Master Thesis Master's Programme in Applied Environmental Science, 60 credits

Investigation of sex-based differences in responses to artificial light of the greater wax moth (Galleria mellonella)

Degree Project in Environmental Science, 15 credits

Halmstad 2021-07-02 Prasoon Singh HALMSTAD UNIVERSITY Investigation of sex-based differences in responses to artificial light of the greater wax moth (Galleria mellonella) Prasoon Singh

Halmstad University, School of Business, Engineering and Science, Master's Program in Applied Environmental Science

Project in collaboration with Research Institutes of Sweden AB (RISE)

Supervisor: Annika Jägerbrand, Halmstad University Supervisor: Petter Andersson, Calluna AB

ABSTRACT

Artificial light at night (ALAN) is becoming a greater threat to nocturnal species. Aside from the overall increase in light output, replacing outdated monochromatic street lighting with light-emitting diode (LED) lights with a broad emission spectrum could raise this issue more. However, studies evaluating the effect of artificial lights on nocturnal species, such as moths, are scarce. This study examines any variations in moth attraction as well as any other sex-based behavioural differences (mating behaviour – wing fanning, trajectory-circular movement, overall movement time) between male and female moths in the presence of warm-white light. This study used warm-white light LED (2718 K) and Galleria mellonella moth, reared in the lab and completed the experiment under standardised conditions in an enclosed setting. I found the male moths (100%) were significantly (p = 0.024) attracted to warm-white light LED compare to female moths (37%). While other behaviours such as wing fanning, circular movement, overall moving time, and favoured light illuminance for rest weren't significantly different (p > 0.05). In the future, it will be important to investigate the effects of warm-white light LED with different lower CCT (< 2718K) on moths, as well as the sex differences in their behaviour both in a controlled and an open environment. This will help authorities to decide on outdoor lighting systems in different countries and continents.

Keywords: Light pollution, Galleria mellonella, warm-white light, moths behaviour, attraction.

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Table of Contents Introduction ...... 1 Background ...... 1 Literature Review ...... 1 Artificial light can play a part in moth population decline ...... 2 Ecosystems function disrupt due to artificial light at night ...... 3 Effect of artificial light spectral composition on moth ...... 3 Effect of artificial light at night on moth sex ...... 4 Aim and goal of this study ...... 5 Methods and materials ...... 5 Study species ...... 6 Experimental design ...... 7 Experiment procedure ...... 8 Data collection ...... 8 Statistical analysis ...... 10 Results ...... 11 Discussion ...... 14 Conclusion ...... 16 Acknowledgement ...... 16 References: ...... 17

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Abbreviation

ALAN Artificial Light At Night BLC Blue Light Content

BGLC Blue Green Light Content CCT Correlated Colour Temperature

CIE International Commission of Illumination CMOS Complementary Metal Oxide Semiconductor EOS Electro-Optical System

HPS High-Pressure Sodium

IDA International Dark-Sky Association LED Light-Emitting Diode

LMK Let Me Know LPS Low-Pressure Sodium

UV Ultra-violet UNOOSA United Nations Office for Outer Space Affairs

3 Introduction

Background

For millions of years, life on Earth has responded to predictable light cycles, natural day- night cycles. Natural day length variation is observed due to the season and the lunar cycle. Early in the 20th-century till the present, 2021, anthropogenic light sources, such as street lights, playground lights, car parks, airports, and vehicle headlights, for human needs have altered the night light's natural patterns. (Longcore et al., 2004; Degen et al., 2016). It is estimated that approximately 22 % of the globe encounters a light-polluted sky (Bruce-White et al., 2011; Falchi et al., 2016), and artificial brightness increased 2.2% between 2012 and 2016 (Kyba et al., 2017). Hölker et al. (2010) found that the average amount of artificial light at night (ALAN) rises by 2 to 20% per year, depending on the geographical location. Consequently, ALAN may have an adverse impact on the ecological and evolutionary implications on flora and fauna population (Rich et al., 2006). For instance, it alters the behaviour of several nocturnal species, including moths (Nowinszky, 2004; Salmon, 2006) ALAN is thought to be a severe hazard to nocturnal animals, particularly insects, who are vulnerable and whose biodiversity is being impacted (Macgregor et al., 2016; Owens & Lewis, 2018). Although numerous studies revealed the negative physiological and behavioural effects of ALAN on nocturnal insects (Botha et al., 2017; Owens & Lewis, 2018; Owens et al., 2020), further exploration into the effects of ALAN is needed.

