Life History Traits of a Key Agricultural Pest, Helicoverpa Armigera (Lepidoptera: Noctuidae): Are Laboratory Settings Appropriate?

Katsikis Christina (Orcid ID: 0000-0003-4585-1127)

Life history traits of a key agricultural pest, Helicoverpa armigera (Lepidoptera: Noctuidae): are laboratory settings appropriate?

Christina I Katsikis, Peng Wang and Myron P Zalucki1

1School of Biological Science, The University of Queensland, St Lucia, QLD 4072, Australia.

Running Title Life history and nutrient self-selection of Helicoverpa armigera

Abstract Helicoverpa armigera is intensively researched in laboratory settings, yet developmental rates can vary considerably even under controlled conditions. Here, dietary choice and light spectra were tested as possible factors influencing this variability, a range of fitness indicators were collected, and dietary choice behaviour in early instars was observed. We show that early instars of H. armigera exhibited self-selection of nutrient intake, a novel finding. Larvae given a choice between two artificial diets varying in macronutrient ratios were heavier, exhibited a higher relative growth rate, shorter developmental time and longer eclosion time compared with larvae reared on a single diet. Wing size relative to body mass was higher for larvae on extreme no-choice treatments and smallest for those on the choice diets, indicating a potential adaptation to escape poor nutrient landscapes. Light spectra had an effect on the size of pupae, with H. armigera reared under white LED light having larger pupae than those reared under fluorescent white light. Larvae reared under LED light took longer to emerge from pupation. Giving larvae a choice of diets with a range of nutrients may reduce developmental variability rather than assuming that one diets suits all.

Key words

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/aen.12441

Lepidopteran bioecology, nutrient choice, geometric framework, light spectrum, developmental variability, polyphagous pests, developmental rate, insect laboratory rearing, wing size, diet deficiency compensation.

INTRODUCTION Helicoverpa armigera (Hübner) is a polyphagous insect pest of significant global importance (Kriticos et al. 2015). It is reported as feeding on over three hundred plant species (Cunningham and Zalucki 2014), including many agriculturally and commercially important crops, and accounts for over $2 billion dollars per year in damage to agriculture worldwide (Tay et al. 2013). Its geographic range has been expanding, and in the last decade it has expanded into a range of Latin and Central American countries (Sosa-Gómez et al. 2016). It has also been sporadically detected in the USA though permanent populations have not yet been established (Gonçalves et al. 2019). In addition, it has evolved resistance to a number of key chemical pesticides, making management difficult (Ahmad 2007; Downes et al. 2017).

Not surprisingly, H. armigera is the subject of intense study worldwide both in the laboratory and in the field (e.g. Zalucki et al. 1986; Armes et al. 1992; Jallow and Matsumura 2001; Downes and Mahon 2012; Tay et al. 2013; Gregg et al. 2019). Laboratory studies have ranged from chemical and molecular to physiological and behavioural (e.g. Armes et al. 1992; Wei et al. 2002; Xu et al. 2005; Luong et al. 2017). Potential pest control and mitigation strategies have been proposed on the basis of these experiments (Reddy and Manjunatha 2000).

Development rate in the laboratory is highly variable at 25 C°, the most common temperature at which H. armigera is raised, with nearly as much variation at this temperature as there was across temperatures (Unpublished data, Puhl de Melo and MP Zalucki, UQ, see Appendix A). Such high variability presents a problem of reproducibility, applicability to natural environments, and raises questions about what factors may affect the development of H. armigera in laboratory settings. While any laboratory experiment runs the risk of abstracting and isolating the subject of study from its natural conditions, the effect this can have on a subject’s life history is often poorly understood (Shields 1989). Causes of high variation in

developmental rates may include a wide range of factors, such as protozoan, fungal, bacterial(Armes et al. 1992) or viral infections (Hanzlik et al. 1993), inbreeding depression (Cacoyianni et al. 1995) and nutrition (Armes et al. 1992).

In laboratory studies H. armigera are often reared from hatching to pupation on artificial diet (Teakle 1991). This is done for a variety of reasons, such as taking potential plant- invertebrate interaction out of equation, and yielding specimens of higher fitness and fecundity, and general ease and convenience (Wu and Gong 1997). However, most diets have not been optimized to correspond to the diet a caterpillar would naturally encounter or choose in the field, which may be quite different in macronutrient content (Behmer 2009). Diets may also differ between research facilities (Ritter and Nes 1981; Wu and Gong 1997; Hamed and Nadeem 2008).

Lepidoptera in natural settings, including H. armigera, regulate their intake of a wide range of nutrients by changing food source and the amount eaten (Raubenheimer et al. 2009). The nutrient landscape that Lepidoptera find themselves in is highly heterogeneous, even in crop monocultures (Deans et al. 2015). Many caterpillars show a natural preference for a specific ratio of different nutrients, termed their intake target (IT), of which carbohydrate and protein composition in particular have been intensely studied (Behmer 2009). A self-selected intake target can be different depending on the life stage of the caterpillar, and is hypothesized to reflect an evolutionary compromise between food availability, maximum fitness, avoidance of secondary plant metabolites and minimization of danger due to predation (Lee et al. 2002; Despland and Noseworthy 2006; Behmer 2009). Food intake preferences can shift even over the course of a single instar, due to changing energetic and developmental needs of the caterpillar (Cohen et al. 1987). Larvae of H. armigera raised on an artificial diet with a single defined protein:carbohydrate composition are therefore unable to optimize their food intake, which may alter their development compared to their wild counterparts, or to larvae raised on an artificial diet with a choice of different macronutrient composition (Deans et al. 2015). Further, a range of behaviours (such as shelter seeking or relocating in order to pupate) may interact with nutrient self-selection and further modify the natural diet of H. armigera compared to their stationary laboratory-raised counterparts (Perkins et al. 2009).

Remarkably, all nutrient choice experiments documented for Lepidoptera have been conducted exclusively on late or final instar caterpillars (Lee et al. 2002; Lee et al. 2004; Lee et al. 2006; Behmer 2009; Deans et al. 2015) including H. armigera (Raubenheimer and Browne 2000; Browne and Raubenheimer 2003; Tessnow et al. 2018). It is not known whether nutrient choice differs between instars, or whether early instars are capable of self- selecting their food.

Early instars show different feeding behaviour to other stages (Johnson & Zalucki 2007). In Helicoverpa zea (Boddie), first and second instar caterpillars prefer different plant parts to later instars (Cohen et al. 1988). Early instars of H. armigera have been shown to exhibit specialized feeding patterns, preferring soft terminal leaves and buds (Perkins et al. 2008), and location of larvae on reproductive plant parts have been linked to adaptive differential survival (Bahar et al. 2019)First instars have a higher relative growth rate, and distribute their time between feeding, resting and moving differently compared to later instars (Johnson and Zalucki 2007). Fourth and fifth-instar H. armigera also exhibit dynamic changes in feeding behaviour as they grow (Raubenheimer and Browne 2000; Browne and Raubenheimer 2003).

The ability to choose between different nutritional options may therefore affect caterpillar development compared to their non-optimizing conspecifics restricted to a single diet. Some of the variation found in the laboratory may be explained by the lack of choice provided to larvae, combined with variability in ingredients such as type of flour (Naseri et al. 2010) and macronutrient content (Behmer 2009).

As H. armigera are generally reared in constant environment cabinets on artificial diet and under artificial light (Armes et al. 1992), the type of light used may also be pertinent. Artificial lighting spectra depend strongly on the type of illumination used. Incandescent, fluorescent and LED lights all have highly distinct spectra, and all differ from natural light, including in their effects on Lepidoptera (Shields 1989; Johansen et al. 2011; Longcore et al. 2015; Degen et al. 2016).

