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Photoresponses of second-instar Chaoborus larvae
J. Insect Ph.wiof., Vol. 28. No. 2. pp. 183-187, 1982.
0022-1910/82/020183-xm03.00/0 Q 1982 Pergamon Press Ltd.
Primed in Greaf Britain.
PHOTORESPONSES OF SECOND-INSTAR CHAOBORUS LARVAE MICHAEL C. SWIFT and RICHARD B. FORWARD, JR. University of Maryland-CEES, Appalachian Environmental Laboratory, Frostburg State College Campus. Gunter Hall. Frostburg, MD 21532, U.S.A. and Duke University Marine Laboratory, Beaufort.
NC 28516. U.S.A.
(Received 9 June 198 I : revised 26 August 198 1)
die1 vertical migration of Chaoborus larvae varies with larval instar. Although light is involved in the control of vertical migration the contribution of larval photoresponses is unknown. In order to describe ontogenetic changes in larval photoresponses we measured photoresponses of second-instar Chaoborus puncfipennis larvae in the laboratory. The response spectrum of these larvae had peaks in sensitivity at 420 and 620 nm with a wide plateau of lower sensitivity from 460 to 600 and 640 to 680 nm. Dark adapted larvae were positively phototactic at intensities from IO-’ to 10’ Wme2 at 420 nm. The level of response decreased somewhat above 10m4 Wm-‘, and above IOmz Wm-* a small proportion of larvae shifted to a negative phototaxis. At 420 nm the threshold intensity was about 10m7 Wme2 for positive phototaxis and lo-? Wm-’ for negative phototaxis. Light adaptation increased the threshold intensity for positive phototaxis. The differences in larval photoresponses between second- and fourth-instar larvae suggests that the young instars are adapted to the photoenvironment of the water column and older larvae are adapted to avoid the water column except at very low light intensities. These predictions match the die1 distribution of these larvae. KeJ Word Index: Chaoboruspunctipennis, second instar, response spectrum, positive phototaxis, negative phototaxis. threshold intensity for phototaxis, ontogenetic changes in phototaxis Abstract-The
responses is not known. Thus, the object of this study was to measure photoresponses of ‘young’ larvae in the laboratory in order to determine the extent of ontogenetic changes in larval photoresponses.
INTRODUCTION
THE DISTINCTIVEdie1 migration of Chaoborus larvae has been widely documented. Larvae are initially planktonic and the amplitude of the vertical migration usually increases as the larvae progress through the four larval instars (TERAGUCHIand NORTHCOTE, 1966; FEDORENKO and SWIFT. 1972; LEWIS, 1975). The typical migration pattern is fully developed in fourthinstar larvae, and consi$s of a rise from the sediments into the water column at dusk and return at dawn (JUDAY, 1921; EGGLETON, 1931; BERG, 1937; ROTH, 1968; PARMA, 1969a). Light appears to control the timing and pattern of larval migrations, as indicated by the correlation of migration pattern with underwater light intensity (TERAGUCHI and NORTHCOTE, 1966; MALUEG and HASLER, 1967; CHASTON, 1969; GOLDSPINK and SCOTT, 1971). Even though LAROW (1968, 1969) and SWIFT and FORWARD (I 980) have suggested mechanisms for the role of light in timing larval migrations, our knowledge of the interaction between larval photoresponses and underwater light is not sufficient to explain the ontogenetic changes in Chaoborus migration patterns. In general, ‘older’ instars are negatively phototactic (BERG, 1937; CCIOK and CONNORS,1964; LAROW, 1971; SWIFT and FORWARD, 1980) and ‘younger’ instars are positively phototactic (BERG, 1937; COOK and CONNORS, 1964; LAROW, 1971). However, detailed measurements of larval photoresponses have only been made for fourth-instar larvae (SWIFT and FORWARD, 1980). The extent to which ontogenetic changes in migration pattern are related to ontogenetic changes in larval photo-
MATERIALS
AND METHODS
Collection of larvae All experiments were done using second-instar Chaoboruspunctipennis (Say) larvae collected in Crane Pond, Newport, North Carolina. Larvae were collected in the late evening using a 240 pm mesh net hauled horizontally behind a rowing-boat. Samples were seived immediately through a 500 pm mesh strainer to remove third- and fourth-instar larvae and other large plankton. Bulk samples of larvae were returned to the laboratory and sorted under dim white light into plastic weighing pans containing about 4 ml of pond water which had been filtered to remove particles larger than 5 pm. Larvae were sorted individually by pipette; each pan received 20-25 larvae. The larvae were either dark or light adapted for at least 1 hr before being used in experiments. Larvae were collected about 2100 hr, sorted from about 2200 to 0100 hr, and tested from about 0100 to 0300 hr. Larvae were always used in experiments the night they were collected. Phototaxis Phototaxis was measured as movement towards (positive) or away (negative) from a directional light source. Details of the light source are given in SWIFT and FORWARD (1980). The stimulus light was obtained IX3
