Journal Pre-proof Thermal ﬁtness costs and beneﬁts of developmental acclimation in fall armyworm

Global increases in mean temperatures and changes in precipitation patterns due to climate change, coupled with anthropogenic pathways, have intensified biological invasions of pest insects. Continuous exposure to bouts of acute and chronic heat and fasting stresses (during e.g., droughts) might improve performance under recurring stresses, therefore enhancing/reducing fitness within- or across- life stages (i.e., ‘carry-over’ effects). Here, we examined developmental acclimation effects in the invasive fall army worm Spodoptera frugiperda — a highly invasive economic insect pest of cereal crops, particularly maize — using standardized heat tolerance metrics. Specifically, we assessed the effects of acute (3h) and chronic (3 days) heat treatments (at 32 °C, 35 °C, 38 °C), as well as fasting (48h), on 3 rd instar larvae, and tested fitness traits (critical thermal maxima [CT max ] and heat knockdown time [HKDT]) at a later life stage (4 th /5 th larval instar). Acclimation to heat stress and fasting had significant fitness costs (lower CT max ) across majority of treatments. However, both heat and fasting acclimation improved HKDT (except for 35 and 38°C [acute acclimation] and 35°C [chronic acclimation]). Our results suggest context-specific developmental acclimation costs and benefits in S. frugiperda. In particular, heat and fasting acclimation potentially have fitness costs and benefits for subsequent developmental stages facing high temperature stress. These results are important for estimating the effects of prior stressful events on future survival of invasive insect species and may be significant in predicting pest population dynamics under changing environmental conditions.