Light plays a key role in regulating insect development, circadian rhythms and diapause initiation/termination, flight and ovipositing (Shimoda and Honda 2013). Photoperiod, the

specific spectrum (wavelength profile) of the light, the intensity of light and its polarization have been shown to significantly affect insects (Johansen et al. 2011).

Investigation into the effects of light type on larval behaviour is limited (Philogene and McNeil 1984; Geffen et al. 2014). Larvae are able to react to certain wavelengths of light, – for example, H. armigera larvae can potentially distinguish between UV, blue and green wavelengths of light (Perkins et al. 2008) and respond differently to different wavelengths (Perkins et al. 2009). Different wavelengths of LED light have been shown to affect caterpillar growth rates and pupation (Geffen et al. 2014), as well as a range of behaviours (Tu et al. 2012). White fluorescent and white LED light have dramatically different spectral distributions (Shields 1989), and there has been no research on whether this difference may affect caterpillar life history. Moreover, artificial lights (apart from incandescent bulbs) consist of very fast flickering, which can be detected by a range of insects, and which can cause a wide variety of behavioural and stress responses (Inger et al. 2014). Unfortunately there is very little reporting of standard light conditions for the rearing of H. armigera, and it is very difficult to compare the light that larvae were reared under to account for possible effects.

Here, we investigate two key factors which may affect rearing of H. armigera in laboratory settings: nutrient choice and light regime. The developmental milestones of caterpillars given a choice of two foods with different defined protein: carbohydrate (p:c) ratios were assessed compared to caterpillars raised on a single artificial diet (no choice).

A secondary aim was to investigate whether early instars of H. armigera are indeed capable of self-selecting their nutrient intake, by comparing early instar development across the choice treatments.

In a separate experiment, spectral distribution of artificial lighting was tested for effects on H. armigera life history, with caterpillars raised under fluorescent lighting compared to caterpillars raised under LED lighting.

MATERIALS AND METHODS Larvae

Both strains of H. armigera used in experiments were reared in the School of Biological Sciences, The University of Queensland. The H. armigera used in the diet experiment were sourced from the Australian Cotton Research Institute, Narrabri, NSW, Australia. The H. armigera used in the light experiment were sourced from AgBiTech, Queensland, Australia. Insects were reared as described by Downes and Mahon (2012).

Artificial Diet

The artificial diet was developed by Ritter and Nes (1981) and modified by Jing et al (2013). Total macronutrient content was 42% of all artificial diets, which is the most suitable ratio for effective metabolism and resembles what larvae would encounter in the wild (Behmer 2009). The relative contribution of protein (p) and carbohydrate (c) to total macronutrient content of 42% was then varied among four diets. A high protein diet (p35:c7), high carbohydrate diet (p7:c35), medium protein diet (p28:c14) and medium carbohydrate diet (p14:c28) were prepared with only protein and carbohydrate macronutrients varying between diets and all other constituents, including water content, held constant (Appendix B). The four different ratios were derived from analysis of cotton tissues that H. armigera might naturally encounter in the field and greenhouse (Deans et al. 2016). Diet cubes were replaced every two days to prevent drying out.

Choice Experiment

Upon hatching, neonates were weighed (Mettler Toledo Excellence XS Balance) and randomly assigned to one of three choice treatments with two diets of differing p:c ratios: a high-protein/high-carbohydrate choice treatment (p35:c7/p7:c35), a medium-protein/medium- carbohydrate choice treatment (p28:c14/p14:c28), and a mixed high-protein/medium- carbohydrate choice treatment (p35:c7/p14:c28 ). A cube (1 cm3) of each diet type was placed on a damp filter paper 2.5 cm apart on a Petri dish (5 cm diameter). Each choice treatment had 50 replicates.

Neonates within each treatment were assigned to either a control group (only weighed as neonate and pupa) or treatment group (weighed in each instar). A t-test was performed to determine that there was no difference between the groups as a result of regular handling. Larvae were weighed after moulting and before they moved or ate in the following instar. Weighing was done immediately post-moult rather than interpolating growth rate between fixed sampling periods in order to resolve the complex non-linear and non-exponential growth rate exhibited by larval Lepidoptera (Tammaru and Esperk 2007). Larvae were inspected three times a day to monitor for moulting. Pupae were inspected twice a day for eclosion.

Upon pupation, pupal mass and sex were determined, and pupae transferred to individual 50 ml plastic sample cups with perforated lids until eclosion. Duration to pupation and time to eclosion (in days) was recorded for each individual larva. Relative growth rate was

determined using the formula RGR = (ln(wt1)-ln(wt0)) / (t1 – t0) (Kogan and Cope 1974)

where wt0 and t0 are the initial weight (e.g. in mg) and start time, and wt1 and t1 are a subsequent weight and end time.

Upon adult eclosion, specimens were left for a period of 24 h, then anaesthetized. The right forewing of each specimen was collected, and wing length and wing width measured using ROK digital callipers to an accuracy of ±0.01 mm. Wing length measurements were performed as outlined in Shi et al. (2015).

Experiments were undertaken under uniform light:dark cycle of 14h:10h at a temperature of 23±2 C° under fluorescent light, Phillips T8 TLD 36W/840 Cool White, suspended at a height of 2 meters.

No Choice Experiment

Upon hatching, neonates were weighed and assigned to one of the four diets. Each treatment consisted of two cubes of identical diets placed as in choice experiment. Larvae were raised under the same conditions as in choice experiment above, and all measurements were performed in the same way.

Movement experiment

Larvae of the choice experiment were observed at the first, second, and third instar for a period of 300 minutes every time, and the location of larvae on diet were recorded every twenty minutes. If any part of the larvae was in contact with either diet cube, they were considered to be on the diet, otherwise they were recorded as ‘off-diet’. In all three choice diet combinations, the larval location on the diet higher in carbohydrate was designated ‘C’ and the diet higher in protein was recorded as ‘P’.

Light Experiment

Upon hatching, neonates were weighed and randomly assigned to one of two light treatments – development under illumination by a Fluorescent 840 light source, or a Valoya NS1 LED light source, in an ADAPTIS A1000-IN CHAMBER at the CSIRO, Queensland Biosciences Precinct, Brisbane. The LED spectrum (NS1) and fluorescent spectrum (840) are shown in Appendix C.

Both treatments were placed on a shelf 84 cm away from the light source, and maintained at 27 °C (in order to facilitate faster growth due to experimental time constrains) with a 14:10 (L:D) cycle and constant light intensity as programmed into the growth chamber. Ambient day light was blocked out by black curtains when opening the chambers. All larvae were rotated daily to ensure even light exposure. In addition, temperature readings were taken with a thermometer daily at the location where caterpillars were placed. A t-test was conducted on the temperature readings to test that local temperature was not significantly different between cabinets.

Artificial diet based upon Perkins et al. (2009) was provided ad libitum. Caterpillars were weighed upon hatching and then placed into a tray with artificial diet, and covered by a clear plastic tray lid with breathing holes. Developmental time until pupation and pupal mass were measured for each insect, and pupae were sexed, and larvae were checked daily for moulting and pupation.

Data Analysis

All data sets met the assumptions of independence, normality and homogeneity of variance. All analyses were done using R and RStudio for windows (Version 1.0.136).

For the choice and no-choice diet experiments, larval weights from second to last instar (choice only), as well as pupal weights were compared across diets using ANCOVA. The relative growth rate between instars and over the larval lifespan were also analysed using ANCOVA. The ANCOVA model included either instar/pupal weights or relative growth rate as the dependent variable, diet and sex as fixed factors, and initial larval weight as a covariate. ANCOVA was also used to analyse wing length, wing width, and wing length:width ratio, which were included as dependant variables, and sex and diet were included as fixed factors, and pupal mass as covariate.