MICHAEL C. SWIFT AND RICHARD B. FORWARD. JR
184
by filtering light from a projector through heat, interference and neutral density filters. The experimental chamber was a lucite trough (13.8 x 2.4 x 3.9 cm) with 3.2 mm thick end walls. The chamber was divided into 5 equal sections. by lucite partitions which could be raised and lowered simultaneously. The protocol for all experiments was the following: (1) The trough was filled with about 60 ml of filtered pond water to a depth of 2 cm, the partitions were lowered, and the trough was positioned in front of the light source. (2) One pan of pre-sorted larvae was poured into the central section of the trough. (3) After 1 min in darkness, the partitions were gently raised, the stimulus light turned on for 30 set, and the partitions lowered. (4) The number of larvae in each section of the trough was recorded, the larvae and water were poured out, and the trough was re-filled for another trial. (5) Interference and/or neutral density filters were changed between trials when necessary. The protocol for the ‘dark control’ was the same except that the stimulus light was not turned on. The proportion of larvae showing positive phototaxis was defined as the number of larvae in the section of the trough closest to the light source divided by the total number of larvae in the trough. Similarly, larvae in the section furthest from the light were considered to show negative phototaxis. In view of the relatively large size of the trough with respect to the size of the larvae and the low number of larvae tested per trial, it is unlikely that the larvae caused much light scattering in the water or acted to substantially block the light when aggregated at the stimulus end of the trough. The mean (f S.D.) percentage positive and negative responses at each wavelength or intensity was calculated from the percentage data following arc sin square root transformation. These values were then plotted against wavelength or light intensity. Analysis of variance and multiple comparison techniques were used to examine the effect of wavelength or intensity on the level of response and to test for significant differences among treatment means. Light intensity was measured using a radiometer (EG & G, model 550-l) with the sensor located at the position where the end of the trough would be during experiments. Measurements were made in energy units (Wm -*). The quanta1 level was calculated using the energy level of a quantum at the wavelength of maximum transmission for each interference filter. In practice, the light intensity used in each
Table 1. Quanta1 intensities
experiment was considere&obe the value at the centre of the trough. This value was calculated from the measured intensity at the entrance to the trough and from the attenuation coefficient of filtered pond water at each test wavelength. Measurements of the pond water were made with a spectrophotometer (Cary, Model 14) having a cell with a 10 cm path length. Spectral attenuation resulted from both scattering and absorption and increased with decreasing wavelength. Attenuation coefficients ranged from 0.008 (10 cm path length) at 660 nm to 0.108 at 380 nm. The light intensity of each wavelength to be tested on a given day was measured the night before the experiments were run. The actual optical densities of the neutral density filters were measured for each wavelength. Spectral sensitivity A response spectrum was produced by measuring the per cent positive phototactic response to stimulation with approximately equal quanta1 intensities of light at each test wavelength. Fifteen wavelengths were tested (400-680 nm in 20 nm steps). Positive phototaxis increases with stimulus intensity over a range of intensities between the lower threshold intensity and the intensity at which the maximum response occurs. Thus, the test intensity must be between these two extremes. Variations in response level among equal quanta1 intensities of different wavelengths indicates variations in sensitivity to those wavelengths; the greater the response the greater the sensitivity. Preliminary measurements indicated that a quanta1 intensity of about 5.0 x lOi quanta me2 see-i produced an appropriate level of response. Each wavelength was neutral density filtered to approximately this value. The mean quanta1 intensity was 4.6 x lOi quanta me2 set-i with a standard deviation of 0.5 x lOi quanta me2 sect’; actual values for each wavelength are given in Table 1. Five replicate sets of dark adapted larvae were tested at each wavelength, and 13 dark controls were tested. Intensity sensitivity To determine the pattern of phototaxis at different light levels we measured larval response to 12 intensities. All measurements were made at 420 nm except the two highest intensities. For these measurements a Corning 5-56 blue filter was used; the filter transmits light between 400 and about 555 nm. The 420 nm stimulus wavelength was chosen because of the peak in sensitivity at that wavelength.
