert (2014) found that lovage essential oil and its main components, limonene, impaired the ventilation of locusts at low concentrations. Lahsen et al. (2015) found that rosemary essential oil displayed a remarkably toxic effect against the morrocon locust (Dociostaurus maroccanus). In all of these studies, essential oils were found to affect locusts in different ways, but oils have not been tested on non-target species, such as beetles that are known to play important roles in the ecosystem. 40

In this study, we developed a novel mixture of plant oils that has highly toxic effects on two target species of gregarious locusts: the desert locust Schistocerca gregaria and the migratory locust Locusta migratoria. The intension was to coat the locusts with a thin layer of an oil that is difficult for the insect to remove. We used linseed oil, a drying oil that becomes viscous over time. In order to speed up the drying process, we mixed it with a saturated solution of sodium bicarbonate, which may have an additional benefit in that it is used for the control of fungal plant diseases (Horst et al., 1992; Kuepper et al., 2001). In order to increase the toxic effect of the linseed emulsion, we added three essential oils that were found to be toxic for desert locusts in a screening study. The toxic effect of the combination of effective essential oils sprayed at a low concentration on two locust species was studied 24 hours after single spray treatment. To test the toxic effect of this botanical pesticide on a beneficial non-target species and an alternative non-target species, we also sprayed adult ladybirds and their larvae, as well as adult mealworm beetles. We selected ladybird beetles as a non-target species because of its important role in the ecosystem (e.g., control of plant lice) and also treated mealworm beetles to determine whether the pesticide has a possibly harmful effect on another small beetle species. To study the effect of this formulation on the growth of grass, we sprayed it on wheat seedlings and exposed them to sunlight.

3.3. Material and methods

3.3.1. Insects

Desert locusts (Schistocerca gregaria) and migratory locusts (Locusta migratoria) were purchased in the gregarious phase from a breeding stock of the Buchner company in Austria. Locusts were maintained in a crowded colony at the Institute of Zoology in Graz. About 100 locusts were kept in a glass terrarium with dimensions of 60 x 30 x 30 cm. Individuals used in this study were taken from crowded colonies about two or three weeks after their last moult. The light:dark cycle was 12:12 h, and the average temperature in the terrarium was 26 ± 2°C (mean ± SD) at night and 33 ± 2°C (mean ± SD) during the day. The relative humidity was 45– 60%. The locusts’ diet consisted of organic wheat seedlings and organic wheat bran (DM- Drogeriemarkt, Karlsruhe, Germany), both in the breeding stock and in the crowded colony.

Adults and larvae of the mealybug ladybird (Cryptolaemus montrouzieri) were purchased from a breeding stock of the Biohelp company (Vienna, Austria). Ladybird adults were kept in plastic boxes with dimensions of 20 x 12 x 14 cm, each containing 30 individuals. About 100 ladybird larvae (final larval instar) were kept in a plastic box with dimensions of 20

41 x 12 x 14 cm, covered with transparent fabric, which contained a small piece of paper towel (12 x 12 cm). The light:dark cycle was 12:12 h, the temperature was 22 ± 2°C (mean ± SD), and the relative humidity was 40 ± 16% (mean ± SD). The diet for ladybird adults and larvae consisted of sweetened water gel (2 ml of 50% (g/V) glucose solution added to 10g water gel (Trixie Heimtierbedarf, Tarp, Germany)).

Adult mealworm beetles (Tenebrio molitor) were taken from the laboratory breeding stock maintained at the Institute of Biology in Graz. Beetles were exposed to a light:dark cycle of 12:12 h and maintained at an ambient temperature of 22°C and 50% relative humidity. Their diet consisted of dry bread, apples, fish food and oatmeal (SPAR Österreich Warenhandels AG, Salzburg, Austria).

3.3.2. Feeding plants

Seeds of organic wheat (DM-Drogeriemarkt, Karlsruhe, Germany) were planted in plastic pots that had been filled halfway with perlite (Plant!t, HydroGarden Wholesale Supplies Ltd, Binley, Coventry, CV3 2NT, UK) and covered with soil (Organic soil from EuFlor Inc., Munich, Germany). Pots were watered daily and plants were exposed to a light:dark schedule of 12: 12h. After one week, the wheat seedlings were placed in the terrarium to feed the locusts.

3.3.3. Oil emulsions and suppliers

In this study, we used linseed oil as insecticidal agent to impair the motion of locusts by means of an oil that becomes viscous over time. In order to accelerate the drying process of this oil, the linseed oil was mixed with a saturated solution of sodium bicarbonate.

The linseed/bicarbonate emulsion consisted of: Cold-pressed organic linseed oil (Linum usitatissimum), (Natur-Pur brand, Spar Österreich) and

saturated sodium bicarbonate solution (IUPAC name: sodium hydrogen carbonate NaHCO3, commercial name: baking soda), purchased from Dr. Oetker, Villach, Austria. We assessed the hardening process of the linseed/bicarbonate emulsion (56:44 % (v/v)) in a petri dish for one day (see Fig. 1). Linseed oil was stored in a dark bottle at 4°C.

Figure 1. Hardening of the linseed-bicarbonate emulsion. (a) Image of the emulsion after mixing linseed oil and bicarbonate solution. (b) Image of the viscous film formed after 24 hours.

To increase the harmful effect caused by the hardening of the linseed oil emulsion, the following organic essential oils were added individually and tested for their insecticidal effects: Peppermint oil (Mentha piperita), orange peel oil (Citrus aurantium dulcis), ginger oil (Zingiber officinale) and eucalyptus oil (Eucalyptus globulus). These oils were purchased from Primavera (Oy-Mittelberg, Germany). Basil oil (Ocimum basilicum) and wintergreen oil (Gaultheria procumbens), both purchased from Naissance (Neath, UK). Garlic oil (Alium sativum), purchased from Dragonspice Naturwaren (Reutlingen, Germany). Caraway oil (Carum carvi), purchased from Bombastus-Werke (Freital, Germany). Clove oil (Eugenia carophyllus), purchased from Greenmade (V03 Trading GmbH, Willich, Germany). Birch oil (Betula lenta), purchased from Laboratoire Centiflor (Entrechaux, France).

All essential oils were 100% pure and were stored at room temperature in a dark location. The selection of these essential oils was based on the findings of previous scientific studies in which some of these essential oils (or oils with similar effective components) were investigated for their effects against various insects such as termites, locusts, moths and beetles (Tripathi et al., 2009; Aly et al., 2012; Halawa & Hustert, 2014; Tak et al., 2016, 2017).

3.3.4. Preparation of the linseed/bicarbonate emulsion

The saturated sodium bicarbonate solution was prepared by dissolving 1 g NaHCO3 powder in 10 ml distilled water (10%). The supernatant was then added to the linseed oil using a 50 ml plastic centrifuge tube. Essential oils were added to this emulsion using an automatic calibrated pipette (Eppendorf Inc.). All oil emulsions were mixed with a vortex device (VF2, Ika labortechnik, Janke & Kunkel, Germany) before spray treatment.

3.3.5. Screening for effective essential oils

To screen for essential oils that are effective against desert locusts (Schistocerca gregaria), we sprayed 4.9 ml of different oil mixtures (equal to five pump sprays) into each box containing 10 individuals. Since the preliminary experiment had demonstrated a higher robustness of desert locusts against linseed/bicarbonate treatment as compared with migratory locusts, we selected the former for this screening study. The number of living and dead individuals was counted after 24 hours by gently shaking the insect boxes. Individuals that were unable to move and breathe normally (absent telescopic abdominal movement) were regarded as dead in this study, because ‘frozen’ individuals did not recover from this state during the observation period of 40 hours. The effects caused by essential oils were compared with three control situations.

Controls and treatments used in the screening experiment: Control 1: no treatment Control 2: spraying pure linseed oil Control 3: spraying saturated sodium bicarbonate solution (10%) Treatments: Linseed/bicarbonate emulsion: 10 ml linseed oil (55.6%) + 8 ml aqueous 10% sodium bicarbonate solution (44.4%).

1 ml pure essential oil (garlic, caraway, clove, peppermint, orange, ginger, birch, eucalyptus, or basil), each was added to 18 ml linseed/bicarbonate emulsion (resulting proportions: 5.3% essential oil, 52.6% linseed oil and 42.1% sodium bicarbonate solution).

Adult desert locusts (mature: about two weeks after last moult) were placed in plastic boxes with dimensions of 20 x 12 x 14 cm. Individuals used in experiments appeared healthy and were able to jump. The boxes were covered with transparent fabric. Each box contained ten locusts (half females and half males), a small piece of paper towel (one layer of paper tissue of 12 x 12 cm) and some wheat grass (about 35 stalks). After shaking the oil emulsion, 4.9 ml was sprayed on the insects and their food using a pump sprayer (hand spray device; Kläger Plastik, Neusäss, Germany; nozzle diameter = 0.6 mm). The food was also sprayed to screen for the oral toxicity and contact-dependent toxic effects caused by essential oils. The insect boxes were turned by 90° to spray the locusts from top (distance between the sprayer and locusts ≈ 20 cm) and keep the aerosol inside the box. The same amount of oil emulsion that was applied to insects was also sprayed on a weighing dish to quantify the applied volume. The paper towel in the boxes soaked up the remaining oil emulsion.