Introduction
Biological invasions are a growing ecological and economic threat worldwide (Bellard et al., 2016;Diagne et al., 2021). The success and impact of invasive alien species may interact synergistically with environmental changes, such as increasing temperature for improved survival chances of invasive species, but potential synergies between these processes remain largely unknown (Ricciardi et al., 2021). Insects are among the most impactful taxa worldwide when they invade, owing to a myriad of introduction pathways and rapid humanmediated dispersal, driving some of the highest economic costs worldwide among invasive species (Cuthbert et al., 2021;Venette & Hutchinson, 2021). The fall armyworm (FAW) Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is an economic invasive insect pest of South America origin (Sparks, 1979) that attacks cereal crops, particularly maize. It first invaded the African region through west Africa in 2016 (Goergen et al., 2016;Stokstad, 2017), and by 2017, it had spread to the whole of eastern Africa and parts of southern Africa including Botswana (Day et al., 2017;Stokstad 2017), before spreading to the Middle East and Asia (India) in 2018 (EPPO, 2020;Sharanabasappa et al., 2019).
FAW is a highly multivoltine polyphagous pest, feeding on over 350 plant species across different families (Midega et al., 2018;Montezano et al., 2018;, with a high preference for maize. Larvae feed on leaves, stems, and economic parts of plants e.g., maize cobs, thereby causing economic damage (Rwomushana, 2019). To reach the adult stage, they go through six instar stages, that may take as little as ~11 days (at 32 ℃) (Du Plessis et al., 2020). However, FAW larva has striking host plasticity, varying from five to ten instar stages depending on host plant (Ali et al., 1990;Murúa et al., 2003); reportedly having more `instars on sub-optimal hosts. This host plasticity facilitates development on less favourable hosts (Esperk et al., 2007) or droughts. Given this host plasticity, African invasion by the FAW represent a substantial biosecurity threat. For example, FAW has threatened maize production across the world, e.g., the pest accounted for ~17.7 million tones in maize losses across 12 African countries to date (FAO, 2021). Maize remains nevertheless, a staple food for over 200 millions of people globally (Nuss & Tanumihardjo, 2010) and accounts for 40% of the cereal production in Sub-Saharan Africa (FAOSTAT, 2016) with economic, social and political significance. Thus, FAW continental invasion and associated crop losses exacerbates the food security crises in Africa (see e.g., Sasson, 2012).
Bio-physical environmental conditions experienced during early life stages of an organism are significant determinants of many key fitness life history traits (Chown & Nicolson, 2004).
In particular, factors such as individual diet, feeding frequency, temperature and relative humidity (RH) environments during early-life stages have significant effects on subsequent developmental stages (Mutamiswa et al., 2019;Sasmita et al., 29;Sgro et al., 2016).
Determining the effects of within-developmental stage phenotypic plasticity has been the focus of research for decades (West-Eberhard, 2003;Chown & Nicolson, 2004;Sgro et al., 2016). However, the effects across developmental stages within-generation remain scarce (but see Terblanche & Chown, 2006;Zeilstra & Fischer, 2005), despite their ecological significance. For example, insect developmental stages may be spatially separated, raising questions about how environmental history may differentially shape fitness of subsequent developmental stages within the same generation, but across new environments (e.g., . Indeed, FAW developmental stages may also occupy ≥2 spatially distinct bio-physical environments (Melo et al., 2014) resulting in likely different fitness consequences in subsequent instars. Through adult flight migrations (Nagoshi et al., 2012) and larval silking (FAO, 2018), FAW uses this behavioral adaptation to extend its geographical range into novel areas to circumvent inter-and intra-specific competition.
However, it is not known whether prior environment affects fitness of subsequent developmental stages, or aids invasiveness of FAW. Previous studies have nevertheless documented that overcoming environmental barriers, e.g., of temperature and desiccation tolerance, are critical for invasion success (Richardson & Pysek 2006). For this reason, invasive insects often have high basal stress tolerance, phenotypic plasticity Kelley, 2014;Wan & Yang, 2016;Machekano et al., 2018) and metabolic flexibility (Smit et al., 2021).
Other external stressors, such as lack of food, have also been documented to influence insect thermal tolerance in complex, often in unpredictable ways (Nyamukondiwa & Terblanche, 2009). Fasting can occur when there is a lack of adequate food (due to various environmental perturbations) to meet the energy requirements for biological processes in the insect's body (McCue., 2010). This food deprivation stress is presumed to result in a trade-off in insect thermal tolerance (Scharf et al., 2016) due to cross talk or cross tolerance (Sinclair et al., 2013). Several studies have supported this notion, as fasting pre-treatment often impairs cold tolerance in insects (Gotcha et al., 2018;Kenny et al., 2008). However, fasting acclimation appears not to have any effects on heat tolerance (see Gotcha et al., 2018;Scharf et al., 2016). Nevertheless, how stressful traits in one developmental stage interact with the subsequent developmental stages and environments remains unknown in FAW, despite evidence for phenotypic plasticity within and across ontogeny (see e.g., West-Eberhard, 2003;Sgrò et al., 2016) and across different developmental stages and seasons/environments (i.e., 'carry-over' effects) (Norris, 2005;Harrison et al., 2011;Fayet et al., 2016;Ezeakacha & Yee, 2019).
Despite overwhelming evidence of the effects of prior environment on insect fitness Fayet et al., 2016;Ezeakacha & Yee, 2019), few studies have investigated developmental acclimation effects for invasive insects with a view of making inferences for pest invasiveness. Nevertheless, investigation of the effect of environmental history has large ecological implications for organismal fitness under changing environments. Unravelling developmental acclimation is important in determining how species may react to changes in environment across developmental stages and seasons, and how this may shape their fitness and by extension, their population dynamics. This has downstream implications on designing pest control strategies e.g., through development of early warning systems. Here, we thus examined innate within-generation developmental temperature and fasting acclimation effects on heat tolerance in S. frugiperda following high temperature acute and chronic acclimation, as well as fasting. Given its tropical origin, heat tolerance may remain a key trait that facilitates invasion, and more-so in arid and semi-arid environments such as Botswana. We hypothesise that temperature and food deprivation stress in one instar may have heat tolerance fitness costs or benefits across other subsequent nonacclimated developmental stages (developmental acclimation). Confirmation of positive adaptive developmental acclimation effects may have implications on S. frugiperda invasiveness under heterogeneous stressful environments associated with changing climate.
This knowledge is important in informing spatially-dependant S. frugiperda pest management strategies.