Literature Review

The current understanding of the effect of ALAN on insect behaviour is summarised in Table 1. Table 1: Effects of ALAN on nocturnal species by various authors according to different Perspectives Response to ALAN Example taxon Result Reference

Sex-biased phototaxis Winter moth Effective sex ratio in insect Van Geffen et al., populations is skew due to 2015 excessive attraction to ALAN.

Altered recognition Firefly beetles ALAN obscures visual signals Owens and Lewis, used by insects to locate and court 2018 potential mates. Sex-based phototaxis Small ermine moth, Sex disproportionate, male more Altermatt et al., 2009 scorched carpet attracted to ALAN moth Mate finding Glow‐worm Female capacity to attract males Stewart et al., 2020 is hampered by ALAN, with the effect rising with light intensity.

1 Mate finding Cricket Chronic night-time exposure to Botha et al., 2017 bright light can have an effect on reproductive behaviour and mate selection. Mate finding Glow‐worm ALAN has a significant negative Broeck et al., 2021 effect on glow-worm mate finding success. Sex-based phototaxis All type of moth Niether male nor female has Degen et al., 2016 differences in terms of attraction rate or radius. Positive phototaxis Insect pollinator Insects stuck near or under Knop et al., 2017 artificial lights do not forage normally. Positive phototaxis Macro Moths Insects trapped in the orbit of Somers-Yeates et al., artificial lights are unable to 2013 move forward. Temporal disorientation Crickets ALAN causes rapid, delayed, or Donner et al., 2018 growth retardation in juvenile insects. Earlier life stages Common moth ALAN disrupt larval Boyes et al. (2021) development and pupal diapause. Foraging Tree weta and cave Reduce the foraging time and Farnworth et al., weta increase the risk of starvation. 2018

Artificial light can play a part in moth population decline

Approximate 60% of invertebrates are nocturnal (Hölker et al., 2010), including approximately 80% of Lepidoptera with 20,000 species of butterflies and 140,000 species of moths (New 2004; Kawahara et al., 2018). Moth population tracking programs in Western Europe have revealed a drastic decline in moth populations (Groenendijk et al., 2010). For example, between 1968 and 2002, more than 20% of the 337 moth species studied in Great Britain reported a decline of more than 30% every 10 years (Conrad et al., 2006). Similarly, Van Langevelde et al. (2018) studied nocturnal species in the Netherlands for 30 years (1985- 2015) and found a significant decline in nocturnal species compared to diurnal species due to artificial light.

The reasons for moth population declines are poorly understood. Land-use changes (Fox et al., 2014), climatic changes, and chemical pollution have been suggested as possible causes of it (Groenendijk et al., 2010; Fox 2013). However, there is no actual evidence for such a causal relationship. Moths are attracted to light sources (phototaxis). They are also nocturnally active, so light pollution is often thought to be one of the contributing causes of moth population declines (Groenendijk et al., 2010; Fox, 2013; Van Langevelde et al., 2018). A meta-analysis by Boyes et al. (2021) shows that ALAN disrupts larval development and pupal diapause. Thus, ALAN is suspected of having substantial negative impacts on nocturnal moth populations. ALAN can reduce nocturnal prey moth foraging time and increase the risk of starvation (van Geffen et al., 2014). Farnworth et al. (2018) studied two

2 weta species (nocturnal) and found 72-85 % of foraging behaviour reduced in the presence of artificial lights. Artificial light has the ability to drive some moth with small and dispersed populations to extinction (Kalinkat et al., 2021).

Ecosystems function disrupt due to artificial light at night

ALAN may also alter cross-ecosystem fluxes at local and global scales (Manfrin et al., 2017). ALAN has the potential to increase overall environmental pressure on insect populations. That is especially significant in agroecosystems where insect communities provide essential ecosystem services such as natural pest control, pollination, soil structure and fertility conservation, and nutrient cycling (Manfrin et al., 2017; Grubisic et al., 2018). Reduced moth abundance has a detrimental impact on nocturnal pollen transport, which can have cascade effects on plant and insect herbivore populations (Fox, 2013; Macgregor et al., 2017). Therefore, it is likely that the moth pollination process is affected by ALAN (van Langevelde et al., 2018; Macgregor et al., 2017).