used at each wavelength
in the
response
spectrum
measurements Wavelength bm) 400 420 440 460 480 500 520 540
Quanta 5.0 4.0 4.3 3.7 3.9 4.9 4.5 4.7
mm2 set-’ x x x x x x x x
10’4 IO” 10’” 10’4 10’4 10’4 10’4 IO’J
Wavelength bun) 560 580 600 620 640 660 680
Quanta 4.8 5.6 5.3 4.6 5.1 4.6 4.6
m x x x x x x x
* set _ 1 10’4 1OlJ 10’4 1OlJ 10’” IO” 10’f
Photoresponses
of second-instar
Chaohorus larvae
IX5
The experimental protocol was the same as in the response spectrum measurements except that stimulus intensity was changed between tests instead of wavelength. Dark controls were tested before and after each set of intensities was tested. Five replicate series of intensities were measured. Larvae were either dark adapted for at least 1 hr before being tested or light adapted at least 1 hour under GE cool white fluorescent lamps at a light intensity of about 1.54 Wme2. Light adapted larvae were held in the dark for the 30 set equilibration period before the partitions were raised and the stimulus lamp turned on. RESULTS The positive phototactic response of second-instar Chaoborus punctipennis larvae to equal quanta1 intensities of light is significantly affected (p < 0.001) by the wavelength of that light (Fig. 1). The response of these larvae is significantly greater at 400-440 nm and 620 nm than at all other wavelengths tested. Thus, sensitivity maxima occur at 420 and 620 nm. The response at all wavelengths was significantly greater than the dark controls. The sign of the phototaxis of dark-adapted larvae varied with light intensity. Positive phototaxis was significantly affected (p < 0.001) by changing light intensity (Fig. 2). The percentage of larvae showing a positive phototactic response was significantly greater than dark controls at all light intensities above 2.2 x 10 _ ’ Wm 2, the threshold intensity for positive phototaxis. The degree of response increased as light intensity increased to about 2 x 10e3 Wmm2 and then decreased somewhat as light intensity increased further. Negative phototaxis was also significantly affected by light intensity @ = 0.003) (Fig. 2). Negative phototaxis was significantly greater than the dark controls at intensities greater than 10m2 Wm-‘. Light-adapted larvae were less sensitive to light than were dark-adapted larvae (Fig. 2). There was a significant effect of intensity on the level of positive phototaxis @ = 0.013). The threshold intensity for positive phototaxis in these larvae was 10e3 Wm-? above this intensity the level of positive phototaxis was significantly greater than the dark controls. The level of negative phototaxis was not significantly affected by light intensity.
Fig. 1. Response spectrum for second-instar C. punctipennis larvae. The per cent positive phototaxis stimulated by approximately equal quanta) intensities of each stimulus wavelength is plotted for each wavelength. Data points are means f S.D. C is the control response in darkness.
Fig. 2. Percentage phototactic response of dark-adapted light-adapted second-instar larvae as a function of intensity at a wavelength of 420 nm. Data points means + SD. for positive phototaxis (solid circles. lines), negative phototaxis (open circles, dashed lines), dark controls (points marked ‘Cf.