Insect rearing and maintenance
Field populations of S. frugiperda were collected as 2 nd -5 th instar larvae from infested maize crops in two commercial farms; Talana farms (S22°.13467; E28°.59468) and Motloutse River farm, Bobonong Village, Central district of Botswana, and placed in 50 ml vials containing artificial diet, adopted from Tefera et al. (2010). Both collection areas are within the same region and experience similar climatic environments. Specimens were reared in Memmert climate chambers (Memmert GmbH + Co. KG, Schwabach, Germany) in the laboratory at optimal conditions (28±1 ℃, 65±10% RH) and fed on the same artificial insect diet aforementioned (see Tefera et al. 2010). Both pupae and moths were kept in Bugdorm cages (Megaview Science Co., Ltd, Taichung, Taiwan) in climate chambers. All adult moths were fed on 10% sucrose solution ad libitum. To obtain the next generation of FAW for experimental use, moths were mated in oviposition cages containing a 4-week-old maize plant (as oviposition substrate). Following oviposition, eggs were allowed to incubate and hatch on the host plant. After hatching, 1 st instar neonates were subsequently transferred to vials containing the artificial diet (Tefera et al., 2010). Each vial comprised three larvae, reared up to 3 rd instar after hatching in the laboratory. However, following moulting to the 3 rd instar stage, all larvae were transferred into individual vials in preparation for the experiments and simultaneously to circumvent larval cannibalism, which is usually more apparent from the 3 rd instar onwards (see Rwomushana, 2019). Experiments were run using these lab-reared 3 rd instar specimens from F 1 to F 4 generations, randomised across the treatments. We assumed that laboratory adaptation has insignificant effects on thermal fitness across the three tested generations, as has been observed in similar experiments (Opperman 2018; but see Hoffman et al., 2001). Acclimation treatments were done following moulting of 3 rd instar larvae, and heat tolerance traits (i.e., critical thermal maxima [CT max ] and heat knockdown time [HKDT]) were tested on 4 th instar larvae following acute acclimation and 5 th instar larvae following chronic acclimation (as majority of the larvae moulted twice during the 3day chronic acclimation plus one day recovery period).

Acclimation experiments
Experimental treatments (acclimation) comprised acute and chronic sub-lethal high temperature acclimation and fasting. This was undertaken in 3 rd instar larvae by exposing insects to temperatures of 28.0 (control) 32.0, 35.0 and 38.0 ± 1.0 °C (each under 65 ±10% RH) for 3 hours (acute) and 3 days (chronic) acclimation treatments (see Table 1) in Memmert climate chambers. Temperatures selected for acclimation were ecologically relevant and based on a previous study that showed temperatures across Botswana to reach up to 42 ℃ during heat waves (Moses, 2017;see Fig. 1), and considering that the optimal temperature range of S. frugiperda is 26-30 ℃ (Du Plessis et al., 2020). From the optimum temperature range, 28 ℃ was selected as the control temperature and 3-4 ℃ was added to establish mild high temperatures for acclimation, based on modified protocols from Mutamiswa et al. (2019). Control insects were kept at optimal environmental conditions of 28±1℃ and 65±10% RH during experimental treatments before measuring thermal fitness traits ( Fig. 1). Following both acute and chronic acclimation, insects were allowed to recover at optimal conditions (28±1℃ and 65±10% RH) for 24 hours before measuring physiological traits.
To determine the effects of feeding status on the thermal fitness of S. frugiperda, 3 rd instar larvae were deprived of food (fasted) for 48 hours. All fasting acclimations were done using a constant time period (48 hours), and results were directly compared to those of acute and chronic temperature treatments. Larvae were removed from artificial diet at 3 rd instar and individually placed into empty 50 ml vials without any food, but with a water source (cotton wad, to prevent desiccation associated mortality) for 48 hrs. The larvae were kept under benign conditions (28±1 ℃, 65 ± 10% RH; 12L:12D) to ensure that food deprivation was the only limiting factor. Post 48 hrs, larvae were returned to individual vials with access to food (artificial diet) and water ad libitum for 24 hours to allow recovery. Measurement of thermal traits was conducted 24 hours post-recovery following methods by Gotcha et al. (2018).
Control larvae were provided with artificial diet and kept at optimal temperatures and RH (28±1 ℃ and 65±10%) throughout prior to running experiments