3.3.6. Effect of the combination of essential oils

Essential oils that contributed to a high mortality rate of adult desert locusts (orange peel, birch oil and caraway oil) were combined in different concentrations to test different botanical pesticide formulations as agents against locusts. Garlic oil has a strong smell and was not used in these formulations. We also replaced birch oil by wintergreen oil, since it is more readily available and has the same active ingredient (methyl salicylate). Finally, two slightly different formulations caused a high mortality in desert and migratory locusts (data not shown). The botanical pesticide formulation used in this study (see description below) was found to be the most effective. In order to reveal a possible synergistic effect caused by the combination of three essential oils, we performed a ‘synergism experiment.’ In this experiment, essential oils were sprayed at low concentrations either individually or in combination (termed botanical pesticide treatment) on desert and migratory locusts. We specifically, compared the mortality rate caused by orange peel, caraway and wintergreen oils with a botanical pesticide formulation that contains all of these oils. The treatment of locusts in the synergism experiment was similar to the treatment used during the screening experiment with the exception that fresh grass was provided after the spray treatment (4.9 ml was sprayed in control 2 and each treatment). This

45 allowed us to discriminate between a toxic effect caused by physical contact with the oil emulsion and oral toxicity. The following controls and treatment were tested:

Control 1: no treatment Control 2: Linseed/bicarbonate emulsion: 10 ml linseed oil (55.6%) + 8 ml aqueous 10% sodium bicarbonate solution (44.4%) Caraway treatment: Linseed/bicarbonate emulsion consisting of 10 ml linseed oil (52.6%) and 8.5 ml aqueous 10% sodium bicarbonate solution (44.7%) + 0.5 ml caraway oil (2.6%) Orange peel oil treatment: Linseed/bicarbonate emulsion consisting of 10 ml linseed oil (52.6%) + 8.75 ml aqueous 10% sodium bicarbonate solution (46.1%) + 0.25 ml orange peel oil (1.3%) Wintergreen treatment: Linseed/bicarbonate emulsion consisting of 10 ml linseed oil (52.6%) + 8.75 ml aqueous 10% sodium bicarbonate solution (46.1%) + 0.25 ml wintergreen oil (1.3%) Botanical pesticide treatment: 18 ml linseed/bicarbonate emulsion (10 ml linseed oil (52.6%) and 8 ml aqueous 10% sodium bicarbonate solution (42.1%)) + 0.25 ml orange peel oil (1.3%) + 0.5 ml caraway oil (2.6%) + 0.25 ml wintergreen oil (1.3%).

Each treatment was repeated five times, and the average mortality rates were calculated.

3.3.7. LC50 test of the botanical pesticide

The lethal concentration 50% (LC50) of our botanical pesticide formulation was assessed within 40 hours to reveal whether it had been applied at a concentration that was effective to control desert locusts, which had been found to have a lower mortality rate as compared to migratory locusts. For this purpose, we diluted the botanical pesticide (same composition as in the synergy experiment) with distilled water to create different concentrations and sprayed it on adult desert locusts (Schistocerca gregaria). A similar treatment procedure was used as in the screening test, but fresh grass was added after the spray treatment. Individuals were counted either as dead or alive when they did not move and breathe after gently shaking the box. LC50 experiments were repeated three times, and the average mortality rate was calculated. The dilutions of the botanical pesticide for the LC50 experiment were:

5 ml botanical pesticide (25%) + 15 ml distilled water (75%) 10 ml botanical pesticide (50%) + 10 ml distilled water (50%) 15 ml botanical pesticide (75%) + 5 ml distilled water (25%) 46

18 ml botanical pesticide (90%) + 15 ml distilled water (10%) 20 ml botanical pesticide (100%)

The LC50 was calculated after performing a linear regression on the averaged mortality rates obtained at different concentrations.

3.3.8. Botanical pesticide treatment of ladybirds

Ladybirds play an important role in many ecosystems and were considered to be a beneficial non-target species in this study. To determine whether our botanical pesticide formulation (see above) could possibly harm ladybirds, we sprayed adults and larvae of the mealybug ladybird species Cryptolaemus montrouzieri in an additional experiment. Thirty ladybird adults and 15 ladybird larvae, all retrieved from the purchased colony, were put in separate plastic boxes with dimensions of 20 x 12 x 14 cm. The boxes were covered with transparent fabric. Each box contained a small piece of paper towel (12 x 12 cm) to soak up the remaining oil. Due to the very small size of ladybird species, we reduced the amount of the sprayed volume to only 2 ml of the botanical pesticide formulation to prevent the submersion of individuals. The oil emulsion was sprayed from above in each box (equal to two pump sprays). This experiment was repeated four times with adults. In each box, a small petri dish containing 10 g of water gel (Trixie Heimtierbedarf, Tarp, Germany) with 2 ml glucose solution (50 % (g/V)) was provided as food, initially after the spraying treatment and then on a daily basis.

3.3.9. Botanical pesticide treatment of mealworm beetles

To determine whether our botanical pesticide formulation (see above) had a possibly harmful effect on an alternative non-target species, adult mealworm beetles (Tenebrio molitor) were sprayed in an additional experiment. For this purpose, 16 mealworm individuals were caged in plastic boxes with dimensions of 9 x 9 x 6 cm that had pores on two sides to allow for ventilation. Individuals used in this experiment appeared to be healthy. All boxes contained a small piece of an apple, 3.5 g of oat flakes and 0.5 g of fish food (ASTRA Aquaria GmbH, Bissendorf, Germany). The botanical pesticide emulsion was shaken before spraying it on the beetles and on the food (except apple pieces, which were added later). Because smaller boxes were used for the beetles, only 2.9 ml of the botanical pesticide formulation was sprayed in each box (equal to three pump sprays). After spray treatment, the boxes were closed with a plastic cover (lacking pores). This experiment was repeated four times. Every second day, a

47 small piece of an apple was provided as fresh food. The number of living and dead individuals was counted after gently shaking the boxes.

3.3.10. Effect of the botanical pesticide on the feeding activity

The effect of the botanical pesticide on the feeding activity of adult desert locusts (Schistocerca gregaria) was investigated in a separate experiment where the same amount of grass (36 stalks) was added to three boxes of dimensions 20 x 12 x 14 cm. Each box contained 10 adult locust individuals and a small piece of paper towel (one layer of kitchen paper tissue of dimensions 12 x 12 cm). One box served as the no-treatment control. Locusts in the second box were sprayed together with the grass (4.9 ml of the botanical pesticide formulation). Desert locusts in the third box were sprayed with same amount of the botanical pesticide, but fresh grass stalks were added right after treatment. 24 hours later, images were taken from each box (see Fig. S2).

3.3.11. Ambient conditions during experiments

All experiments were performed on a work bench with medium airflow and a light:dark cycle of 12:12 h. During the experiments, the temperature was 24.5 ± 2°C (mean ± SD), and the relative humidity was 42 ± 11% (mean ± SD).

3.3.12. Effect of the botanical pesticide on plant growth

A botanical pesticide that is based on hardening linseed oil may adversely affect plant growth because of its viscosity. In order to study the potentially harmful effect on plant growth, the botanical pesticide emulsion was sprayed on wheat seedlings that were exposed to sunlight on a balcony in Graz. In this experiment, 4.9 ml of the botanical pesticide emulsion (equal to five pump sprays) was sprayed on each grass pot and grass growth was monitored over 22 days. The wheat seedlings were purchased as “cat grass” from a pet store (Zoo Muser, Graz, Austria). The temperature during this experiment was between 17–30°C, and the relative humidity was 49.2–62.7%. Each control and treatment group consisted of three pots of wheat seedlings that were submersed in water to provide the same amount of water.

3.3.13. Mortality rate calculations

The mortality rates were calculated in Microsoft Excel 2016 using equation 1:

푡표푡푎푙 푛푢푚푏푒푟 표푓 푑푒푎푑 푖푛푑푖푣푖푑푢푎푙푠 Equation 1: 푃푒푟푐푒푛푡푎푔푒 표푏푠푒푟푣푒푑 푚표푟푡푎푙푖푡푦 = ( ) ∗ 100 푡표푡푎푙 푛푢푚푏푒푟 표푓 푡푟푒푎푡푒푑 푖푛푑푖푣푖푑푢푎푙푠

According to the World Health Organization’s (WHO) test procedures for insecticide resistance monitoring (Global Malaria Programme, 2016), equation 2 was used for the calculation of mortality rates that takes the value of the control mortality into account. If the control mortality is higher than 5% and lower than 25%, then the mortality rate of treatment groups was corrected according to the Schneider-Orelli formula (Püntener, 1981) shown in equation 2.

% 표푏푠푒푟푣푒푑 푚표푟푡푎푙푖푡푦 – % 푐표푛푡푟표푙 푚표푟푡푎푙푖푡푦 Eq. 2: 푃푒푟푐푒푛푡푎푔푒 푐표푟푟푒푐푡푒푑 푚표푟푡푎푙푖푡푦 = ( ) ∗ 100 100 – % 푐표푛푡푟표푙 푚표푟푡푎푙푖푡푦

The corrected mortality was calculated for ladybirds and mealworm beetles, since the mortality of locusts in the control group was less than 5%. To cross-check the corrected mortality rates, we calculated them a second time using the Schneider-Orelli method provided on this website: http://www.ehabsoft.com/ldpline/onlinecontrol.htm. The average mortality is given as the mean mortality rate of different replicates (± standard deviation).

3.3.14. Statistical analysis

Mortality rates of the locusts and beetles were tested for statistically significant differences by performing the Z-test with the Yates correction, which takes the sample number into account. Significant differences between the control and the treatment groups are indicated by an asterisk in Figs. 2, 3 and 4. All statistical tests were performed in Sigma Plot version 13 (Systat Software Inc.). The linear regression performed for the calculation of the LC 50% of the botanical pesticide was calculated in Excel (version 2016, Microsoft Inc.). To determine whether a possible synergistic toxic effect was caused by a mixture of four oils, we calculated the expected mortality rate according to what is called the response addition or Bliss Independence (Bliss, 1939) and compared this rate with the observed mortality rate by performing a chi-square test (equation described in Tounou et al., 2008). The expected mortality rate caused by the addition of the toxicity of four oils was calculated by modifying equation two as stated in Cedergreen (2014).