Developmental time to pupation and pupal duration were compared across diets using ANOVA. The model included the time in days as the dependant variable, and the diet and sex as fixed factors.

For the diet preference observation of the choice experiment, a multinomial log-linear model was fitted to the data. The probability of location on a particular diet, as well as standard error of the ratio of predicted probabilities, was calculated using nnet package in R (Ripley et al. 2016). Observed probabilities were tested against expected probabilities fitted to the multinomial model using a likelihood ratio test to check for non-random diet selection, with the probability of location on diet being the dependent variable, and choice treatment as a fixed factor. Probabilities of larval location on a particular diet block were compared across the three choice treatments using analysis of deviance of the multinomial model, followed by pairwise Tukey’s comparison of least square means, with the probability of location on diet being the dependent variable, and choice treatment as a fixed factor.

Mean number of movement events (defined as a change between diets, as well as movement off or on a diet) were added for each 300 minute period of observation per larva per instar and compared using an ANOVA model, where the number of movement events was the dependent variable, and the diet was the fixed factor.

For the light test, pupal weight was compared between light regimes using ANCOVA. Pupal weight was included as a dependant variable, treatment and sex as fixed factors, and initial larval weight as covariate.

Developmental time to pupation and pupal duration were compared across light treatments using ANOVA. The model included the time in days as the dependant variable, and the diet and sex as fixed factors.

A Tukey’s HSD test was used for all ANOVA post-hoc analysis. A Tukey-b test was used for all ANCOVA post hoc analysis. For all between-treatment comparisons, a X2 test was carried out to confirm that there was no significant difference in the ratio of males and females per treatment.

RESULTS Early instar movement

Diet had an effect on caterpillar location in the first (LR4 = 43.35, P < 0.01), second (LR4 =

139.45, P < 0.01), and third (LR4 = 144.86, P < 0.01) instars, indicating non-random diet block selection by all early instar larvae.

The protein: carbohydrate ratio of the diets had an effect on caterpillar preference. In the first instar, the larvae on the mixed high-protein/ medium-carbohydrate and medium- protein/medium-carbohydrate choice diets had a stronger preference for the carbohydrate-rich

2 block than the protein-block compared to the high-protein/ high-carbohydrate choice diet (X 4 = 43.35, P < 0.01), though all first instar larvae were more likely to be found on the protein rather than carbohydrate-rich block (Fig. 1a).

In the second instar (Fig. 1b) the larvae on the medium-protein/ medium-carbohydrate choice diet were most likely to be found on the carbohydrate-rich diet block while those on the high- protein/high-carbohydrate choice diet were more likely to be found on the protein-rich block

2 (X 4 = 139.46, P < 0.01). In addition, the larvae in the high-protein/high-carbohydrate choice treatment were much more likely to be found on the protein-rich block (Fig. 1b).

In the third instar, the larvae in the high-protein/high-carbohydrate choice treatment spent the

2 least amount of time on the carbohydrate block (X 4 = 144.86, P < 0.01), while those in the mixed high-protein/medium-carbohydrate treatment were more likely to prefer carbohydrate to protein (Fig. 1c). Larvae in the medium-protein/ medium-carbohydrate choice spent an equal proportion of time on the protein-rich and the carbohydrate-rich diet blocks (Fig. 1c). In later instars all larvae spent progressively more time off the diet blocks.

The frequency of movements was compared across all three instars. Only in the first instar

was there a difference (ANOVA, F2,148 = 4.33, P = 0.01) between treatments (Fig. 2), specifically between high-protein/high-carbohydrate diet, which showed the highest mean movement events, and the mixed high-protein/ medium-carbohydrate diet, which showed the least (Fig. 2). Other instars showed no difference in the amount of movement between treatments. On average, larvae changed location twice in a 300-minute period.

Early instar growth

Larvae in the choice experiment were weighed and compared across early instars. In the

transition between first and second instar there was a significant difference (ANCOVA, F2,41 = 11.76, P < 0.01) in larval weights between diet treatments (Fig. 3a). The group of H. armigera given the medium-protein/ medium-carbohydrate choice diet exhibited the highest larval wet mass, while those given the mixed high-protein/ medium-carbohydrate treatment and the high protein/high carbohydrate choice were smaller (Fig. 3a).

There was a significant difference in the weight of newly moulted third instar larvae (Fig. 3b) between the high-carbohydrate/high-protein diet being the heaviest and the mixed high-

protein/ medium-carbohydrate diet, which had the lowest weight (ANCOVA, F2,16 = 5.264, P = 0.02). The RGR showed no difference among treatments unlike wet larval mass (P = 0.09), suggesting a difference in developmental time (see below).

Effect of diet on H.armigera mortality

Mortality within and between diet treatments was compared and no significant difference in 2 mortality was found between groups (X 6 = 11.38, P = 0.08). Larvae that died before pupation were not included in any analyses.

Effect of diet on H. armigera growth

Larvae raised on a high-carbohydrate no-choice diet had a significantly smaller pupal mass

(ANCOVA, F6,250 = 5.63, P < 0.01) than all other treatments (Fig. 4a). No difference in sex

2 distribution was found between any treatment (X 6 = 13.958, P = 0.3).

Relative growth rate, which takes into account initial larval weight and developmental time,

(in days) showed a difference (ANCOVA, F6,250 = 5.03, P < 0.01) between the high protein no- choice diet (p35:c7) and the mixed high-protein/ medium-carbohydrate choice diet, as well as between the high-carbohydrate no-choice diet and the mixed high-protein/ medium- carbohydrate and medium-protein/medium-carbohydrate choice diets, with the larvae on choice diets showing a faster growth rate than the no-choice. There was also a difference by

sex, with males showing a slower growth rate (ANCOVA, F1,250 = 3.6, P < 0.05), with the single exception of the high-carbohydrate no-choice diet.

Effect of diet on H. armigera development

The effect of diet on developmental time was compared across diet treatments, both in the duration from hatching to pupation (Fig. 5) and from pupation to eclosion (Fig. 6). Larvae in the choice treatment pupated in a shorter time compared to no choice (ANOVA, F1,261 = 12.258, P < 0.01) with the high-carbohydrate and the high-protein diets taking the longest, and

the mixed high-protein/ medium-carbohydrate choice diet taking the least time (ANOVA, F6,251

= 4.673, P < 0.01). Males took longer on average to pupate than females (ANOVA, F1,251 = 6.3, P = 0.01).

Duration of pupal development showed a significant difference between choice and no-

choice treatment (ANOVA, F1,251 = 83.436, P < 0.01) with a strong effect of diet self-selection

on time to eclosion (Fig. 6a). Adult males emerged later than females (ANOVA, F1,241 =

161.789, P < 0.01). Analysis of diet effect compared only the same sexes between different diet treatments (Fig. 6b).

All significant differences in eclosion time across diet treatments (Fig. 6b) were only between

choice and no-choice treatments (ANOVA, F6,241 = 16.419, P < 0.01). Overall, caterpillars in the choice diet treatment eclosed later than all of the no-choice treatments, with the single exception of the males on the medium carbohydrate no-choice diet (P = 0.1).

Effect of diet on H. armigera relative wing size

Wings of adult H. armigera for both choice and no-choice diets were measured. In order to avoid conflating adult size and mass with their wing metrics, the length and width of wings were divided by pupal weight in order to analyze wing size relative to body in cm/g (Fig. 7).