Heat tolerance metrics
To test the effects of heat and fasting acclimation on heat tolerance, (i) CT maxthe maximum temperature allowing insect activity, and (ii) HKDTthe time taken to knock down an insect following acute heat stress, were measured (Chown & Nicolson, 2004). Both traits are ecologically sound heat tolerance indices (Lutterschmidt & Hutchison, 1997;Huey & Kearney, 2020) and correlate well with insect biogeographical patterns. For CT max , individual 4 th instar (for acute acclimation) and 5 th instar (for chronic acclimation) larvae were placed into an insulated double jacketed chamber with ten 'organ pipes' connected to a programmable bath filled with 1:1 water: propylene glycol, which regulates the flow of liquid around the chamber (Grant GP200-R4, Grant Instruments, UK) (Nyamukondiwa & Terblanche, 2009). Critical thermal maxima experiments started at 28 °C (FAW optimum temperature) from which temperature was gradually increased using a ramping rate of 0.25 ºC/min until the larvae reached upper temperature limit of activity (CT max ) (Nyamukondiwa & Terblanche, 2009) (Table 1). This ramping rate is faster than the natural diel increase in temperature, but nevertheless slower and thus ecologically more relevant than other ramping rates used in literature e.g., 0.5 °C/minute (reviewed in Chown & Nicolson, 2004). A thermocouple (type K, 36 SWG) connected to a digital thermometer (Fluke 54 series II, Fluke Cooperation, China; accuracy: 0.05 °C) was inserted into the organ pipe to record the chamber temperature. The experimental procedure was repeated 3 times to yield n ≈ 30 larvae per treatment (30 replications). In this study, CT max was defined as the temperature at which an individual larva lost co-ordinated muscle function (self-righting) and ability to respond to mild prodding using a thermally inert object.
Heat knockdown time was assessed on 4 th instar larvae (following acute acclimation) and 5 th instar larvae (following chronic acclimation) using standardized protocols (Nyamukondiwa & Terblanche, 2009). We used treatment-specific heat knockdown temperatures, derived from each treatment's CT max value plus 2 ℃. This heat knockdown temperature is ecologically sufficient to elicit heat knockdown effects in insects (see e.g., Hoffman et al., 2003;Mutamiswa et al., 2019). Thus, knockdown temperatures of 53.0, 50.0, 51.4, and 50.7 ℃ were used as acute knockdown temperatures for 28 (control) 32, 35, and 38 ℃ acclimation pre-treatments respectively, whereas 53.0, 51.6, 51.9 and 50.8 ℃ were used as chronic knockdown temperatures for 28 (control) 32, 35, and 38 ℃ acclimation pre-treatments, respectively. Ten individual larvae were placed in 1.5 ml microcentrifuge tubes and placed in a Memmert climate chamber (HPP 260, Memmert GmbH + Co.KG, Germany) set at various temperatures as indicated above. Temperatures above CT max cause heat coma in insects and are often used in HKDT assays (see Nyamukondiwa et al., 2011). A video recording camera (HD Covert Network Camera, DS-2CD6412FWD-20, Hikvision Digital Technology Co., Ltd, China) linked to a computer was connected to the climate chamber and used to monitor knockdown activity and timing. Heat knockdown time was defined as the time (in minutes) at which an individual larva lost activity following acute heat stress.

Data analysis
Data analyses were all performed using R, version 4.1.1 (R Development Core Team, 2021).
The residuals were first checked for normality and variance homogeneity using Shapiro-Wilks and Levene's tests, respectively, and were found to violate normality and homogeneity of variance assumptions. Kruskal-Wallis tests were thus used to examine CT max and HKDT as a function of treatment for each respective acute and chronic exposure treatment (i.e., four separate models). Dunn tests were used post-hoc for pairwise comparisons, with p-values adjusted via the Holm method (Ogle et al., 2021).
We summarized the magnitude of both CT max and HKDT following acclimation using methods by Tarusikirwa et al. (2020) and Mutamiswa et al. (2019). Specifically, we calculated the magnitude of change in thermal fitness conferred by the acclimation treatment using the formula below, where the mean heat tolerance (CT max / HKDT) after each treatment was divided by the mean control CT max or HKDT results of which were tabulated into Table   1.