3.4. Results

3.4.1. Screening for effective essential oils

In a petri dish, the linseed oil/bicarbonate emulsion (56:44% (v/v), Fig. 1a) hardened within 24 hours and became sticky and viscous (compare Fig. 1a with 1b). This emulsion caused a moderately toxic effect when sprayed on adult desert locusts (Fig. 2), whereas spraying either

49 linseed oil or bicarbonate solution failed to harm these insects. Linseed oil/bicarbonate-treated desert locusts that survived 24 hours had difficulties moving quickly and were unable to jump; they appeared as though frozen. The amount of oil sprayed in this screening experiment was too low to create a sticky surface inside the box upon which insects adhered. To enhance the toxic effect caused by the hardening of linseed oil (see Fig. 2), different essential oils were added to this oil emulsion and sprayed on desert locusts in the screening experiment. Figure 2 shows the mean mortality rates of the desert locust one day after a single spray treatment with each of nine different oil emulsions. The mortality rates of desert locusts were equal or exceeded 70% when each of the garlic, orange, caraway and birch oils were added at a concentration of 5.3% to the linseed oil emulsion. The highest mortality rate was found after adding garlic oil. Using the same concentration of clove, peppermint and basil oil emulsions also led to significantly higher mortality rates of desert locusts as compared to the controls (p < 0.05, Z- test). In contrast, the use of 5.3% ginger or eucalyptus oils did not significantly increase the toxicity of the linseed oil emulsion.

3.4.2. Combination of essential oils

Each of the garlic, orange peel, caraway and birch oils mixed with the linseed oil emulsion caused high mortality in the screening experiment. At lower concentrations, caraway (2.6%), orange peel (1.3%) and wintergreen (1.3%) oils resulted in a rather low mortality of desert locusts after spray treatment. However, the combination of these oils in the botanical pesticide treatment increased the mortality rate of desert locusts significantly compared to all other treatments and the control situation. This botanical pesticide was more effective against migratory locusts (blank bars in Fig. 3) compared to desert locusts (blue bars in Fig. 3), where 20 ± 7.1% survived the first day after spray treatment. However, surviving individuals were rather weak and died within in the next 16 hours. The expected additive mortality rate caused by four oils was 56% in Sch. gregaria and 216% in L. migratoria. The result of the chi-square test was 11.7 when we compared the expected with the observed mortality of Sch. gregaria. The critical value of this test defined for three degrees of freedom and an error probability of 0.05 is 7.8. Since the result of the chi-square test is higher than this critical value, a synergistic toxic effect caused by the combination of these oils was evident in Sch. gregaria. The essential oils evaporated and left no trace of scent behind when we counted the number of dead individuals 24 hours after spray treatment. In contrast to the combined effect, spraying only the linseed oil emulsion without essential oils (control 2) had only a minor toxic effect on desert

50 locusts (14 ± 16.7% mortality), but still killed 46 ± 19.5% of migratory locust individuals, which were also more sensitive to individual essential oil treatments.

Figure 2. The mortality rate of desert locusts in the screening experiment. The mortality rate of the desert locusts is shown after 24 hours (N = 10). * indicates the significant difference between controls and treatment (p < 0.05, Z-test). 51

Figure 3. Botanical pesticide experiment. Essential oils were added either individually or in combination to the linseed oil emulsion and the mean mortality rate of desert and migratory locusts was calculated after a single spray treatment. The mean mortality rate of desert locusts (blue bars, N = 10, five replicates) and migratory locusts (blank bars, N = 10, five replicates) are shown after 24 hours. * indicates a significant difference between control 1 and treatment (p < 0.05, Z-test). Ɨ indicates a significant difference between control 2 and treatment (p < 0.05, Z-test). 52

3.4.3. LC50 test and antifeedant effect

The lethal concentration (LC50) of the botanical pesticide formulation observed 40 hours after spraying desert locusts was calculated from the regression line shown in Fig S1. At a concentration of 60%, this botanical pesticide still killed 50% of desert locust individuals, which demonstrates that the toxicity of this botanical pesticide is high enough to control desert locusts effectively. To test for the additional oral toxicity of this botanical pesticide, we sprayed either desert locusts together with grass stalks or added grass stalks after the spray treatment. As can be seen in Figure S2b, the untreated locusts ate all the grass within one day. In fact, the desert locusts refused to feed on grass whether it was sprayed or not (Fig. S2c and d). This demonstrates a strong antifeedant effect of this botanical pesticide.

3.4.4. Botanical pesticide experiment performed with two beetle species

To collect preliminary data about the effect of this botanical pesticide on a beneficial and an alternative non-target species, we sprayed this formulation on ladybirds and on adult mealworm beetles (Fig. 4). Spraying adult mealworm beetles (i.e., the alternative non-target species) did not increase their mortality rates significantly. Even eight days after treatment, the mean corrected mortality of mealworm beetles was only 12.4 ± 7% (green bars in Fig. 4, p > 0.05, Z-test, N = 16, four replicates). However, spraying ladybird adults (i.e., the beneficial non- target species) caused a significant mean mortality rate of 67.7 ± 8.9% as compared to the control group after eight days (blank bars in Fig. 4, p < 0.05, Z-test, N = 30, four replicates). Ladybird larvae were even more sensitive because 56.7 ± 4.7% of all individuals died within one day after the botanical pesticide treatment (p < 0.05, Z-test, N = 15, two replicates). Records on the effect of the botanical pesticide on the ladybird larvae could not be taken after the first 24 h, due to the fact that the larvae cannibalized each other.

3.4.5. Effect of the botanical pesticide on grass growth

We sprayed the botanical pesticide on fresh wheat seedlings to investigate its impact on plant growth. We investigated the growth of the wheat seedlings that were exposed to natural sunlight on a balcony (Fig. 5a). Figure 5 shows the pots of the wheat grass 22 days after treatment. Almost no difference was observed in the shape and growth of grass stalks between the control (Fig. 5b) and the treated grass (Fig. 5c), with the exception of some yellow tips.

Figure 4. Beetle experiment. The mortality rates of two beetle species after single spray treatment with the botanical pesticide formulation. The mean corrected mortality rate of mealworm beetles (green bars, N = 16, four replicates) and the mean corrected mortality rate of ladybird adults (blank bars, N = 30, four replicates) eight days after treatment. * indicates a significant difference between control and treatment (p < 0.05, Z-test).

Figure 5. Spray treatment of wheat seedlings with the botanical pesticide emulsion. (a) Images of wheat seedlings before treatment. (b) Image of the untreated wheat grass taken 22 days later. (c) Image of the treated wheat grass 22 days after spray treatment. 54

3.5. Discussion

Due to the hardening process of the linseed/bicarbonate emulsion (see Fig. 1), locust mobility was strongly restricted after 24 hours. This was obvious as individuals rarely jumped and moved slowly after disturbance. Adding sodium bicarbonate to the linseed oil clearly accelerated the hardening process of this oil. Bicarbonate salts are also known to be effective against fungal plant diseases. For example, Kuepper et al. (2001) provided a brief survey of the use of sodium bicarbonate (NaHCO3) and potassium bicarbonate (KHCO3) to control powdery mildew and other fungal diseases. Similarly, Horst et al. (1992) used sodium bicarbonate to control powdery mildew and black spot disease in roses in greenhouses and field experiments. However, spraying the linseed oil/bicarbonate emulsion on desert locusts was not very effective, since only 40% of treated individuals died within one day (Fig. 2). To enhance the harmful effect of this linseed oil emulsion, several essential oils were added in a screening experiment. The results of this experiment revealed that garlic, caraway, orange and birch oil emulsions were potential candidates for an insecticidal oil mixture that could kill a high proportion of locusts within a short time period. The desert locust mortality caused by these oils applied at concentration of 5.3% was equal or even exceeded 70%. However, garlic oil was excluded from the next experiment because of its strong and unpleasant smell. Furthermore, birch oil was replaced by wintergreen oil, because wintergreen is more readily available and has the same active ingredient (methyl salicylate).

A formulation consisting of linseed oil and low concentrations of orange peel, wintergreen and caraway oils was then sprayed on desert locusts and migratory locusts to test its potential as a novel botanical pesticide. Results shown in Figure 3 demonstrate that the combination of these essential oils had a strong effect on the mortality rates of the two gregarious locust species. Desert locusts were less sensitive as compared to migratory locusts, but individuals surviving the first 24 hours died within the next 16 hours. Although the use of the same concentration of each essential oil had only a low or moderate effect on desert and migratory locust mortality, the combination of the three essential oils resulted in a strong toxic effect. The results obtained with desert locusts demonstrate that these essential oils act synergistically, because the high mortality rate of desert locusts treated with the botanical pesticide cannot be explained simply by an additive toxic effect caused by each essential oil (Fig. 3).

The botanical pesticide formulation developed in this study harmed the target insects in at least two ways: First, the mobility and most likely the ventilation of the locusts were affected 55 due to the hardening of the linseed emulsion (see Fig. 1). Second, an antifeedant effect was clearly observed, since all locusts stopped feeding immediately after the spray treatment (see Fig. S2). These results, taken together, indicate that our botanical pesticide formulation constitutes a novel, promising and effective agent that can be used to combat upsurges and locust outbreaks. This botanical pesticide formulation was then tested on two beetle species in an experiment lasting for eight days (Fig.4). Both beetle species are related to ecologically important beetle species (Jankielsohn, 2018). Within this time span, although mealworm beetles (i.e., alternative non-target species) behaved normally, a significant, toxic effect was observed in ladybird adults (i.e., beneficial non-target species). Even more problematic a strong toxic effect was observed in the ladybird larvae that were studied for just one day after treatment. Mealworm beetles are less likely to come in contact with our botanical pesticide in the field, except when locusts are treated in the Mediterranean region, which is the home of this beetle species. However, given the fact that the ladybird species used in this study is related to other ladybird species in various ecosystems we cannot exclude an unwanted side-effect on non- target insect species with important ecological functions (e.g., feeding on plant lice). However, this botanical pesticide may have only minor negative effects on the vegetation, since the growth of wheat seedlings on a balcony was not significantly affected (Fig. 5). It will be possible to use conventional spraying devices for field applications of this botanical pesticide when the emulsion is stabilized by means of an emulsifier. To avoid a toxic effect on non-target species, such as other insects (e.g., bees, beetles) and birds, it is highly recommended to spray the pesticide after sunset when the locusts rest in crowded colonies on bushes and trees.

