Low temperatures associated with winter can impose strong selective pressure on organisms (Marshall et al., 2020, Sinclair et al., 2003, Williams et al., 2015). Ectothermic animals are particularly susceptible to the stressors associated with winter because their internal body temperature generally reflects that of their environment. Temperatures below 0°C can therefore cause ectothermic animal body fluids to freeze, resulting in damage that is often lethal (Lee, 2010, Toxopeus and Sinclair, 2018). Many studies have documented overwintering biology of various insect species, including adaptations such as cold tolerance and diapause (Hand et al., 2016, Lee, 2010, Sinclair et al., 2015, Toxopeus and Sinclair, 2018, Wilsterman et al., 2021). In addition, there is increasing interest in exploring the overwintering biology of closely interacting species. For example, the relationship between the cold tolerance and diapause phenotypes of parasitoids and their host species may alter interspecific dynamics as climates change or geographic ranges of populations shift (Colinet and Boivin, 2011, Le Lann et al., 2021). It is in this vein that we investigate cold tolerance and diapause in a guild of interacting endoparasitoid wasps and their host, the apple maggot fly, Rhagoletis pomonella Walsh (Diptera: Tephritidae).

Insects that overwinter in temperate climates have evolved physiological cold tolerance strategies to survive the challenges associated with low temperatures. Freeze-tolerant insects survive freezing of their body fluids, while freeze-avoidant insects depress the temperature at which ice formation begins (supercooling point; SCP) to survive sub-zero temperatures in an unfrozen state (Lee, 2010, Toxopeus and Sinclair, 2018). The ability to survive short exposures to extreme temperatures is important for defining an insect’s lower lethal temperature (Sinclair et al., 2015, Toxopeus et al., 2019, 2016). However, many temperate insects overwinter in relatively mild environments (e.g., temperatures close to 0°C in the soil under a layer of insulative snow cover). Therefore, characterizing survival following both short exposure to extreme conditions (acute cold tolerance) and long exposure to milder chilling conditions (chronic cold tolerance) is important for predicting overwintering survival (Roberts et al., 2021, Sinclair et al., 2015).

In addition to cold tolerance, diapause is an important adaptation for insects overwintering in temperate climates, and diapause phenotypes can vary substantially within and among species. Prior to the onset of winter, many temperate insects enter diapause, a state of developmentally-programmed dormancy associated with metabolic rate suppression and enhanced stress tolerance (Hand et al., 2016, Koštál, 2006, Wilsterman et al., 2021). While diapause is obligate (necessary for completion of the life cycle) in some insect species, many species exhibit interindividual variation in diapause phenotypes based on environmental conditions (Hodek and Hodková, 1988, Koštál, 2006). For example, relatively warm fall temperatures may cause some individuals of a diapause-capable species to completely forgo (avert) diapause prior to winter (Hodek and Hodková, 1988, Koštál, 2006). In this study, we refer to these as ‘non-diapause’ phenotypes; their diapause duration is 0 days (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021). In mild winter climates, these non-diapause insects may be winter active (Alfaro-Tapia et al., 2021; Tougeron et al., 2017). However, diapause is often necessary for overwintering survival and for the proper synchronization of growth and reproduction with favorable environmental conditions (Hand et al., 2016, Koštál, 2006, Tauber and Tauber, 1976). Thus, averting or prematurely terminating diapause can cause decreased cold tolerance and overwintering survival (Boiteau and Coleman, 1996, Ciancio et al., 2021, Lee and Denlinger, 1985, Lehmann et al., 2018, Toxopeus et al., 2021).

