Orthodox theories regarding entrainment and the mechanisms underlying the observed patterns of daily activity almost all rely on evidence drawn from experiments that appear to show a single change in light intensity, most commonly light-off, as being responsible for phase-setting. For mosquitoes, perhaps the most widely cited of such theoretical approaches is that of Peterson (1980a, b, c), which was based on observations of Cx.p.quinquefasciatus.

In contrast, the stimulus for the research described in this paper was the discovery that both light-on and light-off have phase-setting effects on the circadian rhythm of flight activity of Ae. aegypti (Taylor & Jones, 1969). Just after the Ae. aegypti findings had been submitted for publication, White (1968) published his observation that the morning peak of hatching of the eggs of a psyllid, Cardiaspina densitexta, was controlled by two "clocks". One clock appeared to be set by light-off 12-14h earlier and the other by light-on to entrain the hatching some 45min to 2h later. These two papers were the first such reports, at least for insects, and both stemmed from observations made using a range of artificial LD regimes. Within the natural range, southern Australia, of C. densitexta, the length of darkness varies from just under 10h to a little over 14h in midwinter. White concluded that the dual timing ensured that hatching took place only when other conditions of temperature and humidity were favourable for the survival of larvae. The suggestion for Ae. aegypti (Taylor & Jones, 1969) was that the co-entrainment fitted the range of summer daylengths, from 12-16h daylight, found within the known geographical range (0-40°latitude) of an otherwise pan-global species.

The significance of "moving" peaks

Tyschen (1978) examined the circadian rhythm of mating in the Queensland fruit fly, Dacus tryoni, and reviewed a number of other reports of circadian rhythms underlying behaviour. His summary followed earlier thinking, in accepting that entrainment of rhythms in the light period follows the dark-light transition and rhythms in the dark period follow the light-dark transition. Almost all the examples he examined were from studies with LD regimes within the range L 6-18h, and none were of the evening (E'-E) pattern. His own D. tryoni results show very tight E'-E mating responsiveness in median LD regimes, but the peak becomes much more diffuse as the regimes become more extreme. In LD 22:2 the peak is markedly forward, and in LD 4:20 the activity continues well into the dark.

Brady & Crump (1978) compared field and laboratory observations of circadian activity rhythms in a tsetse fly, Glossina morsitans. This species shows a V-shaped pattern with all activity in L and almost all of that concentrated in M and E', especially the first hour after light-on. When flies in an LD 12:12 regime experienced a change to LD 18:6 the pattern retained its V-shape. When light-on was advanced 6h the V shifted forward over two 24h-cycles. When light-off was delayed 6h the V remained unaltered, with activity dropping after the E' in the 12th hour of light. They concluded that sunrise was the primary zeitgeber, and that this was not surprising for a species originating from Zimbabwe (17°S), where natural LD variations were only from LD 11:13 to LD 13:11.

Smith (1985) reported that the peak of eclosion of the blowfly, Lucilia cuprina, occurred close to light-on in all photoperiods, but in long L the mean eclosion was after dawn, whereas in short L it was before dawn. He did not comment on the timing mechanism.

Moore & Rankin (1993) studied the honeybee, Apis mellifera ligustica, (from Austin, Texas, maximum LD 14:10). They used LD regimes from LD 4:20 to LD 22:2, and found a totally day-active pattern with a broad, almost flat, peak, somewhat skewed towards afternoon activity (see Appendix). They denoted onset of activity and noted this appeared to follow light-off by some 10h. Their interpretation was that the main peak was entrained by the previous light-off. Their view did not embrace the evidence from long L regimes. In particular, they offered no explanation as to the strong evidence from LD 22:2, which clearly showed an early peak which appeared to be entrained to follow light-on by some 10-14h.

Saunders (1982) reviewed many aspects of "insect clocks" and wrote (pp 116-117) - "Satisfactory evidence for the existence for separate 'dawn' and 'dusk' oscillators is provided by either (1) stably different phase relationships between the activity peaks, established during entrainment to cycles containing different durations of light, and retained during subsequent DD free-run, or (2) the 'splitting' of the activity into two or more components which, at least, for a while free-run with different periods. In the most convincing cases the two components span the entire 360 of mutual phase relationship, and may even cross and recross". After recognising the evidence from Ae. aegypti, he continued - "A systematic study of this nature, however, has yet to be pursued".

Here it is the significance of "moving" peaks that will be explored. Chiba & Tomioka (1992) exposed Cx. p. molestus to a wide range of LD regimes (Figure 8 shows some of their results). They examined the findings in the light of Aschoff's consideration of how insects respond to a change in LD ratio (Aschoff, 1981). This gives three ways where phase angle difference stays in parallel to light-off, light-on, or the middle of light-time. The postulate, however, seems to depend on only one zeitgeber operating in each of the first two ways, and to require that both steps in light intensity have equal weight in the third. It is an advance from earlier thinking (Tyschen, 1978, derived from Aschoff, 1965) where it was thought that, under normal photoperiods, rhythms with peaks in the light period fit straight lines of slope intermediate between the slope of the dark/light transition and the vertical; and, rhythms with peaks in the dark period fit straight lines of slope intermediate between the vertical and the light/dark transition.

From the mosquito observations, therefore, it is important to emphasise that the existence of more than one zeitgeber can be demonstrated for every one of the seventeen or so species. What is abundantly clear is that studies limited to a single regime (such as LD 12:12 for species of tropical origin, or LD 16:8 for species of temperate origin) will conceal more than they reveal, and that use of a range of LD regimes (within the frame work of the natural earthly 24h-cycle) is essential to investigations of entrainment.

A final point of significance, surely, in terms of orthodox entrainment theory and the extensive use of "skeleton" photoperiods (most notably by Pittendrigh, 1981, for instance) is that the presence of anything other than a single zeitgeber would seem (to the present author) as being likely, at least, to impair the theory, or, at worst, to completely invalidate it. The problem, of course, is that a light pulse given during an otherwise continuous period of darkness has both an upward and a downward change in light intensity. T.C.R. White (personal communication, 1969) also felt that skeleton photoperiods could well be "independent dark periods with light periods between them". One of the features of mosquito rhythms that has permitted the observation of possible multiple zeitgebers is that none of the species studied show purely unimodal activity. Most have readily discernible and clear peaks of activity. Flight activity of individuals has proved to be easy to monitor and the pattern can be observed for several successive 24h-cycles. In contrast, much of the orthodox theory has been derived either from animals showing unimodal diurnal or nocturnal activity (often with activity throughout the light or dark), or else from observations of once-in-a-lifetime events (such as eclosion) requiring conclusions to be drawn from populations.

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©1998, 2010 - Brian Taylor CBiol FSB FRES 11, Grazingfield, Wilford, Nottingham, NG11 7FN, U.K. Comments to dr.b.taylor@ntlworld.com

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