Global climate change has played a role in the alarming declines in abundance and diversity of insects (along with other factors; Bálint et al., 2011, Wagner, 2020, Harvey et al., 2022). Increases in mean temperatures over the last century are clearly associated with shifts in distributions and timing of key life history events (Parmesan and Yohe, 2003). It has also become increasingly clear that more frequent climate extremes (Masson-Delmotte et al., 2021, Meehl and Tebaldi, 2004) may have pronounced effects on insects (Buckley and Huey, 2016, Kingsolver et al., 2013, Ma et al., 2021).

Thermal tolerance metrics have provided a compelling approach to connect direct effects of temperature on individuals to climate-associated geographic distributions of diverse organisms, including insects (Addo-Bediako et al., 2000, Sunday et al., 2011). Estimates of the minimum and maximum temperatures at which organisms can optimally function (CTmin and CTmax, respectively) have been used both to track responses to climate change (Geerts et al., 2015; Kellermann and Heerwaarden, 2019) and to model potential long-term impacts of climate change on insect populations (Williams et al., 2015). However, insects are exposed to and are affected by temperatures well above CTmin and well below CTmax (Dillon et al., 2010, Huey et al., 2012, Kingsolver et al., 2013), suggesting that a sole focus on temperatures that lead to whole organism loss of function may be underestimating the impacts of climate change (Ma et al., 2021).

For many insects, reproductive physiology is far more sensitive to temperature than other aspects of behavior, physiology, and ecology (Walsh et al., 2019). The temperatures at which fertility, and therefore the reproductive fitness, decreases may better indicate how populations will respond to changing temperatures (Harvey et al., 2020). Several studies have shown that declines in fertility at temperatures below an insect’s CTmax. In Osmia bicornis, males exposed to long-term exposure of 22-26°C (warm temperatures below CTmax) experienced a disruption of male mating signals (Conrad et al., 2017). In queen and male Apis mellifera and male Tribolium castaneum, short-term exposure to warm temperatures below their CTmax led to significant decreases in spermatozoa viability (Pettis et al., 2016; Sales et al., 2018; Stürup et al., 2013). One way to characterize these types of responses is thermal fertility limits (TFL), or the temperatures at which a proportion of an organism’s gametes become sterile. This methodology has been applied to several insect species using a variety of fertility measurements (Walsh et al., 2019). For example, the TFL in male Drosophila is ∼30°C (10° below their CTmax) and males experience sterility at lower temperatures compared to females (van Heerwaarden and Sgrò 2021). Much of the literature around TFL is focused on the upper thermal limits of organisms, while the impact of exposure to lower TFL are not well understood. Assessment of TFL at upper and lower limits in insect species may explain vulnerability that CTL data has been underestimating.

Bumble bees are key native pollinators in ecosystems across the world (Goulson, 2010, Woodard, 2017) and are also managed for commercial pollination. Over the past several decades, many species have experienced striking range shifts and local extirpations that are clearly associated with changing climates (Kerr et al., 2015, Soroye et al., 2020) above and beyond other factors like land-use changes, pesticides, and pathogens (Goulson et al., 2015). However, the mechanisms by which changing global temperatures are causing declines in bumble bees is not clear (Maebe et al., 2021), particularly given that, relative to other ectotherms (Sunday et al. 2011), they appear to be quite tolerant in particular to heat and, to a lesser extent, cold (Keaveny et al., 2022, Maebe et al., 2021, Martinet et al., 2015, Oyen et al., 2016, Oyen and Dillon, 2018, Pimsler et al., 2020). Most studies have focused on the temperatures that lead to loss of function (CTmin and CTmax) in female workers (Pimsler et al., 2020, Oyen and Dillon, 2018; but see Oyen et al., 2016), with comparatively little work exploring effects of less extreme temperatures on males (Martinet et al., 2021a, 2021b).

Male bumble bees have been regarded as ‘simple and small mating machines’ (Tsuji, 1996).

Though underappreciated, they play an important role in reproduction and therefore colony growth and population persistence (Belsky et al., 2020). Males emerge in late summer to fall when the colony produces reproductives (males and gynes). Males eclose with a fixed amount of spermatozoa that they cannot replenish as their testes become inactive once mature (Baer, 2003). Once they reach sexual maturity (5-8 days post eclosion when spermatozoa migrate to seminal vesicles; Baer, 2003), males disperse in search of mates (Belsky et al., 2020). As they spend all their time outside the thermoregulated nest, males may be more vulnerable than females to gradual shifts in environmental temperatures and to thermal extremes (Pottier et al., 2021). After mating, the queen stores the spermatozoa from one or more males (mon- or polyandrous, depending on the species; Schmid-Hempel and Schmid-Hempel, 2000) throughout the overwintering period. Production of fertilized eggs throughout the following season of colony growth therefore depends on successful storage of healthy spermatozoa, so queens that mate with males with low quality spermatozoa may be burdened with reduced reproduction the following year.

Some work suggests that fertility of male bumble bees may be reduced by heat exposure. For three species of bumble bees, males held at 40°C until reaching heat stupor (∼60-700 min.) had significantly more spermatozoa mortality and degradation of spermatozoa DNA relative to controls (Martinet et al., 2021b). While this is compelling, it is unlikely for a male to mate once experiencing heat stupor, which results in severe stress and often mortality. Whether shorter-term exposure to hot temperatures similarly affects fertility is unclear. Further, little research has explored the effects of ecologically relevant cold exposure on insect fertility (Denlinger and Lee, 1998). Bumble bees are freeze avoidant, have high survival at low temperatures near their super cooling point (SCP; Keaveny et al., 2022), and can recover from chill coma quickly (Oyen et al., 2021), but it is unknown if exposure to temperatures at or below male CTmin reduces fertility.

Here, we present our findings on the effect of sublethal heat and cold exposure on spermatozoa viability in Bombus impatiens, typically a polyandrous species (Bird et al., 2022, Cnaani et al., 2002) historically found across the Eastern United States and Canada and now naturalized in many areas of the western US and Canada due to its use in commercial pollination. Determining potential effects of extreme temperatures on B. impatiens males is an important starting point for establishing the vulnerability of other bumble bee species and could give insight into their decline, in regard to reproduction. Hence, this study measured spermatozoa viability in males before and during the heat stupor, as well as short- and long-term effects of being in chill coma in the seminal vesicles of whole organism and in seminal vesicle in vitro to assess the thermal limits of fertility of male B. impatiens.