We had two major aims in this paper. The first was to evaluate natural variation in ovarian status and reproductive readiness of potential egg-layers, focussing on the brood provisioning phase of the colony cycle. We did this by examining ovarian development in control females, which did not receive a methoprene dose. The second was to evaluate the extent to which methoprene treatment would influence ovarian development, by comparing ovarian development in bees treated with varying doses of methoprene. We also examined the time course of methoprene influence, comparing ovarian development in bees 2 or 4 days following methoprene treatment. Finally, we assessed whether methoprene treatment might influence bee survival.
Patterns of ovarian development in control females
In our experiments, control females were maintained in the lab under the same conditions as methoprene-treated females. Control females appeared to be unaffected by topical treatment with a small amount of acetone, as indicated by their general behaviour in the lab, as well as their longevity. We therefore considered their ovarian development to indicate natural patterns of ovarian development.
Control females exhibited a distinct pattern of seasonal change in ovarian development, with relatively low scores in May (week 3), increasing to a peak in mid-June (weeks 7 and 8), and then declining into early July (week 11). In southern Ontario, eastern carpenter bees exhibit a biphasic foraging season (Richards & Course, 2015). Soon after emergence from overwintering hibernation, dominant females in social nests begin foraging, but use the pollen and nectar supplies that they collect, to feed subordinate nestmates. The nestmate provisioning phase varies in length from year to year, but the transition to brood provisioning usually beginning around mid-May (week 5). This transition coincides with the increase in ovarian development observed here, rising to a peak around weeks 7 and 8, which likely signals peak egg-laying and brood provisioning. Brood provisioning then continues for 3–4 more weeks, ending around week 11 or 12, in early to mid-July, when all females in the nesting aggregations cease foraging, mostly remaining inside their nests until re-emerging in late summer.
The observed decline in ovarian development over the last two to three weeks of the brood provisioning phase suggests that female carpenter bees anticipate the end of egg-laying and cease developing their oocytes. The timing of the end of the brood provisioning period is highly consistent from year to year (L. Duff and MH Richards, unpub. data), suggesting that some external cue, such as photoperiod, regulates and limits ovarian development and brood production. Why do carpenter bees limit brood production rather than continuing to lay eggs while brood provisions are available? The answer likely relates to extended parental care, a common trait of carpenter bees in which females feed newly eclosed adult offspring prior to overwintering (Hogendoorn & Velthuis, 1999; Lewis & Richards, 2017). After egg-laying ceases, adult females remain inside their nests until late summer, when their brood begin to eclose as adults. Adult females then resume foraging to feed their adult brood. In some carpenter bees, such as C. calcarata, adult offspring that are not fed after eclosion do not survive the following winter (Lewis & Richards, 2017). A regulated decline in ovarian development suggests a trade-off in which female carpenter bees cease brood production early, in order to ensure they survive long enough to provide extended maternal care to their adult brood.
Effects of methoprene treatment on ovarian development
Methoprene treatment had no effect on the number of oocytes, but did increase the volume of oocytes, including the largest ones. This was most apparent with VOL3, the most precise and probably the most informative of the three volumetric measures of ovarian development. Given the large size of X. virginica eggs compared to their body size (Michener, 1973), it makes sense that energy diverted towards reproduction would mainly go to eggs most ready for laying. Females responded to methoprene treatment in a dose-dependent manner, with higher dosages producing higher ovarian development scores. We used dosages of 100, 500 and 1000 µg per bee. The lowest dose, 100 µg, was insufficient to raise ovarian development above levels observed in control bees, but dosages of 500 and 1000 µg were effective. Studies of smaller hymenopterans have used doses below 200 µg, and as low as 30 µg (Bortolotti et al., 2020; Huang et al., 2018; Tibbetts & Sheehan, 2012), whereas a recent study on Bombus impatiens, which are almost as large as carpenter bees, used methoprene dosages similar to our study (Wynants et al., 2023).
As bees killed 2 or 4 days after methoprene treatment had similar levels of ovarian development, methoprene effects must have occurred within 2 days. This is consistent with studies in other hymenopterans, in which methoprene induced increases in ovarian development in as little as 3 days (Tibbetts & Sheehan, 2012). The speed of methoprene effects in X. virginica contrasts with results of field experiments in which primary females were removed from nests, creating opportunities for secondary replacement: on average, it took replacements females about five days to start brood-provisioning, suggesting that it took that long to produce a mature, full size oocyte (Vickruck & Richards, 2018). Carpenter bee eggs are among the largest relative to maternal body size (Michener, 1973) and oocytes may be slow to mature. In a Georgia population of X. virginica, the laying interval between consecutive eggs was 2–3 days (Gerling & Hermann, 1978), whereas in X. pubescens, the laying interval between consecutive eggs is up to seven days, and sometimes longer (Blom & Velthuis, 2010). It would be interesting to investigate whether methoprene treatment would shorten the inter-egg laying interval.
The linear models used to detect methoprene effects also revealed a body size effect that was not evident in the analyses of control females alone: larger females had both more and larger oocytes than smaller females. This result also contrasts with previous studies on the same carpenter bee population, in which no such relationship between body size and ovarian development has been detected (Richards, 2011; Vickruck & Richards, 2018). We believe that the association between body size and ovarian development is a subtle effect, that was detectable only with the large sample sizes available when all methoprene treatment levels were included. Whether higher ovarian development scores in larger females translate into increased egg-laying capacity remains an open question, although larger eggs might contain more nutrients promoting larval growth and survival (Kim, 1997).
Effects of methoprene on survival
We compared long-term survivorship of control bees and bees that received 500 or 1000 µg of methoprene. We found no differences in survival, suggesting that methoprene treatment did not exert sublethal toxic effects. Nearly half of all bees in the survival experiment were still alive after one month, suggesting that they would have survived through most of the brood provisioning phase had they remained in the wild. Increases in wing wear throughout their captivity indicated that bees remained active enough to continue damaging their wings. Thus, neither methoprene nor the vehicle control (acetone) had noticeable effects on bee health or survival.