Compared to dark controls, a field experiment using LED lamps showed that illumination decreased nocturnal visits, which reduces its forage, and reduced pollination success (Knop et al., 2017; Farnworth et al., 2018) results in decreased fruit developments. The adverse effects of ALAN may not be limited to nocturnal pollinators and the plants they pollinate but may also spread to the diurnal pollinator population, putting more strain on this community overall. The adverse effects of ALAN on nocturnal pollinator populations have negative implications for plant reproductive success (Knop et al., 2017). Hence, these effects can cascade from the nocturnal to the diurnal pollinator population. Additionally, ALAN could also influence inter-specific interaction; for example, ALAN may indirectly alter the predator-prey relationship (Loncore 2004; Boyes et al., 2021). The overabundance of insect prey at ALAN sources and under them can lead to an increase in scavengers such as spiders and snails (Manfrin et al., 2017).

Effect of artificial light spectral composition on moth

The impact of ALAN on insects depends on the light intensity and the wavelengths of the light (Longcore et al., 2015; Donners 2018). Light is composed of electromagnetic radiation at different wavelengths. Depending on the wavelength and photosensitive cell, the light has a different correlated colour temperature (CCT), specification of a light source's colour appearance, linking its colour to the colour of light from a reference source when heated to a specified temperature (Borbély et al., 2001)). The wavelengths of visible light are ranging from violet at short wavelengths to red at long wavelengths.

Light is not always viewed in the same way by other species (Briscoe et al., 2001). For example, the human eye is able to detect a narrow range of wavelengths between ~380 – 780 nm, while most insects can detect only 300 to 650 nm (see fig.1 ) wavelengths of lights (Arikawa et al., 1987).

3 Figure 1: Spectrum composition of light with visible human light (~380 - 780 nm) and visible insect light (300 – 650 nm).

Longcore et al. (2015) showed that all insects are not attracted to all light sources with the same colour temperature. In general, long-wavelength light (e.g., amber and red light) do not attract moths, while short-wavelength light (e.g., UV, blue and green light) do strongly attract moths (van Langevelde et al., 2011; Barghini et al., 2012). For instance, high-pressure sodium (HPS) lights have been shown to attract moths due to the presence of ultraviolet (UV) wavelengths, while low-pressure sodium (LPS) lights of the same strength but not emitting UV light attract fewer moths (Rydell 1992; Frank, 2006). The landscape of nocturnal illumination is currently moving away from monochromatic street lighting, such as Low-Pressure Sodium (LPS) lamps, and toward white LED (broader spectrum) street lighting (Elvidge et al., 2010). Most insects can distinguish UV, blue, and green light, which have short wavelengths and high frequencies (see fig. 1), and they are highly attracted to them. It is possible to minimise insect attraction by changing the spectral composition of white light. Therefore the use of LEDs with very low or no blue and green emission can help us to reduce the number of attractions. LEDs have various colour temperatures, for example, warm white ((2,700–3,000 K), cool white (4,000–5,000 K), and neutral white (~4,000 K) (IDA, 2016).

Effect of artificial light at night on moth sex

ALAN has been shown with the available literature to interfere with the overall moth population sex ratio and behavioural differences. For instance, one sex may be more attracted to light. Sexes may have different behaviour patterns (Altermatt et al., 2008; Garris et al., 2010). Some moth species are strongly attracted to artificial lights, while others seldom visit these light sources despite living in close proximity (Scheling, 2007). Many field studies have found that males are more likely than females to get caught in light traps (Altermatt et al., 2009; Owens and Lewis, 2018). ALAN can change the intra-specific interaction of moth (Loncore et al., 2004), i.e., disrupt reproductive behaviour, pheromone signalling (van Geffen et al., 2015; Boyes et al., 2021),

4 or courtship and mating behaviour (Botha et al., 2017). Broeck et al. (2021) studied the effects of artificial light on moths. They found that white LED light reduced males capacity to detect females at previously unreported low light levels, whereas colour temperature had no effect. Nowinszky et al. (2015) analysed 32 macrolepidoptera species by light trap and found males moths captured more, i.e., light disproportionate moth sex.