There was a significant difference between treatments in wing length (ANOVA, F6,168 = 7.443, P < 0.01), with the choice treatments generally having the smallest wings in relation to body (cm wing/g of body weight) while the high-cabohydrate and high-protein no choice diets had

the biggest (Fig. 7a). Wing width showed a similar significance (ANOVA, F6,167 = 8.889, P < 0.01) and pattern, with the medium protein no–choice also having significantly wider wings compared to the medium-protein/medium-carbohydrate choice diet treatment (Fig. 7b). No sex difference was observed for any wing measures (ANOVA, F1,167 = 2.634, P = 0.10).

Effect of light spectrum on H. armigera growth

Larvae grown in two growth chambers under LED and fluorescent light were compared in pupal weight, time to pupation, and eclosion. No difference in sex distribution was found

2 between two treatments (X 2 = 2.33, P = 0.31). There was no difference in the time it took larvae to pupate between treatments (ANOVA, F1,85 = 0.908, P = 0.34). Pupal weight (Fig. 8) showed a significant difference between light regimes (ANCOVA, F1,84 = 0.908, P = 0.03)

though RGR did not (ANCOVA, F1,84 = 0.011, P = 0.91). Time from pupation to eclosion (Fig.

9) also showed a significant differences between the light regimes (ANOVA, F1,75 = 6.23, P = 0.01), with H. armigera reared under LED light taking longer to eclose than those reared under fluorescent light.

DISCUSSION A caterpillar’s diet during its development can affect its fitness as a reproductive adult (Roeder and Behmer 2014). Nutrient content and amount of food eaten can strongly influence the population dynamics and ecology of moth populations (Battisti 1988). Food regimes are therefore directly related to many ecological traits which are of interest in research on any lepidopteran (Taylor 1984). Many studies on insect life history traits are undertaken under laboratory conditions, often on artificial diets. This risks the danger of abstracting from the diet self-selection processes operating in early instars in the wild.

Diet

Comparing diets across laboratory protocols is often difficult – frequently, the source or brand of ingredients is not detailed, or the components such as flour are not standardized (Meng et al. 2009; Mahon and Young 2010) leading to differential outcomes in fitness and fecundity (Naseri et al. 2009a; Naseri et al. 2009b; 2010).

Using defined artificial diet choice (Appendix B) had an effect on caterpillar location in the first three instars of H. armigera, indicating that the feeding was non-random and that early instar H. armigera self-select their dietary intake (Fig. 1) with shifting preferences across instars.

Overall, it appears that while early instar H. armigera are able to self-select nutrient intake, first instar larvae did not appear to adjust their intake based on macronutrient concentration of their diets compared to the second and third instars (Fig. 1a, b, c). While they were more often found on the protein-rich than the carbohydrate-rich block, this was location preference was uniform regardless of macronutrient ratios. First-instar H.armigera may have limited ability to adjust for discrepancy in macronutrient intake, possibly as a result of low amylase but not protease activity in the first instar (Kotkar et al. 2012), limiting their ability to process carbohydrates of any ratio, and explaining their preferred location on the protein-rich block. Later instars of H. armigera can vary the expression of key protease, lipase and amylase

enzymes depending on host plant, and this may be an underlying physiological mechanism that is coupled with feeding behaviour to adjust feeding between diets with different macronutrient constituents (Sarate et al. 2012).

There was differential preference of diet in instars two and three, with larvae of the high- protein/ high-carbohydrate choice treatment (Fig. 1 b, c) located more frequently on the high- protein block over the high-carbohydrate block. For the medium-protein/ medium- carbohydrate larvae, the protein location and carbohydrate location became roughly equal, while for the mixed high-protein/medium-carbohydrate group protein location gradually decreased and carbohydrate location rose, especially in the third instar (Fig. 1 b, c).

The trend of shifting away from protein towards carbohydrate in later instars is consistent with building up energy reserves (Cohen et al. 1987, Stockhoff 1993). Early instars of H.armigera have low levels of amylase, a key sugar-catabolising enzyme, which may explain their general lack of preference for cabohydrate in the first instar (Kotkar et al. 2009) and difficulty processing high carbohydrate concentrations (Telang et al. 2003). The latter may explain their avoidance of the carbohydrate-rich diet block in the high-protein/high- carbohydrate treatment across instars (Fig. 1b, c) and increased movement (Fig. 2).

Larvae of medium-protein/ medium-carbohydrate choice treatment were heavier in second instar (Fig. 3a) and the high-protein/high-carbohydrate choice heavier in the third (Fig. 3b). The latter were heaviest despite being highly protein-focused in their diet in the second instar (Fig. 1b), potentially compensating for low carbohydrate intake by increased protein uptake and processing efficiency (Woods 1999). A potential follow up study may be to investigate the choice behaviour in larvae raised on a no-choice diet in a previous instar and then exposed to a choice diet, to account for differences in the presence or absence of a choice in earlier instars.

There was no difference in movement frequency between the three instars although a number of studies indicate movement in early-instar H. armigera is an important survival mechanism (Zalucki et al. 2002; Johnson and Zalucki 2007). This may be due to the absence of other stimuli involved in movement, such as plant compounds or geotaxis due to spatial orientation

of the petri dishes, as larvae tend to move less on horizontal surfaces (Perkins et al. 2008; Perkins et al. 2009).

By pupation, significant differences in weight between the pupae of the choice treatments had disappeared (Fig. 4), indicating a well-documented ability to compensate for early instar nutrition deficiency and discrepancy (Taylor 1988; Behmer 2009; Lee et al. 2012), which can help explain the lack of difference in weight and relative growth rate by the time of pupation. Helicoverpa armigera also increase their rate of ingestion in later instars (Browne and Raubenheimer 2003) ingesting more per unit time, which gives them a bigger window to adjust for any early-life changes.

For no-choice treatments, both female wet pupal mass (Fig. 4a) and female RGR (Fig. 4b) indicate that the extreme diets (high-carbohydrate and high-protein) had the lowest mass of all diet treatments, while the mixed high-protein/medium-carbohydrate choice performed the highest in terms of relative growth and wet weight (Fig. 4b). This may be due to females having more stringent intake requirements due to their reproductive needs (Liu et al. 2004). Body weight in females is a good indicator of fitness and an indirect indicator of fecundity (Liu et al. 2004), which suggests that having a diet choice may directly affects the fitness of developing H. armigera.

The extreme no-choice diets had the lowest outcomes of indirect fitness indicators for body size, with the high-carbohydrate having the lowest mass (Fig. 4a) while both the high-protein and high-carbohydrate no-choice diets grew significantly slower than other treatments. Overall, having the ability to vary intake seems to have allowed larvae of the choice treatments to overcome the limitations of both the high-carbohydrate and high-protein portions of their diet (Fig. 4b), a factor that is integral to their feeding in the heterogeneous nutrient landscape they encounter in the wild (Raubenheimer et al. 2009), where both diet selection or preference (see Fig. 1) as well as diet switching at later instars (see Fig. 1b, c) are crucial to growth and maintenance of caterpillars (Waldbauer and Friedman 1991).

Developmental time from hatching to pupation (Fig. 5) shows that the no-choice extreme diets both took longer to pupate. Out of all the no-choice treatments, only the medium-

carbohydrate treatment approached the developmental time of the three choice treatments (Fig. 5). Faster developmental times, as exhibited on average by the choice treatments (Fig. 5) may result in higher fecundity, survival rate, and more generations per year (Liu et al. 2004), which is likely a result of self-selected intake approaching the optimal intake target - as hypothesized by the geometric framework paradigm (Raubenheimer et al. 2009).

