Body size and diapause decision
Accumulating the materials to build an adult body takes time; an
organism reaching adulthood within a shorter period of time must
therefore either mature at a smaller size, compensate for the lost
growing time by accumulating mass at a faster rate, or some combination
thereof (Abrams et al., 1996; Davidowitz & Nijhout, 2004). P.
aegeria skews strongly toward the latter option: the considerable
variation in development rates between the treatments was matched to a
large extent by variation in growth rates (defined as average weight
gain per unit time) (Fig. S1), resulting in comparatively small
variation in final size.
To the extent that body size differences between treatments emerged,
they were mostly found in the Öland population. This is consistent both
with earlier observations (Aalberg Haugen & Gotthard, 2015) and with
adaptive expectations. The Stockholm population is univoltine, rarely
expressing the nondiapause pathway in the field, hence the body
size-diapause correlation is presumably not maintained by selection
(Aalberg Haugen et al., 2012; Aalberg Haugen & Gotthard, 2015). While
both of the other two populations are bivoltine, Öland experiences
shorter summers, and only produces a partial second generation
(Lindestad et al., 2019). Such seasonal time stress at voltinism
boundaries is precisely the condition under which non-diapausing
individuals are most expected to trade off fast development for smaller
size (Mousseau & Roff, 1989).
The difference in body size between long-day and short-day Öland
individuals was not detectable at any life stage earlier than pupae
(Fig. S2), suggesting that it emerges at some point during the fourth
instar. (An apparent difference between long-day and short-day
individuals can be seen as early as the third instar in Fig. 4, but this
was due to a coincidental hatching size difference between the two
control groups; no overall photoperiod effect was seen when considering
all six treatments groups.) These results are reminiscent of those
obtained in scarce swallowtail butterflies, where the size polyphenism
is reversed (non-diapause individuals are larger), but the size
difference arises from higher growth rates only at the end of the last
larval instar (Esperk et al., 2013). Intensive studies of the mothManduca sexta have revealed that body size is determined by the
interplay of three parameters: basal growth rate, a critical weight, and
the delay period from when a larva reaches the critical weight to when
the resulting hormonal cascade arrests growth and triggers preparations
for the molt to the pupal stage (Davidowitz & Nijhout, 2004; Nijhout et
al., 2006; Callier & Nijhout, 2013). While each larval instar increases
in size by the same multiple, so that molts between instars occur at
predictable weights, the final instar “overshoots” the critical weight
by continuing to grow during the delay period (Nijhout et al., 2006). IfP. aegeria functions along similar lines, it is possible that the
plastic size difference between developmental pathways is achieved by
modulating the length of the delay period; this would leave the size at
each previous molt the same for both pathways, as was observed.
When comparing the results for body size to those obtained for larval
development rate, an apparent paradox emerges. One may expect that, if
the size difference is only established late in the larval period, it
should be responsive to adjustment by changes in photoperiod earlier
during life. However, this was not observed: final sizes tended to
correspond to the initial photoperiod regime, and Öland individuals
switching to diapause development in response to shortening days pupated
at a smaller size than those reared in constant short days (Fig. 4). In
other words, body size diverged between photoperiods later in life, but
was nonetheless less adjustable to the environmental conditions
experienced late in life, than development rate.
A possible interpretation is that the between-pathway size divergence
that occurs in the fourth instar is driven by physiological mechanisms
that are primed earlier during life, resulting in a delayed and
inflexible effect of photoperiod on weight accumulation. Growth rates in
general were seen to be highly flexible and responsive to photoperiod
treatment (Fig. S1b), but if the diapause-pathway size difference
utilizes a separate mechanism (as discussed above), this need not be a
contradiction, and may explain how the development rate polyphenism can
be shared across Scandinavian populations (Lindestad et al., 2019) while
the body size polyphenism is not (Aalberg Haugen & Gotthard, 2015).
Another possible explanation is that subjecting larvae to such
unnaturally drastic shifts in daylength (and hence imposing rapid shifts
in developmental strategy) resulted in physiological stress, which may
have manifested as decreased final size, at least in individuals forced
to change to the more resource-demanding diapause pathway. As growth
rate was only coarsely measured here, with a single weighing per instar,
a more detailed investigation of these mechanisms will need to await
further study.