Insects can exhibit variation in the depth that they enter diapause (e.g. degree to which metabolic rate is suppressed) and in the intensity that they maintain diapause, i.e., the tendency to remain in diapause despite favourable growth conditions (Wilsterman et al., 2021). For example, the apple maggot Rhagoletis pomonella exhibits two diapause phenotypes with the same depth (metabolic rate suppression) and different intensities (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021). A small (< 25%) proportion of R. pomonella enter ‘weak diapause’ (also called ‘shallow diapause’) R. pomonella pupae and remain in this state for a short duration (35 – 70 days) if exposed to warm conditions prior to winter. In contrast, many R. pomonella enter a higher intensity diapause program (also called ‘chill-dependent’) that does not easily terminate at warm temperatures until after a period of prolonged chilling (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021). Populations containing a mix of weak (low intensity; short duration) diapause and high intensity (longer duration) diapause individuals can exhibit bi- or multi-modal distributions of diapause termination and resumption of post-diapause development over time (Calvert et al., 2022, Dambroski and Feder, 2007, Masaki, 2002). The weak diapause phenotype and its impact on overwintering biology has not been documented in many insect taxa (Masaki, 2002, Wilsterman et al., 2021).

Several studies have examined the cold tolerance and diapause of parasitoids, often with a focus on their potential as biocontrol agents (Colinet and Boivin, 2011, Le Lann et al., 2021). Endoparasitoids feed and develop to adulthood within a single host organism, usually killing the host in the process (Godfray, 1994, Hood et al., 2021). The parasitoid cold tolerance literature is dominated by studies of wasps (Hymenoptera) that parasitize insect pests from many different insect orders, including Diptera (e.g., drosophilids; Amiresmaeili et al., 2020, Li et al., 2015, Murata et al., 2013), Lepidoptera (e.g., pyralid moths; Carrillo et al., 2005, Foray et al., 2013), Coleoptera (e.g., emerald ash borer; Hanson et al., 2013), and Hemiptera (e.g., aphids; Alford et al., 2017, Colinet and Hance, 2010, Tougeron et al., 2018). However, studies that examine multiple parasitoids that attack a single host are rare (e.g., Hanson et al., 2013), and in these cases the immature life stages of multiple parasitoid species may never interact due to distinct geographic distributions (e.g., Murata et al., 2013) or strong differences in life history timing (e.g., Le Lann et al., 2011). Rhagoletis pomonella is a pest of commercial apples that is parasitized by multiple wasp species with overlapping geographic distributions and life history timing (Feder, 1995, Hood et al., 2012). This system provides an opportunity to study the cold hardiness and diapause of species both within a guild of parasitoids and across trophic levels between parastiods and their host flies.

The phenology of R. pomonella is closely linked to its host fruits throughout its range in North America. Adult R. pomonella lay their eggs in ripe fruit of hawthorns (Crataegus spp.) and introduced, domesticated apples (Malus domestica) in many regions of North America during the late summer or fall (Dean and Chapman, 1973, Feder et al., 1993, Hood et al., 2013). Larvae develop within those fruit, burrow out of fruit that have dropped to the ground, and then pupariate in the soil, where they remain for 10 to 11 months of the year (Boller and Prokopy, 1976, Dean and Chapman, 1973). Most of these pupae in the northern part of the flies’ range enter a high-intensity diapause and remain recalcitrant to diapause termination and post-diapause development until late summer of the following year (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021), resulting in a univoltine life cycle. Changes to the timing of diapause induction or termination in R. pomonella can cause mortality, especially if post-diapause adults are active when host fruits are not available.