Up until now, no previous study has examined sex-based differences in response to artificial light in a fully controlled experiment. However, detailed knowledge on sex-based differences to ALAN is essential to give correct recommendations for avoiding detrimental impacts on insects, such as road lighting. This is because if one sex is more sensitive to ALAN, recommendations must be adjusted to incorporate this variation to be effective fully. Consequently, this study will result in new knowledge, which will help make new recommendations for avoiding negative impacts on insects from ALAN. This is highly relevant on a national level in Sweden, but it is also important for forthcoming international recommendations on mitigating the ecological impacts of ALAN.

Aim and goal of this study

The main objective of this study is to investigate if there are sex-based differences in responses to the artificial light of the greater wax moth (Galleria mellonella). The study aims to answer the following research questions:

1. Is there a difference in wax moth attraction to warm-white light based on sex? 2. Is there any sex-based behavioural (i.e., wing fanning, trajectory movement, overall moving time) difference between male and female wax moths exposed to warm-white light? Methods and materials

The Swedish Transport Administration provided financial assistance for this study under project number TRV 2020/86363. Figure 2 depicts the study's step-by-step process. Starting with deciding on a topic, obtaining information through a comprehensive literature study, and finally completing the study with a moth experiment. During the process, a thorough literature search was carried out, using the online scientific databases Wiley online library and Web of Science. When searching by topic, the keywords used were "artificial light at night" or "outside lights" or "LED" were used, as well as "nocturnal species" or "moth behaviour" or "flight-to-light behaviour" or "moth decline" or "Galleria mellonella". The searches covered between the years 2010 to present, 2021, all document types, open access and exclusively English documents. There were 103 hits in total from the systematic searches. Titles were screened for relevance, then the abstracts were reviewed for a second filter, and complete texts were only studied if they were considered appropriate to the topic of this work.

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Figure 2: A diagram of the thesis approach

Study species

A greater wax moth (Galleria mellonella) was used as a model species. Galleria mellonella is a cosmopolitan and ubiquitous pest of the honeybee (Kwadha et al., 2017). The greater wax moth flies from May to October in temperate parts of the earth, such as the Netherlands and Belgium (Abidalla, 2017). G. mellonella has a distinct sexual dimorphism, as shown in figure 3. Moth's sex was determined by examining external morphology. Female G. mellonella is longer than males, and female individuals have an average body length of 15-20 mm with 31 mm wingspan and a weight of 120-169 mg (Williams, 1997; Ellis et al., 2013). Male moths are smaller and have a lighter colouration than females. The male moth has distinctly scalloped apical margins of the forewings (fig. 3B). Further, the female wax moth has a pointed nose by the forward projection labial pulp (see fig. 3C), whereas the male has snub-nosed (see fig 3A). In the female moths, the ninth to eleventh abdomen segments have been modifying into ovipositor (Williams, 1997). Both sexes vary considerably in size and colour according to larvae diets and developmental duration (Kwadha et al., 2017).

Figure 3: Greater wax moth (Galleria mellonella) - (A) male moth with snub-nosed (B) male moth with scalloped apical margins (C) Female moth under a glass jar with a pointed nose.

6 Experimental design

The experiment was carried out in a light-tight rectangular box, 60 x 45 x 90 cm (W x H x L) (fig.4), developed by the staff at RISE. It has several lights, but just one warm-white LED sphere-based luminance source with a mechanical shutter mounted on one of the box's short sides was used to examine moth behaviour, while the rest were infrared lights. The light source was connected to a power supply with several controllers for controlling the intensity of light. Video recordings captured the operation during the experiment. Infrared LEDs were used to light the objects inside the box during the dark, which the camera was supposed to record. Inside the enclosure, a photometer (Hagner S4) was used to measure the light intensity, which was recorded horizontally as 0.07 lux and vertically. It was 0.22 lux, and a thermometer was used to measure the temperature and humidity. The entire experiment was conducted in a controlled environment with a temperature of ~23°C.

Figure 4: Experimental set-up. Experimental box connected with power supply and laptop connected with the camera to capture videos inside the box.