To understand the synergistic toxic effect caused by this botanical pesticide, it is necessary to focus on its active ingredients. The main component of orange peel oil is limonene (87.9 –92.5%; Njoroge et al., 2009), but caraway oil also contains this cyclic monoterpene (1.5– 51.3%; Raal et al., 2012). A recent study demonstrated that the ventilation in the migratory locust is adversely affected by limonene in its pure form or as the major component of lovage essential oil (Halawa & Hustert, 2014). Wintergreen oil mainly contains methyl salicylate (97– 99.6%; Gurung, 2007), which was found to have insecticidal activity against the adzuki bean weevil Callosobruchus chinensis (Park et al., 2016). Raal et al. (2012) identified carvone (44.5– 95.9%) as the main component of caraway oil, and Wawrzyniak and Lamparski (2006) found that aqueous extracts of caraway inhibited the feeding activity of the Colorado potato beetle Leptinotarsa decemlineata and its larvae. This suggests that caraway may be responsible for the antifeedant effect found in locusts. Additional studies, such as the one performed on the cabbage looper (Trichoplusia ni Hübner, Noctuidae, Akhtar et al., 2012), are necessary to reveal 56 the mechanism behind the antifeedant effect of our botanical pesticide. The insecticidal effects of caraway and wintergreen oils on locusts shown in this study is novel and have not been described before.

The main component of the botanical pesticide formulation is the linseed oil, which contains omega-3 fatty acids and has a higher linolenic acid content than other plant oils. For this reason, linseed oil is healthy and may be used as an alternative to fish oil in the human diet (Kolodziejczyk et al., 2012). Since locusts are collected and consumed as a protein source in some countries in Africa, Asia, Central and South America, Australia (Paul et al., 2016), locusts that have been stunned or killed by our botanical pesticide can be collected and used as a protein source in the human diet and the food industry. The other components of this botanical pesticide formulation are often used as preservatives and biological agents in many applications and in alternative medical treatments for human, animals and plants (Elshafie & Camele, 2017).

Linseed oil becomes viscous over time when mixed with the bicarbonate solution, but it may also have an additional adverse effect on locust swarms due to the unsaturated fatty acid content. Yao et al. (2009) reported that certain unsaturated fatty acids like oleic and linoleic acid constitute necromones that are released by dead insect bodies. Necromones can be recognized by other individuals of the same or different species, where they evoke distinct behaviour. For example, social insects remove the dead bodies from their nests (necrophoric behaviour), whereas semi-social species avoid contact with dead or injured individuals (necrophobic behaviour). Therefore, Yao et al. (2009) suggested that fatty acid necromones might be considered in many contexts, including pest management. Linseed oil contains approximately 9–11% saturated (5–6% palmitic acid and 4–5% stearic acid) and 75–90% unsaturated fatty acids (50–55% linolenic acid, 15–20% oleic acid; Bayrak et al., 2010), which suggests that this oil may evoke necrophobic behaviour in swarm mates. This might disrupt swarm formation after a certain percentage of individuals have come into contact with linseed oil. Current studies aim to reveal whether linseed oil has a possible swarm disrupting effect.

In conclusion, we were able to develop a potent novel oil mixture consisting of linseed oil and plant essential oils that displayed toxicity against locusts. This botanical pesticide contains caraway, wintergreen and orange-peel oils at low concentrations. Due to a combined toxic effect, it is effective against two species of gregarious locusts (Fig. 3) while it did not harm mealworm beetles. Due to the toxic effect on the ladybird adults (fig. 4) and its highly toxic effect on their larvae, the application of this botanical pesticide should be restricted to dense locust colonies where other non-target insects are rare. It was interesting to note that our 57 botanical pesticide formulation did not adversely affect the growth of wheat seedlings (Fig. 5). These results suggest this formulation is an effective botanical pesticide that can be used as an alternative to chemical pesticides in the future. The components of our botanical pesticide formulation can be obtained easily at low prices, and no laboratory equipment is needed to mix the ingredients before the pesticide is applied. However, further research under field conditions is necessary to determine the effects of this novel insecticidal formulation on locust swarms and the local ecosystem.

Supplementary materials

Figure S1. LC50 of the botanical pesticide formulation. The average mortality rate was calculated 40 hours after single spray treatment (N=10 desert locusts, 3 replicates).

Figure S2. Anti-feedant effect of the botanical pesticide formulation. (a) Image of 36 stalks of grass that were added to each group. (b) Control: untreated desert locusts ate all grass stalks provided as food 24 hours before. (c) Treatment 1: The desert locusts and the grass stalks were sprayed with the botanical pesticide 24 hours before taking this picture. (d) Treatment 2: Desert locusts were sprayed and grass stalks were added right after this treatment. The image shown in b, c and d were taken 24 hours after spray treatment (N= 10 individuals in each group). 58

Chapter 4

Linseed oil affects aggregation behaviour in the desert locust Schistocerca gregaria - a potential swarm disruptive agent

In special Issue "How to Manage Migratory Pests and Potential Food Crises: Locusts Plagues in the 2020’s"

4.1. Summary

Gregarious desert locusts constitute very destructive agricultural pests. They aggregate and form collectively moving swarms that devastate vegetation and reduce crop production. To combat gregarious locusts, a botanical pesticide formulation that contains linseed oil as the main component was described recently. Since linseed oil is rich in fatty acids, some of which function as necromones that indicate injury or death in various insect species, we investigated the influence of linseed oil on the aggregation behaviour of sexually mature gregarious desert locusts. For this reason, we performed a series of aggregation experiments with six individuals of the same sex and brushed the wings of one individual (target individual) with linseed oil. The time the oil brushed target males spent close to any other individual was reduced in 76% of trials (average reduction of 18%), whereas the time target females spent in groups with members of the same sex did not alter. These results suggest that linseed oil may act as a bioactive agent that has the potential to disrupt swarm formation.

4.2. Introduction

Desert locusts (Schistocerca gregaria Forsskål, 1775) are considered to be among the most serious agricultural pests because of their polyphagous feeding behaviour, rapid reproduction rates and quick migration patterns (Steedman, 1988; Cressman, 2016). Like other locust species, this species displays density-dependent phase polyphenism, which means that they can transform reversibly between two phases in response to population density: the solitary and gregarious phases. Individuals in either phase differ in terms of their morphology, physiology and behaviour (Uvarov, 1966; Steedman, 1988). Behavioural changes can occur quickly, appearing within just a few hours (Gaten et al., 2021). If the population density is low,

59 locusts exist in a solitary phase and avoid each other, except when they are ready to mate. If the population density increases, even over a short time period, the behavioural transition to the gregarious phase is evoked (Pener & Simpson, 2009; Ma et al., 2014). This is the case, for example, after rainfalls that end long periods of drought, resulting in nymphs hatching from eggs laid in burrows in the ground (Latchininsky, 2013). As the population density increases, the locusts become social, aggregate and form marching bands on the ground (wingless hoppers); later, they form collectively moving swarms that can migrate over long distances (Steedman, 1988). Locust swarms can devastate entire fields and cause extensive crop damage over very short time periods. A small swarm of locusts contains thousands of individuals that spread out over several hundred square meters, but large swarms contain up to 80 million individuals per square kilometre. Since such swarms can cover a distance of 100 km per day (Steedman, 1988), farmers regard gregarious locusts as one of the most destructive plagues on earth.

Locusts can sense swarm members using olfactory, visual and tactile cues (Simpson et al., 2001; Pener & Simpson, 2009; Ma et al., 2014). Physical contact was found to be the most potent stimulus, causing solitary locusts to gregarize. Simpson et al. (2001) discovered that touching the hind legs of others in a repetitive way induces phase transition. A patchy distribution of food plants also increases the probability of physical contact between locusts and boosts aggregation (Roessingh et al., 1998). On the contrary, visual or olfactory stimulants have lesser or incompetent effects on phase transition in the desert locust (Roessingh et al., 1998; Simpson et al., 2001), whereas the combination of them can lead to a behavioural gregarization (Simpson et al., 1999). In addition, seeing other locusts for a longer period (24 h) can also mediate phase change, at least partially (Simpson et al., 1999). Agents that disrupt the formation of swarms have not yet been established on the market but may represent alternative measures that can be used against locust outbreaks.

The authors of this study recently developed a highly effective botanical pesticide formulation against two species of locusts that is mainly composed of linseed oil as a carrier oil and three essential oils (Abdelatti & Hartbauer, 2020). A single spray treatment of locusts with this formulation killed all adults and nymphs of desert locust (Schistocerca gregaria) and migratory locust (Locusta migratoria) within 30 hours (Hartbauer & Abdelatti, 2019; Abdelatti & Hartbauer, 2020). In addition to this toxic effect, this botanical pesticide formulation may also exert a change in group formation behaviour after individuals have come in contact with linseed oil that contains 75-90% unsaturated fatty acids (50-55% linolenic acid, 15-20% oleic

60 acid and 11-20% linoleic acid, Bayrak et al., 2010). Yao et al. (2009) described oleic and linoleic acid as necromones, substances that are associated with the injury and death of insects. These often evoke distinct behaviour patterns in other individuals of the same or different species. For example, eusocial and some semi-social species show necrophoric behaviour, including the removal of dead bodies from the nests (observed in bees, ants, spiders and aphids), burial (covering the dead with soil and/or other materials in ants and termites) and cannibalism (intraspecific necrophagy in ants and termites) (Wilson et al., 1958; Yao et al., 2009; Sun & Zhou, 2013). In contrast, solitary and some sub-social species avoid dead or injured individuals (necrophobic behaviour) (Yao et al., 2009; Sun & Zhou, 2013). In this study, we performed aggregation experiments to test the following hypothesis: Linseed oil evokes a change in aggregation behaviour in gregarious desert locusts once one individual (“the target individual”) has come into contact with this oil. To test this hypothesis, we evaluated the time the target individual spent in close proximity to others in a group of six individuals before and after brushing its wings with linseed oil. Moreover, we tested the behavioural responses of desert locusts towards a stationary linseed oil target, dead and crushed locust bodies.