and light are solid and
DISCUSSION The response spectrum we measured for secondinstar C. punctipennis larvae is characterized by peaks in sensitivity at 420 and 620 nm and a lower but approximately constant sensitivity at wavelengths between these peaks. Our data differs a great deal from the only other measurements of spectral sensitivity of young larvae. LAROW (1971) showed that ‘immature’ C. punctipennis larvae were positively phototactic to 350 and 400 nm light and negatively phototactic to light at his test wavelengths above 440 nm. A response spectrum specifies the level of response stimulated by light of equal quanta1 intensity at each wavelength, and it will, therefore, vary as quanta1 intensity varies. LAROW (1971) apparently determined a response spectrum but didn’t report the quanta1 intensities used in his experiments. Because the sign of larval phototaxis varies with light intensity (SWIFT and FORWARD, 1980; this study), the differences between our measurements of spectral sensitivity and those of LAROW (1971) are probably due to differences in the light intensity used in our respective experiments. The spectral sensitivity of second-instar larvae differs from that of fourth-instar larvae. Fourth-instar larvae have a single peak of sensitivity at about 400 nm, a wide region of lower sensitivity (480420 nm), and a sharp decrease in sensitivity above 640 nm (SWIFT and FORWARD, 1980). Although the spectral sensitivity of fourth-instar larvae was determined from a true action spectrum and that of second-instar larvae from a response spectrum, there should be little qualitative difference between the results of these two methods (FORWARD and CRONIN, 1979). The consistent sensitivity to light in the violet part of the spectrum seen in both instars is probably an adaptation to the visual demands of the adult terrestrial environment. Since the attenuation coefficient of Crane Pond water increases dramatically
186
MICHAYLC. Swtrr
AND
in the u.v.-violet region, the amount of violet light underwater is proportionately very low compared to other visible wavelengths. Several other insects with aquatic larvae and terrestrial adults exhibit the same pattern of larval sensitivity (HORRIDGE, 1969; AUTRUM and KOLB, 1978). The peak in sensitivity in the orange-red region of the spectrum found in second-instar larvae is not present in fourth-instar larvae. This difference may be related to differences in larval habitat. Second-instar larvae are typically found in the water column during the day while fourth-instar larvae are not (BERG, 1937; LEWIS, 1975). Because Crane Pond is highly coloured with humic material the spectral composition of underwater light is skewed towards the yellow-range part of the spectrum. Thus, the sensitivity maximum at 620 nm in second-instar larvae may be an adaptation to the light regime in the water column. Dark-adapted organisms generally exhibit a positive phototactic response to low light intensities which reverses to a negative phototaxis as the intensity increases (FORWARD, 1976). Dark-adapted secondinstar larvae in our experiments were positively phototactic at low light intensities, but there was no clear shift to a negative phototaxis as light intensity increased. At light intensities above about 10eJ Wrnm2 the percentage of larvae showing positive phototaxis decreased, and the percentage showing negative phototaxis increased. However, even at the highest intensity tested (50.0 Wme2) more larvae were positively phototactic than were negatively phototactic. The threshold intensity for positive phototaxis was about 2 x IO- ’ Wmm2 for a 30 set stimulus. This value is lower by about one log unit than the thresholds for planktonic crustaceans and fish larvae (FORWARD, 1976) and it is about the same as the threshold level for positive phototaxis in fourth-instar larvae when they are subjected to a 30 set stimulus. The response of dark-adapted second-instar larvae to increasing light intensity was very different from that of fourth-instar larvae. In second-instar larvae the percentage of larvae showing a positive phototaxis increased to a peak at about 10m4 Wm-* and declined somewhat as light intensity continued to increase. In fourth-instar larvae, however, there was a striking shift in the sign ofphototaxis from positive to negative at about 10m4 Wm-’ (SWIFT and FORWARD, 1980). Because of the difference in wavelength at which these two sets of experiments were run (420 and 540 nm) there is a difference in the number of quanta available at any given intensity between the two experiments. However, this difference is equivalent to only about one-half log unit in intensity. Thus, during larval development there is a striking shift in the sign of phototaxis at higher light intensities from positive to negative. Light adaptation generally decreases sensitivity so that the threshold intensity for phototaxis is raised to a higher value. Second-instar larvae responded as expected when light adapted. The percentage of the larvae showing a positive phototaxis increased as intensity increased but the threshold intensity was higher than for dark-adapted larvae. The light intensity in these experiments was apparently never high enough to stimulate a negative phototaxis.