Time from pupation to eclosion shows the clearest difference of the effect of choice and no- choice upon development for both sexes (Fig. 6a). This is a notable difference with previous studies where larval food type and rate of growth were not correlated with pupal duration (Twine 1978; Jallow et al. 2001). Notably, the standard error rates for Figure 6 are quite low. Generally, pupal eclosion was not random but followed a circadian pattern, where most eclosion was usually observed mostly in the morning, with little eclosion observed for the rest of the day (observations were done in the early morning and late afternoon, and eclosion in the afternoon was counted for the same day as eclosion in the morning). The low standard error rates therefore chart a real tendency where eclosion between groups differed by at least half day or more.

Longer pupation time may reflect a larger body mass on average of the choice treatment (Fig. 4), but shorter pupation times also can result in reduced fitness (Wang et al. 2009). The effect of having a choice in diet is significant (Fig. 6b) by treatment and by sex. The medium- protein no-choice treatment (p28:c14) has a p:c ratio that is similar to that used in many artificial diets when raising H. armigera in laboratory conditions (Ritter and Nes 1981; Armes et al. 1992), and it is important to note that pupal duration on this diet differed compared to all choice treatments, indicating that using this artificial diet may result in larval performances that systematically deviate from their natural counterparts in field conditions.

Wing size in insects has been linked to flight time, dispersal activity, and flight performance (Betts and Wootton 1988; Kingsolver 1999; Shi et al. 2015) as well as fitness (Kölliker‐Ott et al. 2003). Previous studies of H.punctigera showed a strong effect of environmental stress on wing shape (Hoffmann et al. 2002). Food choice has also been linked to wing size in H. armigera (Khiaban et al. 2010).

Our results showed that choice treatments had relatively smaller wings (less width and length per gram of body mass) compared to the high-carbohydrate and high-protein no-choice diets which had the biggest wings relative to their body (Fig. 7), with a general trend for the medium-protein and medium-carbohydrate no-choice wings to be slightly bigger than those of the choice population. Poorer diet (very high in protein or very high in carbohydrate with no means for the larvae to alleviate the disparity) appears to result in longer and wider wings relative to the body.

Smaller wing size in H. armigera has been postulated to result from lower host preference for food plants and therefore potentially reduced fitness (Khiaban et al. 2010). Other studies have linked poor survival and smaller body mass to smaller wing size (Singh and Rembold 1988). This differs from our results where the choice treatment had the greater size and growth rate, yet comparatively shorter wings – though the measurements used here differed from the centroid measurements used in the above studies and hence measurements may not be entirely concordant (Fig. 4, 7).

A potential explanation to account for the wing data discrepancy found between diet groups may lie in the observation that not every trait associated with fitness will linearly decline with poor diet. It has been shown that Lepidoptera may compensate for poor diet in various ways. For example, Gelechia monella (Busck) (Lepidoptera: Gelechiidae) develops a stronger immune response when diet quality declines (Krams et al. 2015), and Bicyclus anynana (Butler) (Lepidoptera: Nymphalidae) females exposed to a limited diet showed increased thorax ratio and increased flight performance in forced flight tests (Saastamoinen et al. 2010). Such a phenotype response to environmental stresses is theorized to be an adaptive strategy to alter hormone levels or metabolic activity to respond to an unexpected environment (Zwaan et al. 2008). A key response may be to increase dispersal ability (Meylan et al. 2009) to escape a poor nutritional environment. As larger wings and wing muscles are energetically costly, a propensity to develop larger wings in poor nutritional surroundings in order to migrate may be an evolutionary strategy (Roff and Fairbairn 1991; Saastamoinen et al. 2010). This tendency persists for the medium-protein no-choice diet relative to the mixed high- protein/ medium-carbohydrate choice diet (Fig. 7b), which may indicate that this is not an all-

or-nothing switch, and could have subtle effects depending on the type of diet H. armigera is raised on. Further investigation of the effect of diet on wing morphology by wing centroid measurement may yield important additional insights (Hoffmann et al. 2002).

Overall, self-selected nutrient intake, which is a component of H. armigera life history, is missing from many studies seeking to define its basic physiology, behaviour and life history (Behmer 2009; Roeder and Behmer 2014; Tessnow et al. 2018). When larvae can self-select diet, they appear to improve their performance even on sub-optimal diets, as is the case with the high-protein/high-carbohydrate choice diet treatment, which had much higher fitness indicators than either the high-protein or the high-carbohydrate no-choice treatment. Light

There is a lack of reporting or standardization of light conditions during rearing across studies which may impact the validity and reliability of H. armigera research. Even in experiments investigating the response of larvae to certain light conditions, the type of light used during other life stages, artificial or otherwise, is not always reported (Zhou et al. 2000; Perkins et al. 2008; Perkins et al. 2009). In other cases, it is reported that larvae were reared in incubators (Luong et al. 2017) or under fluorescent light (Tessnow et al. 2018), although fluorescent light sources can differ from manufacturer to manufacturer in the peaks of their wavelength emissions, which are sharp and narrow compared to natural light (Longcore et al. 2015). Other studies have reared larvae using LED light (Geffen et al. 2014), but the difference of rearing caterpillars in LED light compared to fluorescent light is not well known (Gaston et al. 2013; Shimoda and Honda 2013; van Grunsven et al. 2014). Our results seem to indicate that spectral distribution of white light may affect the development of H. armigera.

The evaluation of spectral types of white light on caterpillar growth in this study is a first, and the mechanisms have not been studied before in caterpillars, though specific wavelengths have been shown to affect behaviour and RGR with sex-specific effects (Perkins et al. 2008; Perkins et al. 2009; Tu et al. 2012; Geffen et al. 2014). Comparison of pupal weight shows a significant difference between H. armigera larvae grown under LED compared to those grown under fluorescent light (Fig. 8). The females in the LED treatment in particular tend to

be larger (Fig. 8). Helicoverpa armigera raised under LED light eclosed significantly later than those raised under fluorescent light (Fig. 9).

It may be the case that the broader distribution of LED spectrum compared to the narrower peaks of the fluorescent light (see Fig. 10) incorporates a broader range of wavelengths that stimulate or inhibit behaviour such as ingestion movement (Tu et al. 2012). An alternative explanation may be that H. armigera did worse under fluorescent light compared to LED due to negative effects of fluorescent light, due to adverse effects of visible blue light close to UV spectrum (Hori et al. 2014) or peaks in the UV-A, UV-B, and blue wavelengths where the LED light has troughs (Fig. 10) leading to oxidative stress (Meng et al. 2009, Zhang et al. 2011).

Finally, it is important that a secondary effect of artificial light is a flickering which may be detected by insects, rather than the specific light spectra, and this may be primarily responsible for the variation in data (Inger et al. 2014). Further study may elucidate any effect of spectra relative to the frequency of flicker.

In conclusion, this paper presents a novel finding that larval H.armigera are capable of nutrient self-selection, with a significant effect on their natural history. This is a theoretical insight that should be incorporated into future work on H.armigera early life and developmental processes. Raising H. armigera on a single artificial diet without providing the caterpillars a choice also removes an important physiological control mechanism routinely utilized by wild generalist Lepidoptera (Behmer 2009), and therefore the incorporation of a routine choice of diet for raising H. armigera may yield important insights about H.armigera physiology that may be overlooked when raising larvae on a single-composition diet. Incorporating an optimal diet but without choice, such as one based on the p:c ratio determined in previous nutrient intake target studies on H. armigera (Tessnow et al. 2018) runs the risk of not being optimal for all stages of H. armigera life history. Larvae were significantly heavier and had a higher growth rate on all the choice diets by the time of pupation (Fig. 4), indicating significant changes in feeding and potentially macronutrient requirement between instars. Incorporating an element of choice between at least two diets

with differing p:c ratios into H. armigera rearing protocols may therefore reduce the high variability in developmental rates observed across a wide range of published studies (Appendix A) and yield further interesting insights into H.armigera behaviour and physiology in the wild.