4.3. Materials and Methods

4.3.1. Insect species and oil

Desert locusts (Schistocerca gregaria) were purchased in the gregarious phase from a breeding stock provided by the Buchner Company in Austria. Locusts were maintained in a crowded colony at the Institute of Zoology in Graz. About 100 locusts were kept in a glass terrarium with the dimensions of 60 x 30 x 30 cm. The light:dark cycle was 12:12 h, and the average temperature in the terrarium was 28°C at night and 35°C during the day. The relative humidity was 45-60%. The individuals used in the behavioural experiments were of the same age (mature: about four or five weeks after their last moult).

Organic linseed oil (Linum usitatissimum, Natur-Pur brand) was purchased from Spar Österreich. It was stored in a dark bottle at 4°C during testing.

4.3.2. Insects food

The locusts’ diet consisted of organic wheat seedlings and organic wheat bran (DM- Drogeriemarkt, Karlsruhe, Germany). Pots of wheat seedlings (i.e. “cat grass”) were purchased from Zoo Muser, a local pet shop. These pots of grass were watered daily and exposed to a

61 light:dark schedule of 12: 12h. The wheat seedlings were replaced every two days in the terrarium.

4.3.3. Experimental setup

Aggregation experiments were performed inside an anechoic chamber, using an arena with the dimensions of 85 x 65 x 45 cm. The floor of the arena was covered by a piece of paper that was replaced after each trial. We placed a heater (model: PF320LCD, ewt) to maintain a constant temperature of 30°C inside the anechoic chamber. To illuminate the arena, we use two lamps, an 8W inspection lamp (Electronic Montage Lampe, SLV Elektronik GmbH: Löhne, Germany) mounted on the top of the arena and a standard lamp (type: 160312, lamp/Bulb:

65×LED, 8W, SLV Elektronik GmbH). A top-view video camera (CB-38075, GKB: Taichung, Taiwan, China) was used to record the movement of locust individuals inside the arena.

4.3.4. Insect isolation

Before performing aggregation experiments, we isolated gregarious desert locusts for three days to simulate swarm disruption that has to be expected after treatment of a swarm with the linseed oil based botanical pesticide. Therefore, we caged individuals in plastic boxes with dimensions of 9 x 9 x 6 cm and placed them at a distance of 2 cm from one another. Boxes were located near to a glass terrarium that contains crowded-reared locusts. All individuals used in experiments were taken from isolation boxes, behaved normally and were able to jump. The walls of insect boxes provided air exchange and olfactory communication among the individuals (ninety-eight pores with a diameter of 1 mm). These boxes were placed on sheets of soft kitchen paper towel to absorb the vibrations generated by the locusts. Additionally, white paper tape was applied to the walls of the plastic boxes to prevent visual contact. To study the aggregation ability of locusts three days after isolation, we evaluated the time individuals came close to any other individual in our aggregation experiments (see Table 1). During the isolation period, the same amount of grass was offered to all individuals. On the third day of isolation, a small piece of reflecting tape was mounted on the pronotum of one individual to mark the target locust. This reflecting tape was fixed in place using a small drop of super glue (Loctite, Henkel Central Eastern Europe GmbH, Vienna, Austria), which is harmless for insects such as bees and locusts.

4.3.5. Behavioural experiments

Three days after their isolation, the locusts were transferred to the arena to study group formation during the light phase of their day cycle. In all trials, we studied the group formation of six individuals of the same sex to prevent premating and mating behaviour. All individuals were inspected for dual sexual characteristics (gynandromorph) to prevent an influence arising from mixing different sexes. The inter-individual distance between the target individual and any other individual was observed over a time period of 30 minutes before linseed oil application. Then the wings of the target locust were brushed with a thin film of linseed oil (provided at room temperature), and the locusts were monitored for another 30 minutes. The movements of locusts were recorded under bright conditions using the top-view video camera.

This camera was connected to a frame grabber (Pixelsmart Inc.: Lewiston, NY, USA) that captured images in intervals of 5 seconds, resulting in 12 images per minute. In total, 26 trials were performed with 13 female groups and 13 male groups. Since tactile stimulation may affect the target locust behaviour, we also performed sham operation experiments to exclude an influence of being handled and brushed. In sham operation trials, locusts were monitored over a time period of 30 minutes before treatment during the light phase of their day cycle. Then, the wings of the target locust were brushed with a clean brush, and the locust group was monitored for another 30 minutes after treatment. In total, 24 trials were performed with 6 female groups and 8 male groups. The ambient temperature and relative humidity were measured with a hand- held device during the trial. On average, the ambient temperature was 28.7 ± 0.9 °C (mean ± SD), and the mean relative humidity was 36.3 ± 2.9 % (mean ± SD).

4.3.6. Behavioural responses towards linseed oil and dead insect bodies

Experiments were performed on mature desert locusts of both sexes to study the attraction/avoidance behaviour after exposing locusts to linseed oil-soaked paper and dead locust bodies. Five males or females were taken from the crowded-rearing arena and were placed in a test arena with a dimension of 48 x 24 x 28 cm. Two clean pieces of plastic foil (dimension of 8 x 3.5 cm) were placed on opposite corners of the arena. The movement of locust individuals was monitored for 30 minutes using a USB-camera that captured images in intervals of 5 seconds. Then a filter paper saturated with 0.5 ml of linseed oil (dimension of 7 x 2.5 cm) was placed on a randomly selected plastic foil of the arena and locust individuals were monitored for another 30 minutes. Then, the filter paper was replaced by a clean plastic foil and the locusts were given 5 minutes pause before the next experiment was performed with dead insect bodies. For this purpose, two freshly killed locust individuals (a male and a female) 63 were placed on the clean plastic foil opposite the linseed oil corner and the locust individuals were monitored for another 30 minutes. Locusts were killed by putting them into a freezer for one hour. In total 11 trials were performed with 5 female groups and 6 male groups.

4.3.7. Behavioural responses to crushed male bodies

While intact dead bodies can be an indicator of contagion in wood lice, the crushed bodies of conspecifics can be an alert of an injury caused by a predator (Yao et al., 2009). Therefore, we exposed male locusts to the crushed bodies of conspecific males in a similar setup. Two males were killed by freezing and then their bodies were crushed. A clean piece of plastic foil was placed in a randomly selected corner of the arena and the movements of locusts were monitored for 30 minutes by means of a top view USB-camera. Then, the foil was replaced by another foil to expose locusts to crushed conspecific males foil and to monitor locust movements for another 30 minutes. Ten trials were performed with groups of 5 males.

4.3.8. Data evaluation and statistical analysis

The obtained video frames were imported into ImageJ (version 1.51j8, National institutes of Health: Bethesda, MD, USA) to measure the distances between the target locust and the next individual. Frame-by-frame distance measurements allow us to quantify the tendency to form groups. We regarded two or more individuals as a group when the inter- individual distance from the target individual was shorter than or equal to its body length (see red scale bar in Fig. 1, on average: 5.8 cm for male groups and 6.2 cm for female groups). Such a small distance allowed individuals to see and smell each other. Gillet defined the grouping of locusts by the distance of two body-lengths to each other (Gillett, 1973; 1975). To analyse the group formation over a period of 30 minutes, 360 images were imported into ImageJ, and the time the target locust spent in groups (aggregation time) was evaluated by hand. The distance between the body of the target individual (see blue circle in Fig. 1) and the closest individual was measured manually after spatially calibrating the image. The first five minutes at the beginning of frame grabbing (equals to 60 image) were excluded from the evaluation to remove possible behavioural influences related to insect handling. The time spent in groups was calculated using equation (1):

푆푢푚 표푓 푔푟표푢푝 푓표푟푚푎푡푖표푛 ∗ 5 푇푖푚푒 푠푝푒푛푡 푖푛 푔푟표푢푝푠 (푚푖푛푢푡푒) = ( ) (1) 60

The term “sum of group formation” refers to the number of frames in which the inter- individual distance of the target individual from any group member was shorter than or equal to its body length. To calculate the number of minutes individuals spent in a state of aggregation, we multiplied the “sum of group formation” by 5, which refers to the inter-frame interval of 5 seconds, and divided the result by 60. Percentages of aggregation relative to an observation period of 25 min were calculated using equation (2):

푇푖푚푒 푠푝푒푛푡 푖푛 푔푟표푢푝푠 푃푟표푏푎푏푖푙푖푡푦 표푓 푎푔푔푟푒푔푎푡푖표푛 (%) = ( ∗ 100) (2) 25

Figure 1. Snapshot of the top view camera. Blue circle: the target individual with the reflecting tape on the thorax. The red scale bar indicates the body length of the target individual, which was used as minimum distance to define group formation.

If the duration of group formation changed after the application of linseed oil or after sham operation by more than 10% (2.5 min), we considered this as a change in aggregation time. This threshold definition allowed us to discriminate between trials in which group formation either increased or decreased after linseed oil treatment.

To study the general tendency of the individuals to form groups after three days of isolation, the time any individual spent in groups of at least two individuals was quantified by measuring the distances between the closest parts of their bodies. We regarded two or more

65 individuals as a group if their inter-individual distance was shorter than or equal to the average of the body lengths (on average: 5.6 cm for male groups and 6.2 cm for female groups) of all six locusts. The time any individual spent in groups was calculated according to equation 1 (results are shown in Table 1).

To quantify the activity of the target individual before and after the application of linseed oil, the distance covered by the target individual was measured with the help of the MTrackJ plugin offered in ImageJ. The time spent in the target corner was measured by counting the frames in which any individual was within one body length to borders of the plastic foil. The time spent in the target corner was converted into minutes by using equation (1).

