The data suggest that sexually-produced A. pisum
embryos undergo a period of diapause, although a diapause different to that commonly presented in the literature. Diapause typically involves as a period of developmental arrest during which an insect is resistant to environmental influences on developmental progression [2
]. Sexually-produced A. pisum
embryos do not demonstrate developmental arrest. Whilst there is little increase in overall body size between anatrepsis and katatrepsis, there is continued growth of the legs and further differentiation of body organs. A. pisum
embryos do, however, show resistance to environmental influences on developmental progression. Growth rate up to katatrepsis is the same at 0–4°C, 10°C and 16°C. This is in contrast to asexually-produced embryos where growth rate approximately doubles between 5°C and 10°C and between 10°C and 16°C [19
]. In sexually-produced embryos the period of temperature independent development, or diapause, ends around katatrepsis. After this point development becomes temperature dependent, with more rapid growth at 10°C than at 0–4°C. Katatrepsis, and the end of diapause, however, occurs earlier in embryos maintained at higher temperatures, (10°C and 16°C) and can be bought forward in embryos maintained at 0–4°C by transferring them to 16°C at day 49.
At all the temperatures tested there was no cessation of morphological development. Cell division was evident at all ages and, at 0–4°C at least, there was continued growth of the legs. This appears to be somewhat contrary to the traditional understanding of diapause, which typically involves a distinct 'resting stage' [1
]. Much research has revealed, however, that even during the 'resting stage' there are physiological changes in the diapausing insect, leading to the concept of 'diapause development' [22
]. Nevertheless, such development appears to be primarily physiological rather than morphological, and was defined as physiogenesis by Andrewartha (1952). It is possible that the morphological development observed in diapausing A. pisum
is exceptional. Alternatively, diapause in many other insects may also involve a slowing but not a cessation of morphological development. It is difficult to test this notion with the current literature as timed dissections of diapausing embryos, larvae and pupae have rarely been conducted. There are, however, a few examples of continued morphological development during diapause. Diapausing embryos of the orthopteran Austroicetes cruciata
undergo slow development for the first two months of diapause, before entering a resting stage [24
]. Similarly, caterpillars of Cirphus unipunctata
and Laphygma exigua
continue to feed and develop during diapause, albeit at a much-reduced rate [25
], and caterpillars of the corn-stalk borer Sesamia nonagriodes
undergo non-stationary moults during diapause [26
]. These examples hint that the expression of diapause-controlled dormancy may vary between species [2
]. Consequently, diapause could be considered extreme regulation of developmental rate, rather than a shutting down of morphological development. The mechanisms that control diapause in A. pisum
may therefore be the same as those that control the rate of morphological development at other stages of development.
Several insects that diapause over winter are resistant to environmental conditions conducive to growth only at the beginning of winter [27
]. This prevents premature emergence, be it from egg, larvae or pupae, just at the onset of the cold temperatures the insect is trying to avoid. Once winter has commenced, diapause ends and the insect enters post-diapause quiescence, relying on persistent low temperatures to maintain developmental arrest [27
]. This appears to be the case in A. pisum
. The aphid clone used for the experiment would normally oviposit in mid-October, when the average daily temperature is 9°C with a maximum of 15°C [32
]. By day 28 the embryos would be exposed to average daily temperatures of 3°C. Consequently, whilst A. pisum
maintained at 0–4°C had regained temperature-dependent growth by day 49 (figure ), it would not normally be exposed to warmer conditions until the following spring, approximately 150 days later. By this time the aphid would be fully developed and ready to hatch. In embryos that remained at 0–4°C until day 98, there was no obvious difference in development rate during and after diapause, and this is consistent with observations in other insects [1
]. There must, however, be some change in condition to move development from being temperature independent to temperature dependent [1
]. This change appears to be at or around katatrepsis. Identifying what this change is, and what, if anything, it has to do with katatrepsis, will be key to understanding how the pea aphid controls development during diapause.
The finding that A. pisum
embryos maintained at 16°C showed considerable malformation may be a consequence of surpassing the normal thermal limits of developmental regulation. The genetic pathways involved in the development of A. pisum
embryos must build an animal under two conditions. The first is slow, temperature independent development at low ambient temperatures in sexually-produced embryos, and the second is rapid temperature dependent development at higher ambient temperatures in asexually-produced embryos. Presumably the same pathways are involved in both processes – it would be surprising if A. pisum
were to have different sets of genes for the two modes of development. Diapause may therefore act to limit the speed of reactions that would otherwise occur more rapidly at higher temperatures, possibly through the action of an 'inhibitory factor' [22
]. According to this model, the higher the ambient temperature the greater the required activity of the inhibitory factor [33
]. When temperatures are maintained at abnormally high temperatures (16°C), regulation may be compromised with only some aspects of development slowed. This may result in embryos displaying a mosaic of developmental stages.
Alternatively, the malformed phenotypes seen at 16°C may result form differing levels of temperature independence for different developmental processes. There would be no selection on embryos to make later developmental processes temperature independent, as they normally occur during winter. At unnaturally high temperatures, for example 16°C, these later, temperature dependent processes, may occur prematurely, resulting in embryos displaying a mosaic of developmental stages. This hypothesis suggests that the timing of some developmental processes occurs independently, i.e. that the beginning of one developmental process is not contingent on the ending of another. This appears to be the case for katatrepsis, which occurs in smaller embryos at higher temperatures. Premature katatrepsis did not, in itself, cause the malformations. Embryos transferred to 16°C from 0–4°C on day 49 underwent early katatrepsis but displayed a high hatching success.
It is perhaps surprising that many embryos should develop abnormally at 16°C given that they would encounter temperatures as high, or higher, during winter in their natural environment. However, such high temperatures would be experienced only temporarily, and it may be persistent exposure to high temperatures that caused the developmental abnormalities in our experiment. Further research is therefore necessary to determine the influence of fluctuating temperatures on developmental progression, both with respect to developmental abnormalities and developmental rate.
There has been little progress towards identifying the genetic control of diapause in insects. The one system where diapause has been well elucidated, dauer formation in the nematode C. elegans
, indicates that the insulin pathway plays an important role [34
]. Recent work on mutant Drosophila
that mimic reproductive diapause in adults also suggests a role for insulin [35
]. Insulin, which regulates growth in other stages of development in Drosophila
], may therefore play a similar role during A. pisum
diapause. Work on diapause in the flesh fly Sarcophaga crassipalpis
has identified a second class of genes that may be involved in diapause regulation [39
]. These are the heat shock proteins, which are normally expressed during times of stress [40
] and which provide cryptoprotection during diapause. Since heat-shock proteins are also involved in cell cycle arrest, however, they may play a more central role in the regulation of diapause [1
]. Heat-shock proteins are also typically heat inducible, and so fit with the concept of a temperature dependent 'inhibitory factor' [33
]. Additionally, high levels of heat-shock proteins may disrupt development [41
], which could help explain the developmental defects observed in embryos maintained at 16°C. Further work on the expression of both insulin and heat shock proteins in A. pisum
embryos will clarify their respective roles.