The spectral power distribution of the warm-white light sources was measured with a spectrometer (see fig. 5 b). The warm-white light had a CCT of 2718K and a colour rendering index (Ra) of 83.0. Through the use of an LMK Mobile Advanced imaging illumination photometer (based on a Canon EOS 550D) and the LMK laboratory Software 4 Program version: 20.11.11 (Techno Team Bildverarbeitung GmbH, Ilmenau, Germany), photos of the luminance distribution were taken and created. With the support of a software application, the LMK Mobile Advance can directly translate images into luminance values. The Canon EOS 550D is a digital single-lens reflex camera with an 18.0 effective megapixel CMOS sensor, a working temperature range of 0–40°C, and an operating humidity of less than 85%. The output of it is shown in figure 5a.

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(a) (b) Figure 5: a) Illuminance distribution inside the experimental box. Source photo taken by Annika Jagerbrand. b) The spectral power distribution of the warm-white LED light source used, measured by GL SPECTIS 1.0 touch + FLICKER device.

The light distribution within the experimental box during the light condition was depicting in the image above (see fig. 5a). The area behind the camera is completely dark in this picture. Because the camera was not calibrated to the light source for this experiment, the values are not accurate. As a result, the luminance photo has a false colour presentation, but it shows true illuminance distribution. A logarithmic scale was utilised in this light illuminance measurement.

Experiment procedure

Live specimens of moths were acquired at RISE, Borås, Sweden. Commercially available G. mellonella was bred from purchased larvae rearing in the laboratory. Larvae were kept at ~ 24 °C in the box, and the relative humidity (RH) was 30%. One to six days old G. mellonella were used in the experiment. All moths were in the dark for at least 12 h before being used in the experiment. The moths employed in the experiment were first kept in the dark chamber for a longer length of time. They moved from the storage box to the experimental box with the help of featherweight forceps to pick and a compact red light source to locate the moth in the dark. When the moth had been withdrawn from the dark box, it was placed in a column positioned in the centre of the experiment box and made sure only one moth was present inside the experiment box. The experiment lasted about ten minutes. Identical processes were used in light and dark experiments. For each experiment, a new moth was used. Throughout the experiment, two video cameras recorded the moth activity within the experiment box. With the help of a magnifying glass, the moth's sex was determined after the experiment was completed.

Data collection

Data was initially obtained in the form of video. Later during the statistical analysis, video data was transposed to quantitative data through various viewing parameters and with the

8 help of a stopwatch to measure the time. I converted the video data into moth behavioural activities, and the same is represented in table 2.

Table 2: Data collection chart with response type and recording observation

Variable/Behaviour Explanation Response Observation Attraction Movement towards the Categorical Flight-to-light movement light source (Yes/No) Circular Motion Rotation around a Yes/No Observe the trajectory point behaviour Wing Fanning Moth is seeking Yes/No Observe wing fanning attention Number of circular Motion Number of rotation Number Number of circular movement around a point Total wing fanning time Duration of seeking Time in Total duration of wing fanning attention seconds during the experiment Overall movement time Active action duration Time in Total duration of movements of moth seconds during experiment Rest on light illuminance Inactive states of moth Resting light Preferable light illuminance by illuminance moth for rest

I found one very interesting behaviour: Resting on the light illuminance, as most moths were trying to find a place to rest after a certain time of activity. With reference to the light illumination distribution (see fig. 5a) in the box, male and female moths show different resting patterns on such illuminance. The box has a variation in light illuminance depending on the distance from the source of light. To explain the position of the moth where they stay, I divided the experimental box into six walls and four corners (fig. 6).

Figure 6: Illustration of the open experimental box. Named the wall as W1, W2, W3, W4, W5, W6, and corner as C1(between W1 & W2), C2(between W2 & W3), C3(between W3 & W4),

9 C4(betweenW4 & W1). W1 is the wall with the source of light (L), and W5 and C (central) is the ground surface.

The experiment included 27 samples, including 14 samples (female: 8, male: 6) in the warm- white light presence (light) condition and 13 samples (female: 8, male: 5) in the warm-white light absence (dark) condition (see Figure 7). The sample was taken based on sex. Out of 16 females, two distinct females in different scenarios, presence and absence of light, did not show any response at all. Although these moths were alive. So a total of 12.5% from the collected sample in each of the cases (present & absent of light) had no response, and the rest had a proper response.