Chronic and acute acclimation had contrasting results on the direction of both CT max and
HKDT plasticity (Table 1). Both acute and chronic acclimation, as well as fasting, yielded negative deleterious plastic effects (i.e., negative magnitude; see Table 1) for CT max , whereas positive effects on HKDT were exhibited following chronic exposure and fasting, but not acute exposures beyond 32 °C.

Discussion
Population dynamics of individuals that may occupy multiple spatially-distinct habitat environments can be highly complex (Webster et al., 2002). As such, sub-lethal stressful conditions experienced during early developmental stages e.g., early instars of the larvae, may be important in determining key life history traits, either manifesting as beneficial through e.g., developmental acclimation effects (see Chown & Nicolson, 2004) Given the mobility of its life stages e.g., larva (through silking) and adults (through flight), it is largely unknown how previous environments may shape fitness of the same and/or subsequent life stages, and by inference, invasion propensity thereof. Our results showed that most acute and chronic heat acclimation treatments, as well as fasting, significantly depressed CT max . This result means that sub-lethal food deprivation and heat stress at the 3 rd instar stage larvae may have negative CT max fitness consequences on 4 th and 5 th instar larvae and probably by extension, other subsequent developmental stages. Climate change is often associated with episodes of acute and chronic heat stress, and prolonged droughts that may limit food resources (IPCC, 2014;Stillman, 2019). Thus, the deleterious effects of acclimation treatments recorded here may mean that frequent episodes of environmental heat and food deprivation stress faced in nature may offset heat tolerance of subsequent developmental stages, affecting population dynamics of invasive species. The reason for the negative effects of heat acclimation reported here are largely unknown. However, it may point to the notion that the stress faced during acclimation treatment and CT max assays is additive (see Jorgensen et al., 2021). Moreover, we also speculate that we may have missed certain acclimatory cues or specific treatment combinations that specifically elicits CT max acclimation responses. Thus, future studies may need to explore differential temperature and time combinations at all higher instar stages of the larvae that may elicit acclimation. One more interesting result observed here is that a treatment to one stress may also have negative effects on a divergent stress. For example, 3 rd instar acute and chronic fasting acclimation had deleterious consequences on 5 th instar larval CT max . This may point to the notion that injury associated with diverging environmental stresses may be the same (see e.g., Shen et al., 2015;Farahani et al., 2020), and that divergent stress effects may thus be additive. Nevertheless, the lack of beneficial acclimation effects for heat acclimation reported here is consistent with studies on Tuta absoluta, that reported no beneficial acclimation effects following chronic high temperature acclimation (Tarusikirwa et al., 2020).
By contrast, both acute and chronic 3 rd instar larvae acclimation had beneficial effects on subsequent larval HKDT, albeit for specific treatments (32 ℃ and fasting for acute acclimation, and 32, 38 ℃ and fasting for chronic acclimation). Conditions eliciting acclimation responses are highly complex and often context-dependent (Chown & Nicolson, 2004;Sgro et al., 2016;Mutamiswa et al., 2019). This agrees with our results, that observed positive acclimation responses were specific to certain heat acclimation groups and corroborates with previous reports suggesting that conditions conferring acclimation responses are highly context-dependent (see Mutamiswa et al., 2019). Similarly, acute heat acclimation at 35 and 38 ℃, and chronic heat acclimation at 35 ℃, had no significant effects on HKDT. This result means that heat wave episodes associated with climate change on 3 rd instar larvae of S. frugiperda may have positive or neutral effects on subsequent developmental stages in the context of HKDT but not CT max . Thus, S. frugiperda may have fitness benefits under projected heat stress in terms of enduring long durations of mild to high temperature stress associated with changing climates, potentially translating into greater invasiveness and resilience in high temperature tropical habitats.
