Evaluation of the new system

One of the features of mosquito rhythms which 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. As mentioned above, the stimulus for the research which led to the concept of a multi-clock system 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). The suggestion for Ae. aegypti 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.

In a contemporaneous study, White (1968) concluded that the morning peak of hatching of the eggs of a psyllid, Cardiaspina densitexta, was controlled by two "clocks". Saunders (1982) wrote that - "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 (Taylor & Jones, 1969), he continued - "A systematic study of this nature, however, has yet to be pursued". Perhaps the lack of such a study stems from the fact that other observations of both peaks of a bimodal circadian rhythm remaining overt under constant conditions are quite rare.

Recently, Kyorku & Brady (1994) found such persistence with males, and perhaps females, of a species of tsetse fly, Glossina longipennis, which, interestingly, is one of the few tsetse species known to fly in the dark. Other tsetse species, for instance Glossina morsitans morsitans, show bimodal, V-shaped activity in LD cycles but only one peak persists in DD, or LL (Brady, 1998b). Which of the two peaks persists in DD changes from near subjective dawn (M) in unfed newly emerged flies to near subjective dusk (E') after the first blood-meal (Brady, 1988a). Phase resetting studies have shown that the bimodal rhythm is phase-set by light-on, with the two peaks remaining some 12h apart. In nature, the E' peak tends to be greatest but this is due mainly to the temperatures being optimal in the evening (being too cold at dawn and too hot at mid-day). Also in nature, with an LD varying little from LD 12:12, the dusk activity was found to be a response to an absolute illumination level, under artificial conditions this was 350 mW/m² (Brady 1988b). A problem in studying the 18 of 19 species or subspecies of tsetse fly which are day-active is that they show little activity in DD and conclusions have to be reached from observations of populations rather than single individuals.

Even more recently, Watari & Arai (1997) have reported the bimodalism, or even trimodalism, of the day-active species of Onion Fly, Delia antiqua. As noted earlier, they used a wide range of LD regimes. The major E' peak (f_A in their terminology) was judged to be depend on light-off but to some extent also by light-on. M and a light-off response E were also noted. M was felt to be a direct response to light-on and E a direct response to light-off, although they added that activity did continue into D when the major E' was incomplete when lights went off in short L regimes. In their equivalent of the photoperiodogram, as with other authors, the relatively small upsurges of activity exhibited outside of the framework of the acknowledged three peaks were not plotted. Additionally they studied activity in DD in following several LD regimes, illustrating the results of DD after LD 6:18, LD 12:12 and LD 16:8. In DD from LD 16:8, 14:10 and 12:12 E' freeran but M did not persist (suggesting to Watari & Arai that the latter was solely an exogenous response). In LD 2:22, 4:20 and 6:18 activity before lights-on was noted and in DD following LD 4:20 and 6:18 this peak continued (suggesting to Watari & Arai that this might be part of the "major activity controlled by the endogenous pacemaker").

For most of the mosquito species studied in LD 12:12 or other nearly symmetric LD regimes, the observed peaks of activity have asymmetric shapes, with abrupt rises at light-on and abrupt drops at light-off. In constant conditions (DD or LL), however, the activity rises and falls in a relatively smooth manner (see Figures 22, 27, 31, 33, 34, 36 and 48). The shape (breadth or roundness) of the observed peaks, and perhaps their amplitude, appears to be affected by the degree of separation of the underlying clocks, and that separation is due entirely to the LD regime experienced before the change to constant conditions. Evidence in this paper of the continuance of separated peaks in constant conditions can be seen in Figures 31, 33 and 34, for Ae. aegypti; in Figure 36, for Cx. p. molestus; and, in Figures 48 and 49, for Cx. p. quinquefasciatus. In general, the greater the separation of the clocks the narrower the shape of each observed peak but, when the effects of each clock are clear (for instance, see Figures 34 and 36), a full smoothly curved peak would seem to span some six to seven hours.

Click here for : Figure 22, Figure 27, Figure 31, Figure 33, Figure 34, Figure 36, Figure 48 and Figure 49.

The contribution of the individual clocks to activity in DD following LD 4:20 was calculated from the experiments illustrated in figures 34a and 34b. The results, shown in Tables 1 and 2 (below), reveal how all four clocks contribute. Although, the strongest single contribution to total activity is by the OFF SINE clock it amounts to only some 45% of the activity. Even adding the contribution of the OFF ANTISINE clock brings the total to no more than 70%. Thus, at least 30% of activity stems from the ON SINE and ON ANTISINE clocks; the contribution of these clocks is shown as actually 39-49%. The grand totals, 108-116%, indicate overlapping effects of the clocks, and this can be seen in figures 34a and 34b.

Table 1. Contribution (%) of each sine wave to total activity of Aedes aegypti LSHTM strain in DD following an LD 4:20 rearing regime (over a 72 hour period following light-off). Each sine wave was taken as exerting influence for 3 hours before and after the apogee of the wave.