Figure 7: The percentage number of females and males in the experiment (N = 27).

Statistical analysis

Data collected from different behaviours like attraction, wing fanning, wing fanning time, circular movement, the total number of circular movement, overall moving time and rest on the light illuminance were previously checked for normality distribution (Kolmogorov- Smirnov test ). In statistics, parametric t-test (two groups) used on normally distributed data (p ≥ 0.050). In contrast, the Non-parametric Mann-Whitney U-test (2 groups) or Kruskal- Wallis (≥ 2 groups) used for not normally distributed data (p<0.050). Therefore, I used Mann-Whitney U-test for overall moving time, and for the rest of the other observations, I used the t-test as the recorded values were normally distributed. I segregated the collected data on the basis of sex (male/female) and use behavioural data as independent variable and light condition (light/dark) as a group variable.The significance for all p values was p< 0.050.

All statistical analyses performed in IBM SPSS statistical version 27, and for graphical representation, I used SPSS and Ms-excel 16.42, Microsoft 365 subscription package. SPSS is an data sensitive statistical program. The current experiments did not contain a dataset that was really large enough for a thorough statistical treatment. So, in the result part I used the real raw data and excel for graphical representation and comparison. While I uses SPSS tests to just verify the raw data statistically, but it shows a deviatiation in interpretation from raw data.

10 Results

Attraction Statistical result shows (see table 3), light condition had a significant effect on the attraction of male moths (t-test, t = 2.714, df = 9, p =0.024) compared with female moth (t-test, p = 0.847). In figure (8a), warm-white light attracted 100% of males, and in the dark, only 40% of male moth moving to the light source side (W1 in fig. 6), while an equal percentage of females (37.5%) were attracted to warm-white light presence and absence (see fig. 8b).

Table 3: Sex-based parametric t-test result of moth behaviour Group Sex t-value df p-value

Attraction Female -0.197 13 0.847 Male 2.714 9 0.024*

Wing Fanning Female 0.612 12 0.552 Male 0.208 9 0.840

Circular Movement Female 1.044 12 0.317 Male 0.980 9 0.300

Number of circular movement Female -0.363 12 0.723 Male -1.080 9 0.285

Wing fanning time Female -0.137 12 0.893 Male -0.228 9 0.821 * Shows the significant value p <0.05.

Circular movement and number of circular movement In warm-white light presence and absence, neither male nor female moths showed any significant differences in trajectory behaviour such as circular movement and number of circular movement (t-test, p = 0.353, p = 0.317, p = 0.723, p = 0.285) (see table 3) respectively.

The graph (8a, 8b) represents 50% of male moths showing circular movement in the presence of warm-white light, while 20% show such movement in the absence of light. In the case of the female moth, it also had similar results during the presence and absence of light. In the presence of light, 50% of female moths show a circular movement, while in the absence of light, 25% of female moths have such movement. In figure 8c, male moths showed more number of circular movement in warm-white light presence than in the absence of light. Females also showed more number of circular movement in the presence of light than in the absence of light.

11 Wing fanning and wing fanning time in second Statistical result shows, neither male nor female moths were show significant difference in their wing fanning behaviour and wing fanning time in second in the warm-white light presence or absence (t-test, p = 0.8, p = 0.0552, p = 0.893, p = 0.821) respectively (see table 3). However, in the graph (fig, 8a and 8b) in the dark, slightly more female moths (75%) showed wing fanning behaviour than warm-white light presence (62.5% female moths). In comparison, 40% of male moths showed wing fanning behaviour in the absence of light and 33% male moth presence of warm white light—similarly, overall wing fanning time in second (total duration of wing fanning during the observation period). Male and female moths showed slightly more wing fanning time in warm white light presence than in the absence of light (see fig. 8d ).

Figure 8: Moths behaviour in ALAN (warm white light, 2718 K) sex-based. (a) Percentage of male moth behaviours in presence and absence of light, (b) Percentage of female moth behaviour in presence and absence of light, (c) Number of circular movement in male and female moth in the presence and absence of light, (d) Wing fanning time in second of moths during different light conditions based on sex.