The time spent by individuals in groups or in the target corner before and after the treatments was tested for statistically significant differences by performing a paired t-test. If data distribution deviated from a normal distribution, a Wilcoxon signed-rank test was performed. To test for statistically significant differences between the percentages of trials in which either an increased or decreased group formation time was observed after the application of linseed oil, I performed a z-test with Yates correction. All statistical tests were performed in

Sigma Plot version 14 (Systat Software Inc.: Erkrath, Germany). The average amount of aggregation time and the proportion of aggregate formation relative to the observation period are given as the arithmetic mean ± standard deviation.

4.4. Results

4.4.1. Group formation after isolation

To study the general tendency of individuals to form groups after three days spent in isolation, we quantified the amount of time (see equation 1) during which any individual maintained a distance to other individuals that was equal to or smaller than the average body length of all locusts in a trial (Table 1). Neither males nor females changed the time spent close to any other individual before and after the wings of target individuals were brushed with linseed oil (females: 21.2 ± 2.2 minutes before vs. 22 ± 2.7 minutes after treatment, p = 0.137, paired t-test, N = 13; males: 23.0 ± 2.1 minutes before vs. 21.8 ± 1.9 minutes after treatment; p = 0.112, paired t-test, N = 13).

Table 1. Time any individual spent in groups of at least two individuals (including the target one) before and after the application of linseed oil. Males and females were tested in separate experiments. Total observation time = 25 minutes, Group size = 6 individuals, N = 26.

4.4.2. Linseed oil treatment of male and female groups

The average amount of time target males spent in groups with members of the same sex was significantly reduced by 4.6 ± 5.3 minutes (18.2 ± 21.2% of the observation time, Fig. 2A, p = 0.010, paired t-test, N = 13) after brushing their wings with linseed oil. In 76.7% of trials, males reduced the time spent in aggregation by more than 10%. In these trials, the average aggregation time among males significantly decreased by 6.8 ± 3.2 minutes (27 ± 12.6% of observation time) after oil treatment (red horizontal stripes in Fig. 2B, p <0.001, paired t-test, N = 10). In only two trials, the aggregation time among males increased by 4.9 ± 1.9 minutes (20 ± 7.8% of observation time, blue vertical stripes in Fig. 2B), and no change was observed in one trial (black area in Fig. 2B). Furthermore, the proportion of trials exhibiting a decreased aggregation time after treatment was significantly higher compared to the proportion of trials showing an increase (Fig. 2B, p = 0.006, z-test, N = 13). Before the application of the linseed oil, the target males sometimes mounted other males or were mounted by other males. Interestingly, male-male mounting involving the target individual was never observed after the target locust’s wings were brushed with linseed oil. The average distance covered by the target males (activity) within 25 minutes did not change after linseed oil application (Table S1, p = 0.818, paired t-test, N = 13). Tracking brushed males revealed that in 92.3% of trials, the target males were very active and moved towards other individuals in the arena, but other individuals were avoiding them.

The average amount of time that the target females spent in groups with members of the same sex did not significantly differ before and after the application of the linseed oil (0.2 ± 6.2 minutes, Fig. 2C, p = 0.962, paired t-test, N = 13). In six trials out of 13, the target females spent on average 5.0 ± 3.5 minutes longer in groups after oil treatment (blue vertical stripes in Fig. 2D). However, the proportion of trials exhibiting an increased and decreased aggregation time did not differ significantly (Fig. 2D, p = 0.687, z-test, N = 13). Egg pods laid by females were often found in the isolation boxes and one time inside the arena after the trial. The average distance covered by the target females did not change after linseed oil application (Table S1, p = 0.814, paired t-test, N = 13). Observations performed after linseed oil application revealed that in 69.2 % of trials performed with females, six females including the target one, were active, behaved normally and moved towards each other. In the other 30.8%, the target females were less active and only walked along the borders of the arena.

Figure 2. Sex differences in group formation after linseed oil treatment. Time the target males (A) and the target females (C) spent in groups. Red lines: time spent in groups decreased. Blue lines: time spent in group increased. Black lines: time spent in groups did not change by more than 10%. Total observation time = 25 minutes, N = 13 experiments. (B, D) Percentage of experiments in which aggregation time decreased (red horizontal stripes), increased (blue vertical stripes) or was left unchanged (black area) after linseed oil treatment. * indicates a p-value < 0.05 (paired t-test, N = 13). Ɨ indicates a significant difference between the percentage of experiments with a decreased and increased aggregation time related to the application of the linseed oil (p < 0.05, z-test, N = 13).

4.4.3. Pooled aggregation time after linseed oil treatment

In more than half of all trials performed with either males or females, the total amount of time that the target individual spent in groups of at least two individuals decreased by more than 10% after the application of linseed oil (Fig. 3A, B). An increase in the amount of aggregation time, however, was found in only about one-third of the trials. Averaging over all trials and sexes, the time spent in an aggregation decreased by only 2.2 ± 6 minutes (8.7 ± 24.5% of the observation time), which indicates that the application of linseed oil did not significantly change the total aggregation time (p = 0.077, paired t-test, N = 26). However, in 53.8% of trials in which the target individuals spent less time in groups, the aggregation time was significantly reduced by 7 ± 2.9 minutes after the application of linseed oil (red striped

69 segment in Fig. 3B, p < 0.001, paired t-test, N = 14). In only 30.8% of trials, the target individuals significantly extended the average time spent in aggregation by 5 ± 3.1 minutes (blue-striped segment in Fig. 3B, p = 0.008, Wilcoxon signed-rank test, N = 8) after treatment. In 15.4% of all trials, there was no change in the aggregation time of the target individuals of more than 10% (black segment in Fig. 3B).

Figure 3. Time the target individual spent in male and female groups. (A) Aggregation time of the target individual before and after treatment with linseed oil. Red lines: time spent in groups decreased. Blue lines: time spent in groups increased. Black lines: time spent in groups did not change by more than 10%. (B) Percentage of experiments in which aggregation time decreased (red horizontal stripes), increased (blue vertical stripes) or was left unchanged (black area) after linseed oil treatment. Total observation time = 25 minutes, N = 26 experiments.

4.4.4. Sham operation

To exclude an influence caused by the handling of the target individual, we quantified the total amount of time the target locust spent in groups of at least two individuals before and after the wings were brushed with a clean brush (results are shown in Table S2). Averaging

70 over all trials and sexes, the time spent in an aggregation decreased by only 0.8 ± 6.2 minutes (2.7 ± 20.8% of the observation time), which shows that the clean brush did not change the total aggregation time significantly (p = 0.538, paired t-test, N = 24). The average amount of time target males spent in groups with members of the same sex was reduced by only 1.2 ± 6.2 minutes (3.9 ± 20.7% of the observation time) after sham operation. The average amount of time the target females spent in groups with members of the same sex in this experiment was reduced by only 0.4 ± 6.5 minutes (1.4 ± 21.7% of the observation time).

4.4.5. Group exposure to linseed oil and dead bodies

The average amount of time males or females spent in the target corner did not change significantly after the addition of the filter paper containing linseed oil (Table 2, males: p = 0.460, paired t-test, N = 6; females: p = 0.451, paired t-test, N = 5).

Table 2. Time any individual spent in the target corner of the arena before and after the addition of the linseed oil. Males and females were tested in separate experiments. Total observation time = 25 minutes, Group size = 5 individuals, N = 11.

The average amount of time males or females spent in the target corner did not change significantly after the addition of the dead bodies (Table 3, males: p = 0.894, paired t-test, N = 6; females: p = 0.381, paired t-test, N = 5).

Table 3. Time any individual spent in the target corner of the arena before and after the addition of the intact dead bodies. Males and females were tested in separate experiments. Total observation time = 25 minutes, Group size = 5 individuals, N = 11.

4.4.6. Responses of males towards crushed bodies

In an observation period of 25 minutes, the average amount of time males spent in the target corner was significantly decreased by 6.4 ± 4.9 minutes (table 4, p = 0.003, paired t-test, N = 10) after the addition of the crushed male bodies.

Table 4. Time any individual spent in the target corner of the arena before and after the addition of the crushed male bodies. Total observation time = 25 minutes, Group size = 5 individuals, N = 10.

4.5. Discussion

After isolating individuals for three days, we observed a general tendency for these individuals to form groups during the observation period (see Table 1), which is in contrast to solitary-reared locusts that are less active and freeze when they sense movement in the arena (Simpson et al., 1999; Pener & Simpson, 2009). Furthermore, the amount of time any group member spent in groups was not influenced by brushing the wings of the target individuals with linseed oil (Table 1). Brushing the wings of the target locusts with linseed oil, however, affected the amount of time these individuals spent in proximity to other individuals in most trials (see Fig. 3). In contrast, brushing the wings with a clean brush in the sham operation experiment did not change the average group formation time significantly (see Table S2). This suggests that the reduced group formation time observed in males is caused by linseed oil and less likely by physical stimulation. Brushing the wings of the target individuals with linseed oil did not change the average distance covered by the target locusts (see Table S1), which suggests that the reduced group formation time observed in males is not caused by an overall reduction in the activity of the target locust. Linseed oil is rich in unsaturated fatty acids, and some of these acids (especially oleic and linoleic acid) have been described as necromones that are released by injured and dead insects. These necromones are known to trigger distinct behaviour patterns in other individuals of the same or different species (Wilson et al., 1958; Yao et al., 2009; Sun & Zhou, 2013). Therefore, we tested whether linseed oil could be used as an agent to control the aggregation behaviour of gregarious locusts by investigating the aggregation behaviour of members of both sexes in controlled experiments.