Mosquito	No. 1	No. 2	No. 3	No. 4	No. 5	No. 6	No. 7	No. 8	No. 9	mean % activity	SD+/-
Clock
OFF SINE	45.3	41.2	56.4	39	23	53.5	66.7	61.1	15.4	44.6	16.1
OFF ANTISINE	15.1	24.7	12.2	29.9	45.7	25.6	8.2	13	46.2	24.5	13.3
ON SINE	36	10.3	13.3	20.9	30.5	23.3	5.9	13	48.3	22.4	13
ON ANTISINE	5.8	23.9	21.8	6.4	11.9	7.8	28.1	27.8	22.6	17.3	8.8
Total	102.2	100	103.7	96.2	111.1	110	108.8	114.8	132.5	108.8	10

Table 2. Contribution (%) of each sine wave to total activity of Aedes aegypti LSHTM strain in DD following an LD 4:20 regime (over a 72 hour period following light-off); mosquitoes reared in LD 12:12 and regime changed by delaying light-on. Each sine wave was taken as exerting influence for 3 hours before and after the apogee of the wave.

Mosquito	No. 1	No. 2	No. 3	No. 4	No. 5	No. 6	No. 7	No. 8	No. 9	mean % activity	SD+/-
Clock
OFF SINE	58.3	30.1	30.1	70	53.5	41.2	44	58.2	34	46.6	13.4
OFF ANTISINE	14.3	24.4	28.2	6.3	21.1	14	31	12.8	32.7	20.5	8.7
ON SINE	22.1	29.6	39.7	13.8	21.8	8.1	29.4	8.2	28.3	22.3	10.1
ON ANTISINE	21.2	21	27.6	56.3	22.9	47.8	8.2	17.3	20.1	27	14.4
Total	115.9	105	125.6	146.3	119.4	111	112.5	96.4	116	116.5	13.2

A very thorough examination of light-dark thresholds was undertaken by Dreisig (1980). Although his work centred on nocturnal moths, the adjective "nocturnal" may be misleading, as his results showed emphatically that the illumination threshold for commencement of activity is highly species-specific and the "gating" for all individuals of a species can be as narrow as ten minutes in duration.

Experiments involving low intensity LL were not carried out with Ae. aegypti and, so, it is not possible to say definitely that there is not a gradual transition in t from total dark to bright light. Use of artificial dusk and dawn, however, did not materially affect the activity patterns of Ae. aegypti (Rowland & Lindsay, 1986) and it has been shown for other species, for instance Cx. p. quinquefasciatus (Jones, 1982), that the peaks did not shift significantly. For the latter, which is a dark-active species, there is a definite threshold of light intensity, albeit very low, above which t lengthened, from < 24h in DD to about 25h. Gibson (1985) used a light-intensity of about 1 lux for her studies of swarming in Cx. p. quinquefasciatus, remarking that this is about the intensity at which the species swarms in the field, and similar to the intensity of a starry moonless night in Europe.

Rowland (1989) examined the effect of light intensity on the activity of An. stephensi. In separate experiments, he used both simulated gradual dawn and dusk transitions, and a regime of 12h bright light (40 lux) and 12h dim light (0.3 lux, simulating moonlight). For males and females in most physiological states (virgin, inseminated, blood-fed inseminated), the gradual transitions made no significant difference, although the M peak was less than that found when light-on was abrupt. The moonlight regime, used only with inseminated females, produced a markedly different pattern. There was an overall increase in activity, with no E peak but considerable activity in the second half of the night, giving a broad, symmetrical N peak, and most activity ceasing at least an hour before the upward light change. Rowland concluded that the E and N peaks probably represent the behaviour of constituent oscillators of the underlying pacemaker system. Some of the individual activity patterns showed that an effect of low light appeared to be a narrowing of the phase of the two peaks, with E moving towards N. An interesting comparison can be made between the moonlight (LM 12:12) result and the activity patterns shown in Figure 44. Although E, which appears to be underlain by the OFF SINE, is strong in all five LD regimes, the LD 20:4 pattern (see Figure 44e) shows the ON ANTISINE underlying activity in the moonlight broad N.

The question of light thresholds as they affect tsetse fly activity was examined by Brady (1987). With males of Glossina morsitans a sharp light-off led to a burst of activity but simulated gradual dusk gave a broader but still quite sharp peak. He concluded, as did Dreisig (1980), that there is a threshold of absolute light intensity. In the case of G. morsitans this was between 1700 mW m^-², above which no activity would take place, and about 300 mW m^-², when all the flies would take off. In Zimbabwe (16°10'S) the latter intensity occurs perhaps 15 mins after sunset. The expression of light intensity as watts per square meter was used as giving as better representation of actual total light energy. The more commonly used lux (lumens m^-²) is a photographic term based on the light spectra visible to the human eye and, thus, has less accuracy when used for insect studies (Young et al., 1987). On a logarithmic scale, the range of natural light intensity is from 1 W m^-² in full daylight (sun and sky, about 355 lux); through sunrise and sunset intensity of 0 mW m^-²; to full moon at -3 W m^-². Thus, there is strong evidence that there are precise thresholds of light intensity and that there is no need to attempt to simulate dusk and dawn in laboratory studies of activity.

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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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