Genetic, evolutionary, and physiological studies in R. pomonella have established that overwintering, diapausing pupae have relatively low and invariant SCPs, but their diapause development can be disrupted by high temperature (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021). Overwintering R. pomonella pupae from apple fruits are freeze-avoidant down to c. -20°C and have similar acute cold tolerance regardless of diapause status (Toxopeus et al., 2021). Although high-intensity (longer duration) diapause is likely important for overwintering survival in natural environments, both non-diapause and weak (short duration) diapause pupae have been observed when R. pomonella are held in warm (>20°C) conditions for several weeks in the laboratory (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021). These weak diapause individuals differ in allele frequencies from those that enter a high-intensity diapause or avert diapause altogether (Calvert et al., 2022). In some populations, more than half of the individuals may avert or prematurely terminate diapause under extended warm conditions in the laboratory, and the proportion that do so can vary with latitude and host fruit (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021). The incidence of non-diapause and weak diapause pupae under field conditions have not been well documented, but occasionally non-diapausing adult flies can emerge late in the season (JLF personal observation). However, both non-diapause and weak diapause phenotypes are likely to be lethal in nature. For example, pupae that terminate diapause prior to simulated winter have poor (< 20 %) survival following 24 weeks at 4°C (Toxopeus et al., 2021). Even if they survive winter conditions, non-diapause and weak diapause individuals typically eclose as adults more quickly post-winter than high-intensity diapause individuals (Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021), which may result in mismatches between adult activity and host fruit availability. Combined studies applying artificial selection and population genetic surveys suggest that natural selection is acting on these diapause phenotypes in populations that experience relatively longer and warmer pre-winter conditions (Doellman et al., 2019, Dowle et al., 2020, Egan et al., 2015, Hood et al., 2020, Powell et al., 2020, Ragland et al., 2017), with potential implications for their overwintering survival.

Three endoparasitoid wasps (Hymenoptera: Braconidae: Opiinae) attack R. pomonella in the northeastern and midwestern United States: Utetes canaliculatus Gahan, Diachasma alloeum (Muesebeck), and Diachasmimorpha mellea Gahan (Forbes et al., 2010, Hood et al., 2015, Hood et al., 2012, Wharton and Marsh, 1978). In these regions, D. alloeum is usually the most prevalent parasitoid of R. pomonella that infest apples and hawthorn fruits, followed by U. canaliculatus and D. mellea (Feder, 1995, Hood et al., 2015, 2012). Each parasitoid’s life history is closely linked to that of its host but varies slightly among species. Utetes canaliculatus oviposits into R. pomonella eggs, and has an earlier but overlapping eclosion phenology with D. mellea and the later emerging D. alloeum, which both oviposit into second and third instar larvae of R. pomonella (Forbes et al., 2010, Hood et al., 2015). Although multiple species may infest the same immature fly (Hood et al., 2012), only one wasp larva will survive to completely consume the host within its puparium (tanned outer case) soon after host pupariation (Forbes et al., 2010, Hood et al., 2015, Lathrop and Newton, 1933). While most of these wasp larvae are assumed to overwinter in diapause (Forbes et al., 2009, Hood et al., 2015), the diapause phenotypes and ability to survive extreme low temperature of each parasitoid species have not been systematically characterized to the same detail as their fly host (cf. Calvert et al., 2022, Dambroski and Feder, 2007, Toxopeus et al., 2021).

Our goal in the current study is to determine whether the guild of endoparasitoid wasp species that attack R. pomonella fruit flies exhibit similar or different overwintering cold tolerance compared to their host, and whether the parasitoids might also be susceptible to diapause disruption under warm pre-winter conditions. Although many R. pomonella studies compare populations that infest apple and hawthorn fruits (e.g., Calvert et al., 2022, Doellman et al., 2019, Dowle et al., 2020, Egan et al., 2015, Hood et al., 2020, Powell et al., 2020, Ragland et al., 2017), here we focus on hawthorn-infesting populations only, which tend to have higher rates of parasitism from each of the three wasp species than populations attacking apple-infesting flies (Feder, 1995, Hood, 2016, Hood et al., 2015). We characterize and compare the cold tolerance of U. canaliculatus, D. alloeum, and D. mellea to each other and to R. pomonella collected from hawthorn fruits using several metrics, including cold tolerance strategy, SCP, acute cold tolerance, and chronic cold tolerance. In addition, we use respirometry and laboratory eclosion experiments of the wasps to detect variation in diapause duration phenotypes for comparison to their fly host. Given increasingly warmer fall and winter temperatures (Le Lann et al., 2021, Marshall et al., 2020), our study lays the ground work for future investigations of the interaction between diapause phenotype and cold tolerance in these parasitoid species.