A reduced group formation time of males after the linseed oil application (see Fig. 2A and B) may be the result of necrophobic avoidance behaviour, which may be absent in females most likely because of oviposition pheromone release. Males in this study showed a necrophobic avoidance and tended to move away from the target individual that released “the smell of death” in order to avoid possible infections that can be transmitted by swarm mates or indicates an injury from a predator attack (Yao et al., 2009). This aversion to necromone fatty acids (especially oleic and linoleic acids) were reported in a large number of invertebrates (Aksenov & David Rollo, 2017). However, locusts are also known to cannibalize other swarm mates in situations where food is scarce. We have not observed cannibalistic tendencies in our study because all individuals were fed with fresh grass during the isolation period. An interesting observation made in the course of this study was that the target individuals were no longer involved in mounting behaviours after being brushed with linseed oil. This male-male 73 mounting behaviour is rather common in the absence of females, and our results indicate that the observed change in male behaviour was mediated by linseed oil. This result differs from that of Clancy et al. (2017), who observed an increase in the frequency of male-male mounting (MMM) behaviour in desert locust males suffering from Metarhizium acridum, a fungal infection. This reduction in MMM is similar to the effect caused by “phenylacetonitrile” (PAN, also known as benzyl cyanide), which is a mature male volatile that prevents males from being mounted (homosexuality) by other males (Seidelmann & Ferenz, 2002; Pener & Simpson, 2009). It is mainly released from the wings and the hind legs (Seidelmann et al., 2003) and acts as a strong repellent for mature males to hide a female from other competing males (a courtship inhibition pheromone) (Seidelmann & Ferenz, 2002; Pener & Simpson, 2009). Brushing the wings with linseed oil likely dilutes the concentration of PAN on the wings of the target male due to the high hydrophobicity of this pheromone. Therefore, we conclude that the repellent effect related to our treatment is mediated by fatty acids but less likely by PAN. However, Bashir et al. (2010) proved that PAN has a solitarising effect (anti-gregarisation) on the hopper bands and another recent study revealed that PAN serves as an antipredator defence in gregarious migratory locusts as it is converted into a hypertoxic cyanide (HCN) when they are under attack (Wei et al., 2019). Since mature males exclusively produce aggregation pheromones that are attractive to members of both sexes (Obeng-Ofori et al., 1994, 1998; Ferenz & Seidelmann, 2003), a reduction in male group formation caused by linseed oil may have important consequences for the formation and persistence of locust swarms.

We observed no significant difference in the average amount of time the target females spent in groups before and after the application of linseed oil (see Fig. 2C and D). In almost half of all trials, there was a significant increase in the average amount of aggregation time by 5 ± 0.6 minutes. This result can be attributed to the attraction between female individuals mediated by the oviposition pheromone (Ferenz & Seidelmann, 2003). Since the target females were treated after the control recording ended, this might lead to an increase of the amount of oviposition pheromone released in the arena with time. This consequently increases the time the females spent in groups even in the presence of fatty acids necromones. This female-female attraction ensures the spatial aggregation of egg pods, which increases the survival rates and supports gregarious cohesion among members of the next generation. Furthermore, it has been also shown that ovipositing females of desert locusts aggregate responding to a pheromone produced by alive or dead individuals in all development stages (Norris et al., 1970). In 30.8 % of trials performed with females in this study, brushed target females stayed away, were less

74 active and only walked on the borders of the arena. This may indicate that females carrying the smell of death seem to avoid being eaten by conspecifics because it is known that desert locusts show cannibalistic behaviour beginning in the fifth-instar stage, and especially adults may be regarded as an important source of proteins by conspecifics (Whitman et al., 1994; Niassy, 1996). Bazazi et al. (2008) studied collective motion and cannibalism in marching bands of desert locust nymphs and was able to show that individuals that were injured by conspecifics may suffer an increased risk of cannibalism. Since females have a higher demand for the proteins involved in egg development, they may respond to the linseed-oil-treated target individual in a different way than males.

To study possible attraction/avoidance behaviour of the desert locusts to necromones, we tested the time males and females spent in the presence of a stationary linseed oil target and dead bodies. While we found no difference in the average amount of time males and females spent in the target corner after the addition of the linseed oil or dead bodies (see table 2 and 3), there was a significant reduction in this parameter in the experiment performed with crushed male bodies as males spent less time next to them (see Table 4). A response to freshly dead individuals is rather unlikely since the responses to fatty acid necromones increased over time (Akino & Yamaoka, 1996; Yao et al., 2009; Aksenov & David Rollo, 2017). According to our results, responses of some genera of isopods were very weak to intact dead bodies (disease) compared to crushed bodies or body extracts (Yao et al., 2009). The latter indicates injury resulting from predation and swarm mates should avoid them.

Sex-specific responses to fatty acid necromones were also described in the cricket Acheta domesticus where females respond to body extracts less than males, as females might be less risk averse because they seek out singing males and explore oviposition sites (Aksenov & David Rollo, 2017). Another study investigated the repellent effect of various fatty acid necromones on cockroach males and females. In both sexes, the percentage of repellence was strictly dose dependent and the percentage of oleic acid repellence was more significant in cockroach males (70 %) than females (43 %) (Rollo et al., 1994). The results obtained from males in our study indicate that fatty acids from linseed oil may change the aggregation behaviour of desert locusts. A similar solitarising effect has been found in S. gregaria when nymphs were exposed to faeces of crowded locusts (Gillett & Phillips, 1977).

4.6. Conclusion

A novel botanical pesticide formulation uses linseed oil as the main component and was found to be highly effective against gregarious desert locusts as well as migratory locusts (Abdelatti & Hartbauer, 2020). Since linseed oil contains unsaturated fatty acids that have been shown to act as necromone cues in different insect species, it was of interest to determine whether this oil affects group formation in gregarious desert locusts. Brushing the wings of single gregarious desert locust males with this oil significantly decreased the amount of aggregation time they spent in male groups in the majority of the trials (Fig. 2). In contrast, most treated females either did not display alterations in aggregation time or the aggregation time increased (Fig. 2). These sex differences in aggregation behaviour may be explained by the release of different pheromones produced by adult males and females in the gregarious phase. Since a reduction in the amount of aggregation time among males leads to a reduction in tactile stimuli that have been shown to be highly gregarizing (Roessingh et al., 1998; Simpson et al., 2001), linseed oil seems to be a promising candidate agent for the control of aggregation behaviour in gregarious desert locusts, as it may even disrupt swarm formation once a certain percentage of individuals have come into contact with linseed oil in the course of botanical pesticide treatments. Therefore, fatty acids that act as necromones and are contained in linseed oil should be considered in pest management as previously suggested by Yao et al. (2009). Future studies need to reveal the origin of this behaviour by exposing males and females to different concentrations and types of necromones in controlled laboratory conditions.

Supplementary materials

Table S1. Distance covered by the target individual before and after the application of linseed oil. Males and females were tested in separate experiments. Total observation time = 25 minutes, Group size = 6 individuals, N = 26.

Table S2. Time the target individual spent in groups before and after sham operation. Males and females were tested in separate experiments. Total observation time = 25 minutes, Group size = 6 individuals, N = 24.

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List of Figures

Chapter Figures Caption Pages

(1) Fig (1) Preparation of the bushcricket in the neurophysiological 9 experiments.

Fig (2) Caricature for an effective treatment of locusts using a novel 11 botanical pesticide.

Fig (3) Setup and locust arena used in behavioural experiments. 13

(2) Fig (1) Oscillograms of the calling song of the two Mecopoda species 21 (reprinted with permission from Siegert et al. 2013).

Fig (2) Oscillograms of the acoustic stimuli used in SR experiments. 25

Fig (3) Example showing the phase relationship between the 2 kHz signal 27 and the syllables of the trill.

Fig (4) Percentage of TN-1 responses to pure tone signals broadcast at a 29 1 dB subthreshold with different amplitudes of white noise.

The mean percentage of the TN-1 responses of two individuals to Fig (5) repeated presentations of subthreshold pure tone signals at 30 various amplitudes of white noise.

The mean percentage of TN-1 responses to the pure tone signals Fig (6) broadcast at a 1 dB subthreshold together with different 31 amplitudes of the trill.

Fig (7) Within-individual TN-1 response variability and phase 33 relationships.

Fig (8) Spatial arrangement of signallers and a receiver between which 37 SR is possible.

Fig (S1) SR statistics. 37

(3) Fig (1) Hardening of the linseed-bicarbonate emulsion. 43

Fig (2) The mortality rate of desert locusts in the screening experiment. 51

Fig (3) Botanical pesticide experiment. 52

Fig (4) Beetle experiment. 54

Fig (5) Spray treatment of wheat seedlings with the botanical pesticide 54 emulsion.

Fig (S1) LC50 of the botanical pesticide formulation. 58

Chapter Figures Caption Pages

Fig (S2) Anti-feedant effect of the botanical pesticide formulation. 58

(4) Fig (1) Snapshot of the top view camera. 65

Fig (2) Sex differences in group formation after linseed oil treatment. 69

Fig (3) Time the target individual spent in male and female groups. 70

List of Tables

Chapter Tables Caption Pages

(1) Table (1) Taxonomy of orthopteran species that were studied in my 3 thesis.

Time any individual spent in groups of at least two (4) Table (1) individuals (including the target one) before and after the 67 application of linseed oil.

Table (2) Time any individual spent in the target corner of the 71 arena before and after the addition of the linseed oil.

Time any individual spent in the target corner of the Table (3) arena before and after the addition of the intact dead 72 bodies.

Time any individual spent in the target corner of the Table (4) arena before and after the addition of the crushed male 72 bodies.

Table (S1) Distance covered by the target individual before and after 77 the application of linseed oil.

Table (S2) Time the target individual spent in groups before and 78 after sham operation.

Scientific contributions

Articles

Aly, M. Z. Y., Osman, K. S. M., Mohanny, K. M. & Abd Elatti, Z. A. S. (2012). Indoor and outdoor controlling evaluation on the Subterranean Termite, Psammotermes hybostoma (Isoptera: Rhinotermitidae) Using Some Unordinary Natural Oils and Others. Egyptian Academic Journal of Biological Sciences -A- Entomology, 5(2), 175–189.

Abdelatti, Z. A. S. & Hartbauer, M. (2017). The heterospecific calling song can improve conspecific signal detection in a bushcricket species. Hearing Research, 355, 70–80. https://doi.org/10.1016/j.heares.2017.09.011.