The findings have implications not only for laboratory work, but also understanding the behaviour of wild H.armigera larva. Early instars preference for reproductive parts of a plant may cause disproportionate damage to a crop (Perkins et al. 2009). The ability of early instars in the choice treatment to reach the same baseline of development despite different choice nutritional substrates also has implications for the ability of H.armigera to adjust to nutritional changes in the agricultural changes and to different succession of plants. Further understanding of specific early-instar needs may also allow for more accurate agricultural damage modelling.

ACKNOWLEDGEMENTS Cate Paull and Andrew Hulthen of the CSIRO, Queensland Ecosciences Precinct, Brisbane, provided access to ADATPIS A1000-IN Growth Chambers and calibrated them, as well as providing valuable advice.

REFERENCES

Ahmad, M. 2007 Insecticide resistance mechanisms and their management in Helicoverpa armigera (Hübner)–A review. Journal of Agricultural Research 45, 319-335. Armes, NJ, Bond, G & Cooter, R. 1992. The laboratory culture and development of Helicoverpa armigera, Natural Resources Institute Bulleting 57, Chatham, United Kingdom: Natural Resources Institute.

Bahar, MH, Stanley, J, Backhouse, D, Mensah, R, Del Socorro, A & Gregg, P. 2019 Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants. Entomologia experimentalis et applicata.

Battisti, A. 1988 Host‐plant relationships and population dynamics of the Pine Processionary Caterpillar Thaumetopoea pityocampa (Denis & Schiffermuller). Journal of Applied Entomology 105, 393-402.

Behmer, ST. 2009 Insect herbivore nutrient regulation. Annual Review of Entomology 54, 165–187.

Betts, C & Wootton, R. 1988 Wing shape and flight behaviour in butterflies (Lepidoptera: Papilionoidea and Hesperioidea): a preliminary analysis. Journal of Experimental Biology 138, 271-288.

Browne, LB & Raubenheimer, D. 2003 Ontogenetic changes in the rate of ingestion and estimates of food consumption in fourth and fifth instar Helicoverpa armigera caterpillars. Journal of Insect Physiology 49, 63-71.

Cacoyianni, Z, Kovacs, IV & Hoffmann, AA. 1995 Laboratory Adaptation and Inbreeding in Helicoverpa punctigera (Lepidoptera, Noctuidae). Australian Journal of Zoology 43, 83-90.

Cohen, R, Waldbauer, G & Friedman, S. 1988 Natural diets and self‐selection: Heliothis zea larvae and maize. Entomologia experimentalis et applicata 46, 161-171.

Cohen, R, Waldbauer, G, Friedman, S & Schiff, N. 1987 Nutrient self‐selection by Heliothis zea larvae: A time‐lapse film study. Entomologia experimentalis et applicata 44, 65-73.

Cunningham, JP & Zalucki, MP. 2014 Understanding heliothine (Lepidoptera: Heliothinae) pests: what is a host plant? Journal of Economic Entomology 107, 881-896.

Deans, CA, Behmer, ST, Fiene, J & Sword, GA. 2016 Spatio-temporal, genotypic, and environmental effects on plant soluble protein and digestible carbohydrate content: implications for insect herbivores with cotton as an exemplar. Journal of Chemical Ecology 42, 1151-1163.

Deans, CA, Sword, GA & Behmer, ST. 2015 Revisiting macronutrient regulation in the polyphagous herbivore Helicoverpa zea (Lepidoptera: Noctuidae): New insights via nutritional geometry. Journal of Insect Physiology 81, 21-27.

Degen, T, Mitesser, O, Perkin, EK, et al. 2016 Street lighting: sex‐independent impacts on moth movement. Journal of Animal Ecology 85, 1352-1360.

Despland, E & Noseworthy, M. 2006 How well do specialist feeders regulate nutrient intake? Evidence from a gregarious tree-feeding caterpillar. Journal of Experimental Biology 209, 1301-1309.

Downes, S, Kriticos, D, Parry, H, Paull, C, Schellhorn, N & Zalucki, MP. 2017 A perspective on management of Helicoverpa armigera: transgenic Bt cotton, IPM, and landscapes. Pest Management Science 73, 485-492.

Downes, S & Mahon, R. 2012 Successes and challenges of managing resistance in Helicoverpa armigera to Bt cotton in Australia. GM crops & food 3, 228-234.

Gaston, KJ, Bennie, J, Davies, TW & Hopkins, J. 2013 The ecological impacts of nighttime light pollution: a mechanistic appraisal. Biological Reviews 88, 912-927.

Geffen, KG, Grunsven, RH, Ruijven, J, Berendse, F & Veenendaal, EM. 2014 Artificial light at night causes diapause inhibition and sex‐specific life history changes in a moth. Ecology and Evolution 4, 2082-2089.

Gonçalves, RM, Mastrangelo, T, Rodrigues, JCV, et al. 2019 Invasion origin, rapid population expansion, and the lack of genetic structure of cotton bollworm (Helicoverpa armigera) in the Americas. Ecology and Evolution.

Gregg, PC, Del Socorro, AP, Le Mottee, K, Tann, CR, Fitt, GP & Zalucki, MP. 2019 Host plants and habitats of Helicoverpa punctigera and H. armigera (Lepidoptera: Noctuidae) in inland Australia. Austral Entomology.

Hamed, M & Nadeem, S. 2008 Rearing of Helicoverpa armigera (Hub.) on artificial diets in laboratory. Pakistan Journal of Zoology 40, 447-450.

Hanzlik, TN, Dorrian, SJ, Gordon, KH & Christian, PD. 1993 A novel small RNA virus isolated from the cotton bollworm, Helicoverpa armigera. Journal of general virology 74, 1805-1810.

Hoffmann, AA, Collins, E & Woods, R. 2002 Wing shape and wing size changes as indicators of environmental stress in Helicoverpa punctigera (Lepidoptera: Noctuidae) moths: comparing shifts in means, variances, and asymmetries. Environmental Entomology 31, 965-971.

Hori, M, Shibuya, K, Sato, M & Saito, Y. 2014 Lethal effects of short-wavelength visible light on insects. Scientific Reports 4, 7383.

Inger, R, Bennie, J, Davies, TW & Gaston, KJ. 2014 Potential biological and ecological effects of flickering artificial light. PLoS One 9, e98631.

Jallow, MF & Matsumura, M. 2001 Influence of temperature on the rate of development of Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae). Applied Entomology and Zoology 36, 427-430.

Jallow, MF, Matsumura, M & Suzuki, Y. 2001 Oviposition preference and reproductive performance of Japanese Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae). Applied Entomology and Zoology 36, 419-426.

Jing, X, Grebenok, RJ & Behmer, ST. 2013 Sterol/steroid metabolism and absorption in a generalist and specialist caterpillar: Effects of dietary sterol/steroid structure, mixture and ratio. Insect Biochemistry and Molecular Biology 43, 580-587.

Johansen, N, Vänninen, I, Pinto, DM, Nissinen, A & Shipp, L. 2011 In the light of new greenhouse technologies: 2. Direct effects of artificial lighting on arthropods and integrated pest management in greenhouse crops. Annals of Applied Biology 159, 1-27.

Johnson, M-L & Zalucki, M. 2007 Feeding and foraging behaviour of a generalist caterpillar: are third instars just bigger versions of firsts? Bulletin of Entomological Research 97, 81-88.