Overall moving time in second

12 Mann-Whitney U-test result shows (see table 4) that neither male nor female moths showed any significant difference between warm-white light presence or absence (U- test, p = 0.410 and p = 0.848).

Table 4: Sex-based Mann-Whitney U-test result of overall moving time in seconds. Z p Overall moving time in seconds Female -0.192 0.848 Male -0.823 0.410

In figure 9, male moths show higher overall moving time when warm-white light was the presence, i.e., more active (moving), but statically it was not significant. In contrast, the female overall moving time was not affected by light presence and absence of light.

Figure 9: Mean value of total overall move timing of moths during different light conditions on the basis of sex.

Rest on light illuminance Considering the false colour distribution and illumination value depicted in figure 5a and box side nomenclature in figure 6. In presence of warm white light, 50% of male moths prefer to stay on Green colour illuminance inside the box. Which is distributed over different wall of box. While rest prefer to stay equally on black, blue and red illuminance. However female moths in presence of light, prefer to stay equally both in black and green light illuminance. Black illuminance is present at the corner of the box.

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Figure 10: Frequency of male and female moths rest on the different distribution of light illuminance. Discussion

For the experiment, evaluated several behaviours of G. mellonella in an enclosed setting under standardised conditions. To eliminate confounding effects of the environment, used moths were reared from larvae or raised in the lab. This experiment corroborated the observation of sex-based difference in moth all behaviour. Furthermore, the findings were consistent with both sexes of the same moth genus. It is the first single experimental study that I heard of that looked at multiple moth behaviours under completely controlled conditions in the presence of artificial light.

During this analysis, I had two research questions that I attempted to answer through my lab experiment. The first is to see - Is there a difference in wax moth (Galleria mellonella) attraction to warm-white light based on sex? Compare to male and female moths attraction (fight-to-light) behaviour on warm-white light and dark control treatment. With the study, it appears that warm-white light, CCT 2718 K, at night significantly attracts male moths compared with female moths. Similar results were recorded on a small ermine moth (Yponomeuta cagnagella) and a scorched carpet moth (Ligdia adustata) with TL 6W/05, CCT UV; Philips, as the source of light (Altermatt et al., 2009). Thus, individual moth behaviour, physiology, or processes can be affected by ALAN, which could affect moth populations. As my result also show significant male moths attracted to a light source which may be a cause of sex disproportion. Light exposure is often cited as one of the causes of moth declines, despite the lack of direct evidence for such a connection to date (Fox, 2013). But we have two supportive reasons for such a statement (i) Direct mortality may result from a moth colliding with a hot bulb or from fatigue if it continues to circle the light. (ii) Predation of moths, which are amplified in the presence of street lights. Adult moths are only adults for a small portion of their lives (Boyes et al., 2021) and adults are responsible for the

14 reproduction and dispersal of species. As a result, ALAN has a disproportionately high chance of affecting moth populations by processes that affect adults.

Second, in the presence of warm-white light, observe other sex-based moth behaviour, like, circular movement, wing fanning and overall moving duration. With the experimental result, in light, more male and female moths display circular movement and a greater overall number of circular motions by individuals. Moreover, during observation, I found that with reduced distance from illumination, moths show a high speed to move away from the source of light—a similar result described in Gaydecki, 2018 automated analysis of insect trajectories behaviour. Usually, a moth follows a linear trajectory, but some of the moths follow a linear as well as circular trajectory. With the observed record, it appears that a higher number of moths start to follow a circular trajectory in the presence of light.

Further, in the presence of light, it is observed that several individuals moths start doing a higher number of circular movements, which makes the moth trajectory more complex in the presence of light compared to the absence of light. Although, the statistical outcome found in the result didn't show any significant difference in moth trajectory under different light conditions (presence & absence). This might be because of less data availability. But with the observation result (see fig. 8a, 8b), we can see the difference.

Female Galleria mellonella produces a wing fanning display in response to male G. mellonella releasing vast quantities of a distinctive scent combined with bursts of ultrasonic calls (Jones et al., 2002). According to this research, a high number of female moths do wing fanning in the dark. This suggests that in the presence of light, the duration of this behaviour shortens, resulting in a limited spectrum of sexual attraction. According to my observations, male moths have a longer overall time of wing fanning than female moths. The explanation for male wing fanning is unclear to me based on the research available.