Abdelatti, Z. A. S. & Hartbauer, M. (2020). Linseed Oil Affects Aggregation Behaviour in the Desert Locust Schistocerca gregaria—A Potential Swarm Disruptive Agent. Agronomy, 10(10), 1458. https://doi.org/10.3390/agronomy10101458.

Talks

Zainab A. S. Abdelatti & Manfred Hartbauer (2017). Signal detection in a bushcricket: does stochastic resonance improve it? 6th International Scientific Workshop for Egyptian PhD students and researchers, 27 April. Zainab A. S. Abdelatti & Manfred Hartbauer (2018): Biological agents against gregarious locusts. Three Minute Thesis Competition - 3MT organized by the European university network Coimbra Group and hosted by the Doctoral Academy Graz. 22 March. ZainabA. S. Abdelatti & Manfred Hartbauer (2018). Biological control of swarming locusts. Institut für Biologie, Uni Graz. 30 Mai. Zainab Abdelatti & Manfred Hartbauer (2018). Effect of linseed oil on the aggregation behaviour of gregarious locusts. Graduate meeting in Animal Behaviour of the German Zoological Society and Ethologische Gesellschaft e.V. German Zoological Society and Ethological Society in Hannover, 12-13 Juli.

Konstantinos Kostarakos, Zainab A. S. Abdelatti & Manfred Hartbauer (2019). A novel Bio- pesticide for the control of locust outbreaks. I.E.C.T. summer school on entrepreneurship, Wattens. 21.08.2019. Hartbauer, Manfred, Konstantinos Kostarakos & Zainab A. S. Abdelatti (2020). Alternative Treatments of Gregarious Locusts. Virtual practitioners conference on locust management 2020. 10.08.2020. Hartbauer, Manfred, Konstantinos Kostarakos & Zainab A. S. Abdelatti (2020). Alternative treatments of gregarious locusts. Konstanz. CASCB. 07.12.2020.

Posters

Zainab A. S. Abdelatti and Manfred Hartbauer (2017). Stochastic resonance in an acoustically communicating insect. 12th Göttingen Meeting of the German Neuroscience Society (36th Göttingen Neurobiology Conference), 22 – 25 March. Manfred Hartbauer and Zainab A. S. Abdelatti (2017). Stochastic resonance improves signal detection in a bushcricket. 16th Invertebrate Sound and Vibration meeting, Rauischholzhausen, 14-17 September. Manfred Hartbauer and Zainab A. S. Abdelatti (2019). A novel bio-pesticide to control locust outbreaks. 13th International Congress of Orthopterology in Morocco.

Patents

Hartbauer, M. & Abdelatti, Z.A.S. (2018). Pesticidal compositions for pest control, Application number: EP18162806.6A. European patent publication number: EP3542630A1.

Hartbauer, M. & Abdelatti, Z.A.S. (2019). Pesticidal compositions for pest control, Interntional application number: PCT/EP2019/056709. International publication number: WO2019/179945A1.

Hartbauer, M. & Abdelatti, Z.A.S. (2021). Pesticidal compositions for pest control, Application number: EP19711361.6A. European patent publication number: EP3768087A1.

Public media articles

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Bio-Pestizid gegen Wanderheuschrecken an der Uni Graz entwickelt. In: Der Standard. 03.05.2018. Medientyp: Internet.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Duftöl gegen Heuschrecken: Uni Graz entwickelte Biopestizid. In: Salzburger Nachrichten. 03.05.2018. Medientyp: Printmedium.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Universität Graz patentiert Bio-Pestizid. In: oe-journal.at. 03.05.2018. Medientyp: Internet.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Natürlicher Schutz vor Heuschrecken. In: Neue Vorarlberger Tageszeitung. 04.05.2018. Medientyp: Printmedium.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Bio-Pestizid gegen Heuschrecken patentiert. In: Kleine Zeitung, Helle Köpfe. 04.05.2018. Medientyp: Printmedium.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Biologisches Insektizid gegen Heuschrecken patentiert. In: TopAgrar. 09.05.2018. Medientyp: Internet.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Pflanzenöle gegen Heuschrecken. In: Kronen Zeitung. 13.05.2018 - 13.05.2018. Medientyp: Printmedium.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Mit “Gewürzmischung” gegen Heuschrecken. Steirische Wirtschaft. 30 Unternehmer. Nr. 16 – 25 Mai 2018. Medientyp: Printmedium, Internet.

Manfred Hartbauer & Zainab A. S. Abdelatti (2018). Leinöl, Kümmel und Co als Schädlingskiller. Der Standard (ÖkoStandard), 21 Juni 2018. Medientyp: Printmedium.

Zainab A. S. Abdelatti & Manfred Hartbauer (2018). Heuschrecken, rauschfreie Bienen und Bio-Pestizide. In: Spirit of Styria. 01.07.2018 - 30.10.2018. Medientyp: Printmedium.

Manfred Hartbauer & Zainab A. S. Abdelatti (2019). Was kann man von Heuschrecken lernen? In: LetsDogAboutScience. 08.10.2019. Medientyp: Internet.

Hartbauer, Manfred, Konstantinos Kostarakos & Zainab A. S. Abdelatti (2020). Wissenswert: Vorbild Natur. In: ORF - STMK Heute. 03.02.2020. Medientyp: Fernsehen.

Hartbauer, Manfred & Zainab A. S. Abdelatti (2020). What we found when we tested a botanical pesticide to combat locust invasions. The Conversation UK, March 16, 2020.

Statutory Declaration

I herewith declare that I have completed the present thesis independently making use only of the specified literature and aids. Sentences or parts of sentences quoted literally are marked as quotations; identification of other references with regard to the statement and scope of the work is quoted. The thesis in this form has not been submitted to an examination body and has not been published.

Contribution to the publications of my thesis:

1. Abdelatti, Z. A. S. & Hartbauer, M. (2017). The heterospecific calling song can improve conspecific signal detection in a bushcricket species. Hearing Research, 355, 70–80. https://doi.org/10.1016/j.heares.2017.09.011. Chapter 2: 65%. I performed all experiments (except acoustic stimuli generation and sound calibration), data evaluation (except modelling sound propagation and statistics), prepared figures and wrote the draft of the manuscript. 2. Hartbauer, M. & Abdelatti, Z.A.S. (2018). Pesticidal compositions for pest control, Application number: EP18162806.6A. European patent publication number: EP3542630A1. https://patents.google.com/patent/EP3542630A1/en. Chapter 3: 10%. I contributed to four ideas out of ten, performed all experiments and data analyses, prepared tables and figures, and revised and wrote parts of the patent draft. 3. Hartbauer, M. & Abdelatti, Z.A.S. (2019). Pesticidal compositions for pest control, Interntional application number: PCT/EP2019/056709. International publication number: WO2019/179945A1. https://patents.google.com/patent/WO2019179945A1/en. Chapter 3: 10%. I contributed to four ideas out of ten, performed all experiments and data analyses, prepared tables and figures, and revised the draft of the patent. 4. Abdelatti, Z. A. S. & Hartbauer, M. (2020). Plant oil mixtures as a novel botanical pesticide to control gregarious locusts. Journal of Pest Science, 93(1), 341–353. https://doi.org/10.1007/s10340-019-01169-7. Chapter 3: 80%. I established the experimental set-up, performed all experiments, data evaluation and analyses, prepared figures, wrote the draft of the manuscript, and replied to the comments of the reviewers. 5. Abdelatti, Z. A. S. & Hartbauer, M. (2020). Linseed Oil Affects Aggregation Behaviour in the Desert Locust Schistocerca gregaria—A Potential Swarm Disruptive Agent. Agronomy, 10(10), 1458. https://doi.org/10.3390/agronomy10101458. Chapter 4: 80%. I established the experimental set-up, performed all experiments, data evaluation and analyses, prepared

tables and figures, wrote the draft of the manuscript and replied to the comments of the reviewers. 6. Hartbauer, M. & Abdelatti, Z.A.S. (2021). Pesticidal compositions for pest control, Application number: EP19711361.6A. European patent publication number: EP3768087A1. https://patents.google.com/patent/EP3768087A1/en. Chapter 3: 10%. I contributed to four ideas out of ten and revised the patent draft.

Graz, June 2021 Zainab A. S. Abdelatti

Acknowledgement

First of all; the prayerful thanks to merciful Allah who gives me everything I have.

I would like to thank the Egyptian ministry of higher education, cultural affairs and mission sector for providing funding for my PhD external scholarship.

I am deeply indebted to my “Doktorvater”, Assoc. Prof. Manfred Hartbauer, for suggesting the research work, his kind supervision, constructive criticism, faithful encouragement, valuable advice, great help in solving any problem and unlimited guidance during the progress of my doctoral study until the preparation and writing of this thesis. It has been a great honour to be his student.

A very special thank is dedicated to Prof. Heiner Römer for his mentoring and support. Many thanks to Prof. Boris P. Chagnaud for his critical reading and valuable comments on chapter 4.

I am particularly grateful to Maria-Theresia Poschner and Sara Crockett for English proofreading.

Grateful appreciation is also expressed to all colleagues of the Institute of Biology for their support in professional matters and also for the nice moments during work. I am grateful for the support I have received from working group members. Special thanks to Kostantinos Kostarakos, Björn Thorin Jonsson, Birgit Rönfeld, Bettina Erregger, Ruth Gutjahr, Ismene Fertschai, Arne Schmidt, Erik Schneider, Michaela Bodner and Wolfgang Gessl.

I am deeply grateful to Isabel Krobath for her nice and friendly company in the lab and Sylvia Schäffer her kind friendship.

My thanks have to be extended to Valerie Kornschober, Bianca Pichler-Their and Caecilia Grabenhofer

I am indebted forever to My Father, My Mother, My Brother and My Sisters for their support and continuous encouragement. Finally, lovely and special thanks to My kids Nada, Ali and Sara and My Husband Osama N. A. Esmail, assistant professor of electrical engineering – Al- Azhar university, for his unlimited help, endless patience, emotional support and continuous encouragement.

Zainab Ali Saad Abdelatti

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