Comparisons for HKDT experimental traits have often been investigated using a more constant temperature (see Chown & Nicolson 2004). However, here, we used different heat knockdown temperatures to investigate HKDT across different acute and chronic temperature treatments. Thus, differences in results reported here may also be partly due to the treatmentspecific heat knockdown temperature methodology used in our study. Furthermore, the results also showed the positive beneficial effects of acclimation to a divergent stress trait (fasting) on a different stressor (heat tolerance [HKDT]). Such cross tolerance represents shared co-evolutionary response mechanisms to stress traits involved (see Gotcha et al., 2018), and represent another facet that may help invasive species survive highly variable stressful environments, e.g., through integrated stress resistance (see discussions in Renault et al., 2015). Cross tolerance results reported here are nevertheless in contrast with reports on Ceratitis rosa, where fasting resulted in increased CT max and reduced HKDT (Gotcha et al., 2018). These results indicate that fasting has dissimilar effects on different traits used to measure heat tolerance, and that elicitation of acclimation responses are trait dependant.
Results obtained here thus mean that food deprivation (i.e., temporary absence of host plants) during mid-season droughts, and other plant-damaging natural disasters such as hailstorms, may provide a benefit in periods of rapid heat shock, such as heat waves, through cross tolerance developmental acclimation effects. Increased heat tolerance (HKDT) reported here for certain treatments may represent context-specific expression of heat shock proteins and other hormonal heat stress regulators (Hoffmann et al., 2003;King & Macrae, 2015). Heat shock proteins and hormonal activation due to heat stress usually occur over a certain temperature threshold, which are context-dependent but usually range from 39 -41 °C (Qazi et al., 2019). Similar results were observed in Drosophila mojavensis (Patterson & Crow, 1940), where high temperature acclimation at one developmental stage increased HKDT in subsequent developmental stages (Diaz et al., 2021). Indeed, sublethal high temperature stress may influence physiological traits in addition to other life history traits e.g., fecundity and longevity (Nguyen & Amano, 2010). A study on Plutella xylostella (L.) showed that high temperature acclimation on larval stages had effects on adult stages, affecting oviposition patterns and adult life span (Zhang et al., 2015). Our results thus indicate that timing of sublethal high temperature stress such as heat waves may enhance thermal fitness and survival of other life stages of invasive pest insects. Therefore, survival of S. frugiperda may increase in hot tropical areas even during high temperature incidences. Inclusion of effects of extreme high temperature incidences in pest management forecasts will increase accuracy of invasive species biogeographical patterns (Skendžić et al., 2021). Likewise, processes and events at one developmental stage and in a particular season may have far-reaching consequences for other developmental stages in a different season; a phenomenon called 'carry-over' effects (Harrison et al., 2011). While this phenomenon has largely been explored in migratory birds, little is known on how this affects the fitness of migratory insects in general (but see Galarza et al., 2019), and invasive insects in particular. Future work should accordingly examine carry-over effects in S. frugiperda and assess how they may shape population dynamics and pest invasiveness. We also observed that acclimation responses were trait-dependant and observed such positive effects only for HKDT; a more acute heat stress metric compared to CT max . We thus speculate, with caveats, that the differences in chronic exposure to stress during CT max and HKDT assays may also have affected differential acclimation responses reported here. Future studies should thus look at more acute CT max ramping methodologies e.g., 0.5 or 1 °C/minute (see e.g., Terblanche et al., 2007) to try and capture potential acclimation cues that we may have missed.
Costs and benefits of plastic acclimation are difficult to predict and are not uniform across species, metrics tested and acclimation treatments. A study done by Alemu et al., (2017) showed that age, increasing rate of temperature change and duration of heat hardening resulted in a benefit or increase in CT max . Factors such as body mass have also been shown to have a cost on HKDT (Nyamukondiwa et al., 2018 (Zhang et al., 2015). Our results provide a step into understanding costs population dynamics. Future pest forecasting models should thus incorporate developmental acclimation responses and by extension, 'carry-over' and integrated stress resistance effects (see e.g., Norris & Taylor, 2006;Nyamukondiwa et al., 2022) to potentially improve accuracy of model outputs and pest early warning systems.

Availability of data and material
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Submission declaration and conflict of interest statement
This work has not been previously published. Authors declare no conflict of interests.

Consent for publication
Not applicable

Competing interests
All authors declare no conflict of interests.

Funding
Science and Technology (BIUST). RC acknowledges funding from the Alexander von Humboldt Foundation.

Authors' contributions
Project conceptualization and management: BS HM CN.
Formal analysis: RC CN.
Visualization and validation: BS HM RC CN.
Writing, review and editing: BS HM RC CN.