The total moth movement during the experiment was also analysed. The results indicate that male moths travel for more extended periods in light than in the dark. Still, statistical results show no significant association with the light condition and overall moving time. Female moths, on the other hand, do not have a major difference in overall movement duration. In both light and dark conditions, they have a similar duration. With the observed result, I can say, male moths are more active in flight than female moths in the presence of light.

According to the data acquired through observation, females in light favoured staying on the central and corner with low illuminance of light rather than the wall, while in the dark, they favour staying on the wall. Considering two female moths stay in the column during the whole experiment from beginning to end. The placement of the column was in green illuminance. At the same time, male moths prefer to stay equally on the wall and corner in the presence of light, with moderate illuminance. While during dark, they prefer to stay on the wall (see fig.10).

15 During the experiment, I found a few moths were not active, or if someone was active, they started responding very late. A sudden change in light levels was considered to effectively blind moths before their eyes adjust (Frank, 1988), which can take up to 30 minutes in some species (Bernhard et al., 1960). As a result, moths that fly from darkness to light may become inactive, often for the entire night (Frank, 2006). This may be because light triggers the daytime response of ceasing behaviour in insects, which was regulated by the light-adapted and dark-adapted states of the compound eye (Robinson, 1952; Laughlin et al., 1978). This might be the cause of the no response behaviours of moths during my studies.

ALAN with a CCT value lower than 3000 K should have a limited effect on nocturnal animals, according to the UNOOSA, 2020 recommendation. However, in my small-scale experiments, I noticed a notable difference in nocturnal moth behaviour when using warm white light with a CCT 2718K, which had a small portion of blue and green light content, as seen in my spectral power distribution measurement (see fig. 5). Green, orange, and red light were less effective in attracting moth than blue light (Cruz et al., 2011). It means that below 3000K, there is a chance that the moth will not be attracted to the light source. In the study, Deichmann et al. (2021) evaluate insect attraction to three lamp type white (3200 K) with 13% blue light content (BLC) and 29% blue-green light content (BGLC), yellow (2700 K) with 0% BLC and 17% BGLC, and amber (2200K) with 0% BLC AND 12% BGLC. They found amber lamps attract the fewest insects. Amber lamps did not contain blue light (Deichmann et al., 2021) and had the lowest CCT. Hence, low CCT and long-wavelength lights are suitable for the nocturnal insects.

Conclusion In the presence of warm-white light (2718K), more male Galleria mellonella were attracted than female Galleria mellonella. The attraction of moths depends on the spectral composition of light. Shorter wavelength light (UV, blue and green light) attract more insects while longer wavelength light(amber and red light) attract fewer. LEDs used in this study emit a small portion of blue, green lights. However, other Galleria mellonella behaviour based on sex, such as circular movement (trajectory), wing fanning (mating behaviour), overall moving time during the experiment, were not significantly affected by warm-white light. I highly recommend that we change warm-white light to amber, white light with respect to low colour temperature. There is a need to conduct more research with a variety of low CCT light sources. It will help to see if street lights are directly responsible for moth population declines. Acknowledgement

Throughout the writing of this thesis, I got a lot of help and encouragement. First and foremost, I want to express my gratitude to my supervisors Annika Jägerbrand and Petter Andersson, for their invaluable guidance and unwavering encouragement. My

16 appreciation goes out to Maria Nilsson Tengelin, RISE and the Swedish Transport Administration for the financial support I needed to pursue my studies at the RISE. I like to express my sincere gratitude to my examiner David Green for his valuable comments and a special thank you to Aldonna Pruba for being a fantastic opponent. I would like to express my gratitude to all of my classmates for their constructive and valuable input during our class meetings. Thanks to Yahe in general for his assistance in the lab. In addition, I would express my gratitude to my parents for their sound advice and sympathetic ear. You are always willing to support me. Finally, I would like to thanks my husband, Raghwendra Singh, who offered stimulating discussions as well as enjoyable distractions from my study.

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22 Prasoon Singh from Kanpur, Uttar Pradesh, India. Master’s graduate in Applied Environmental Science with another Master’s in Zoology.

PO Box 823, SE – 301 18 Halmstad Phone: +35 46 16 71 00 HALMSTAD E-mail: [email protected] UNIVERSITY www.hh.se

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