Khiaban, N, Haddad Irani Nejad, K, Hejazi, M, Mohammadi, S & Sokhandan, N. 2010 A geometric morphometric study on the host populations of the pod borer, Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae) In some parts of Iran. Munis Entomology & Zoology 5, 140-147.

Kingsolver, JG. 1999 Experimental analyses of wing size, flight, and survival in the western white butterfly. Evolution 53, 1479-1490.

Kogan, M & Cope, D. 1974 Feeding and nutrition of insects associated with soybeans. 3. Food intake, utilization, and growth in the soybean looper, Pseudoplusia includens. Annals of the Entomological Society of America 67, 66-72.

Kölliker‐Ott, UM, Blows, MW & Hoffmann, AA. 2003 Are wing size, wing shape and asymmetry related to field fitness of Trichogramma egg parasitoids? Oikos 100, 563-573.

Kotkar H. M., Sarate P. J., Tamhane V. A., Gupta V. S. & Giri A. P. (2009) Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants. Journal of Insect Physiology 55, 663-70. Kotkar H. M., Bhide A. J., Gupta V. S. & Giri A. P. (2012) Amylase gene expression patterns in Helicoverpa armigera upon feeding on a range of host plants. Gene 501, 1-7. Krams, I, Kecko, S, Kangassalo, K, et al. 2015 Effects of food quality on trade‐offs among growth, immunity and survival in the greater wax moth Galleria mellonella. Insect Science 22, 431-439.

Kriticos, DJ, Ota, N, Hutchison, WD, et al. 2015 The potential distribution of invading Helicoverpa armigera in North America: is it just a matter of time? PLoS One 10, e0119618.

Lee, K, Behmer, S, Simpson, S & Raubenheimer, D. 2002 A geometric analysis of nutrient regulation in the generalist caterpillar Spodoptera littoralis (Boisduval). Journal of Insect Physiology 48, 655-665.

Lee, KP, Behmer, ST & Simpson, SJ. 2006 Nutrient regulation in relation to diet breadth: a comparison of Heliothis sister species and a hybrid. Journal of Experimental Biology 209, 2076-2084.

Lee, KP, Kwon, S-T & Roh, C. 2012 Caterpillars use developmental plasticity and diet choice to overcome the early life experience of nutritional imbalance. Animal Behaviour 84, 785-793.

Lee, KP, Simpson, SJ & Raubenheimer, D. 2004 A comparison of nutrient regulation between solitarious and gregarious phases of the specialist caterpillar, Spodoptera exempta (Walker). Journal of Insect Physiology 50, 1171-1180.

Liu, Z, Li, D, Gong, P & Wu, K. 2004 Life table studies of the cotton bollworm, Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae), on different host plants. Environmental Entomology 33, 1570-1576.

Longcore, T, Aldern, HL, Eggers, JF, et al. 2015 Tuning the white light spectrum of light emitting diode lamps to reduce attraction of nocturnal arthropods. Philosophical Transactions of the Royal Society. B 370, 20140125.

Luong, TT, Zalucki, MP, Perkins, LE & Downes, SJ. 2017 Feeding behaviour and survival of Bacillus thuringiensis‐resistant and Bacillus thuringiensis‐susceptible larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) exposed to a diet with Bacillus thuringiensis toxin. Austral Entomology.

Mahon, R & Young, S. 2010 Selection experiments to assess fitness costs associated with Cry2Ab resistance in Helicoverpa armigera (Lepidoptera: Noctuidae). Journal of Economic Entomology 103, 835-842.

Meng, J-Y, Zhang, C-Y, Zhu, F, Wang, X-P & Lei, C-L. 2009 Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults. Journal of Insect Physiology 55, 588-592.

Meylan, S, De Fraipont, M, Aragon, P, Vercken, E & Clobert, J. 2009 Are dispersal‐ dependent behavioral traits produced by phenotypic plasticity? Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 311, 377-388.

Naseri, B, Fathipour, Y, Moharramipour, S & Hosseininaveh, V. 2009a Comparative life history and fecundity of Helicoverpa armigera (Hubner)(Lepidoptera: Noctuidae) on different soybean varieties. Entomological Science 12, 147-154.

Naseri, B, Fathipour, Y, Moharramipour, S & Hosseininaveh, V. 2009b Life table parameters of the cotton bollworm, Helicoverpa armigera (Lep.: Noctuidae) on different soybean cultivars. Journal of Entomological Society of Iran 29, 25-40.

Naseri, B, Fathipour, Y, Moharramipour, S & Hosseininaveh, V. 2010 Comparative reproductive performance of Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae) reared on thirteen soybean varieties. Journal of Agricultural Science and Technology 13, 17-26.

Perkins, LE, Cribb, BW, Hanan, J, Glaze, E, Beveridge, C & Zalucki, MP. 2008 Where to from here? The mechanisms enabling the movement of first instar caterpillars on whole plants using Helicoverpa armigera (Hübner). Arthropod-Plant Interactions 2, 197.

Perkins, LE, Cribb, BW, Hanan, J & Zalucki, MP. 2009 The role of two plant-derived volatiles in the foraging movement of 1st instar Helicoverpa armigera (Hübner): time to stop and smell the flowers. Arthropod-Plant Interactions 3, 173.

Philogene, B & McNeil, JN. 1984 The influence of light on the non-diapause related aspects of development and reproduction in insects. Photochemistry and Photobiology 40, 753-762.

Raubenheimer, D & Browne, LB. 2000 Developmental changes in the patterns of feeding in fourth‐and fifth‐instar Helicoverpa armigera caterpillars. Physiological Entomology 25, 390- 399.

Raubenheimer, D, Simpson, SJ & Mayntz, D. 2009 Nutrition, ecology and nutritional ecology: toward an integrated framework. Functional Ecology 23, 4-16.

Reddy, G & Manjunatha, M. 2000 Laboratory and field studies on the integrated pest management of Helicoverpa armigera (Hübner) in cotton, based on pheromone trap catch threshold level. Journal of Applied Entomology 124, 213-221. Ripley, B, Venables, W & Ripley, MB. 2016 Package ‘nnet’. R package version 7, 3-12.

Ritter, KS & Nes, WR. 1981 The effects of the structure of sterols on the development of Heliothis zea. Journal of Insect Physiology 27, 419-424.

Roeder, KA & Behmer, ST. 2014 Lifetime consequences of food protein‐carbohydrate content for an insect herbivore. Functional Ecology 28, 1135-1143.

Roff, DA & Fairbairn, DJ. 1991 Wing dimorphisms and the evolution of migratory polymorphisms among the Insecta. American Zoologist 31, 243-251.

Saastamoinen, M, Van der Sterren, D, Vastenhout, N, Zwaan, BJ & Brakefield, PM. 2010 Predictive adaptive responses: condition-dependent impact of adult nutrition and flight in the tropical butterfly Bicyclus anynana. The American Naturalist 176, 686-698.

Sarate P., Tamhane V., Kotkar H., Ratnakaran N., Susan N., Gupta V. & Giri A. (2012) Developmental and digestive flexibilities in the midgut of a polyphagous pest, the cotton bollworm, Helicoverpa armigera. Journal of Insect Science 12, 42.

Shi, J, Chen, F & Keena, MA. 2015 Differences in wing morphometrics of Lymantria dispar (Lepidoptera: Erebidae) between populations that vary in female flight capability. Annals of the Entomological Society of America 108, 528-535.

Shields, E. 1989 Artificial light: experimental problems with insects. Bulletin of the ESA 35, 40-45.

Shimoda, M & Honda, K-i. 2013 Insect reactions to light and its applications to pest management. Applied Entomology and Zoology 48, 413-421.

Singh, A & Rembold, H. 1988 Developmental value of chickpea, Cicer arietinum, soybean, Glycine max, and maize, Zea mays, flour for Heliothis armigera (Lep., Noctuidae) larvae. Journal of Applied Entomology 106, 286-296.

Sosa-Gómez, DR, Specht, A, Paula-Moraes, SV, et al. 2016 Timeline and geographical distribution of Helicoverpa armigera (Hübner)(Lepidoptera, Noctuidae: Heliothinae) in Brazil. Revista Brasileira de Entomologia 60, 101-104.

Stockhoff, BA. 1993 Ontogenetic change in dietary selection for protein and lipid by gypsy moth larvae. Journal of Insect Physiology 39, 677-686.

Tammaru, T & Esperk, T. 2007 Growth allometry of immature insects: larvae do not grow exponentially. Functional Ecology 21, 1099-1105.

Tay, WT, Soria, MF, Walsh, T, et al. 2013 A brave new world for an old world pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil. PLoS One 8, e80134.

Taylor, M. 1984 The dependence of development and fecundity of Samea multiplicalis on early larval nitrogen intake. Journal of Insect Physiology 30, 779-785.

Taylor, MF. 1988 Field measurement of the dependence of life history on plant nitrogen and temperature for a herbivorous moth. The Journal of Animal Ecology, 873-891.

Teakle, R.E. 1991 Laboratory Culture of Heliothis Species and Identification of Disease, pp. 22-29 in M.P. Zalucki (ed). Heliothis: Research Methods and Prospects, Springer-Verlag, New York, 234p.

Telang, A, Buck, NA, Chapman, RF & Wheeler, DE. 2003 Sexual differences in postingestive processing of dietary protein and carbohydrate in caterpillars of two species. Physiological and Biochemical Zoology 76, 247-255.

Tessnow, AE, Behmer, ST, Walsh, TK & Sword, GA. 2018 Protein-carbohydrate regulation in Helicoverpa amigera and H. punctigera and how diet protein-carbohydrate content affects insect susceptibility to Bt toxins. Journal of Insect Physiology 106, 88-95.

Tu, X, Chen, Y, Chen, J, Hu, Z, Jin, Y & Xu, F. 2012 Effects of different LED light sources on the behavior of Brithys crini (Lepidoptera: Noctuidae). Acta Entomologica Sinica 55, 1185-1192.

Twine, P. 1978 Effect of temperature on the development of larvae and pupae of the corn earworm, Heliothis armigera (Hubner)(Lepidoptera: Noctuidae). Queensland Journal of Agricultural and Animal Sciences (Australia).

Van Grunsven, RH, Donners, M, Boekee, K, Tichelaar, I, Van Geffen, K & Veenendaal, E. 2014 Spectral composition of light sources and insect phototaxis, with an evaluation of existing spectral response models. Journal of Insect Conservation 18, 225-231.

Waldbauer, G & Friedman, S. 1991 Self-selection of optimal diets by insects. Annual Review of Entomology 36, 43-63.

Wang, D, Gong, P, Li, M, Qiu, X & Wang, K. 2009 Sublethal effects of spinosad on survival, growth and reproduction of Helicoverpa armigera (Lepidoptera: Noctuidae). Pest Management Science 65, 223-227.

Wei, G, Zhang, Q, Zhou, M & Wu, W. 2002 Characteristic response of the compound eyes of Helicoverpa armigera to light. Acta Entomologica Sinica 45, 323-328.

Woods, HA. 1999 Patterns and mechanisms of growth of fifth-instar Manduca sexta caterpillars following exposure to low-or high-protein food during early instars. Physiological and Biochemical Zoology 72, 445-454.

Wu, K & Gong, P. 1997 A new and practical artificial diet for the cotton boll‐worm. Insect Science 4, 277-282.

Xu, X, Yu, L & Wu, Y. 2005 Disruption of a cadherin gene associated with resistance to Cry1Ac δ-endotoxin of Bacillus thuringiensis in Helicoverpa armigera. Applied and Environmental Microbiology 71, 948-954.

Zalucki, M, Daglish, G, Firempong, S & Twine, P. 1986 The biology and ecology of Heliothis armigera (Hubner) and Heliothis punctigera Wallengren (Lepidoptera, Noctuidae) in Australia-What do we know. Australian Journal of Zoology 34, 779-814.

Zalucki, MP, Clarke, AR & Malcolm, SB. 2002 Ecology and behavior of first instar larval Lepidoptera. Annual Review of Entomology 47, 361-393.

Zhang, CY, Meng, JY, Wang, XP, Zhu, F & Lei, CL. 2011 Effects of UV‐A exposures on longevity and reproduction in Helicoverpa armigera, and on the development of its F1 generation. Insect Science 18, 697-702.

Zhou, X, Coll, M & Applebaum, SW. 2000 Effect of temperature and photoperiod on juvenile hormone biosynthesis and sexual maturation in the cotton bollworm, Helicoverpa armigera: implications for life history traits. Insect Biochemistry and Molecular Biology 30, 863-868.

Zwaan, BJ, Zijlstra, WG, Keller, M, Pijpe, J & Brakefield, PM. 2008 Potential constraints on evolution: sexual dimorphism and the problem of protandry in the butterfly Bicyclus anynana. Journal of Genetics 87, 395-405.

Figure Legends

Fig. 1. Probability of H. armigera larval location on diet during choice experiment in first three instars ±95% CI. Panel (a) shows first instar diet location probability, (b) second instar, (c) third instar. C indicates location on the food cube containing more carbohydrate out of two choices, P indicates location on cube higher in protein, OFF indicates larva is not touching a diet cube. Different letters (A/B/C for location C, a/b/c for location OFF, X/Y/Z for location P) indicate statistical significance at p < 0.05 between different diets. Fig. 2. Mean number of movement events ±SEM in each treatment across the first instar. Different letters indicate statistical significance at p < 0.05 between bars. Fig. 3. Mean larval wet mass ±SEM upon (a) moulting into second instar and (b) moulting into the third instar between three choice diet treatments. Different letters indicate statistical significance at p < 0.05 between bars.

Fig. 4. Helicoverpa armigera pupal mass and growth rate over the course of the choice and no-choice experiments. (a) Mean pupal wet mass in mg ±SEM (b) Mean relative growth rate in ln µg/day ±SEM by treatment and across sexes. Different letters indicate a significant difference at p < 0.05 between females only.

Fig. 5. Developmental time of H. armigera. Duration in days ±SEM until pupation, different letters indicate a significant difference at p < 0.05, with * indicating a difference between females and + indicating a difference between males. Fig. 6. H. armigera developmental times (a) Time in days ±SEM from pupation to eclosion in the choice and no-choice treatments. Different letters indicate a significant difference at p < 0.05 (b) Time in days ±SEM from pupation to eclosion by sex across all diets. Different letters indicate a significant difference at p < 0.05 between each treatment of both males and females

Fig. 7. Helicoverpa armigera adult wing size relative to body weight (a) Wing length to body in cm/g ±SEM (b) Wing width to body in cm/g ±SEM. Different letters indicate a significant difference at p < 0.05 for both males and females of each treatment. Fig. 8. Mean pupal weight in mg ±SEM for H. armigera raised under fluorescent and LED light. Different letters indicate a significant difference at p < 0.05 between treatments.

Fig. 9. Mean time to eclosion in days ±SEM for H. armigera raised under fluorescent and LED light. Different letters indicate a significant difference at p < 0.05 between treatments.

Figures

(a)

(b)

(c)

(a) (b)

4 (a)

(b)

(a)

(b)

7 (a)

(b)