RICHARD B. FORWARD,JR
The ontogenetic changes in photoresponses measured in the laboratory in our experiments parallel ontogenetic changes in eye morphology and the timing and pattern of vertical migration in Chaoborus larvae. Second-instar larvae appear to be adapted to the photoenvironment of the water column. They are sensitive to those wavelengths which can be expected to penetrate into the water column, and they exhibit little negative phototaxis--even at relatively high light-intensities. Fourth-instar larvae appear to be adapted to avoid the water column except at very low light intensities (SWIFT and FORWARD, 1980). They lack a peak in sensitivity to longer wavelengths of light, and they show a strong negative phototaxis even at relatively low light intensities. The role of the compound eye in light reception is not clearly known. It develops and becomes pigmented during the second, third and fourth instar. Visual observation indicated that the second-instar larvae used in our experiments had not developed pigmented ommatidia in the compound eye; they had a pigmented ocellus only. Fourth-instar larvae have fully developed compound eyes. This description of second-instar larvae adapted to the photoenvironment of the water column predicts that these larvae should be planktonic during the day. Second-instar larvae of several species have been described as being planktonic during the day and as undergoing a vertical migration of small amplitude (e.g. PARMA, 1969a, b; TERAGUCHIand NORTHCOTE, 1966; FED~RENKO and SWIFT, 1972; LEWIS, 1975). However, in Crane Pond second-instar larvae were never collected near the surface during daylight. Their exact die1 vertical migration pattern is unknown, but they were abundant near the surface at dusk or later. Perhaps the light intensity near the surface of Crane Pond during daylight was high enough to stimulate a negative phototaxis. It is clear that before we can fully understand the role of light in controlling vertical migration in Chaoborus it is necessary to measure underwater light at those wavelengths to which the larvae are sensitive and at a time when the larvae are responding to light. Acknowledgements-We would like to thank Mr. CARLETON GARNER for unlimited access to Crane Pond and for the use of his rowing-boat. This material is based upon work supported by the National Science Foundation under grant No. OCE 77-26838 to R. B FORWARD and Duke University Research Council Grant No. 453-5926-6634-2290 to M. C. SWIFT. Appalachian Environmental Laboratory Contribution No. 1254 (UM-CEES).
REFERENCES AUTRUM H. and KOLB G. (1978) Spektrale Empfindlichkeit einzelner Sehzellen der Aeshniden. 2. vergl. Physiol. 60, 450-477. BERG K. (1937) Contributions to the biology of Corethru Meigen (Chaoborus Lichtenstein). Biof. Meddr. 13, l-101. CHASTON I. (1969) The light threshold controlling the vertical migration of Chaoborus puncripennis in a Georgia impoundment. Ecofogy 50,916-920. COOKS. F., JR. and CONNORS J. D. (1964) The direct response of Chaoborus astictopus to light (Diptera: Culicidae). Ann.
Photoresponses
of second-&tar
ent. Sot. Am. 57, 387-388. EGGLETON F. E. (193 1) A limnological study of the profundal bottom fauna ofcertain fresh-water lakes. Ecol. Monogr. I, 23 l-332. FEDORENKO A. Y. and SWIFT M. C. (1972) Comparative biology of Chaoborus americanus and Chaoborus trivittatus in Eunice Lake, British Columbia. Limnol. Oceanogr. 17, 721-730. FORWARD R. B., JR. (1976) Light and diurnal vertical migration: photobehavior and photophysiology of plankton. In Photochemical and Photobiological Reviews (Ed. by SMITH K. C.). Vol. 1. pp. 157-209. Plenum, New York. FORWARD R. B., JR. and CRONIN T. W. (1979) Spectral sensitivity of larvae from intertidal crustaceans. J. camp. Phvsiol. 133, 3 1 l-3 15. GOLDSPINK C. R. and SCOTT D. B. C. (1971) Vertical migration of Chaoborus flavicans in a Scottish loch. Freshwater Biol. 1, 41 l-421. HORRIDGE G. A. (1969) Unit studies on the retina of dragonflies. Z. vergl. Physiol. 62, l-37. JU~AY C. (1921) Observations on the larvae of Corethru punctipennis Say. Biol. Bul!. 40, 271-286. LAROW E. J. (1968) A persistent diurnal rhythm in Chaoborus larvae. I. The nature of the rhythmicity. Limnol. Oceanogr. 13, 250-256. LARo~ E. J. (1969) A persistent diurnal rhythm in
C’I~aoho~us larvae
IX7
Chaoborus larvae. II. Ecological significance. Limno/. Oceanogr. 14, 213-218. LAROW E. J. (1971) Response of Chaoborus (Dlptera: Chaoboridae) larvae to different wavelengths of light. Ann. ent. Sot. Am. 64, 461-464. LEWIS W. M.. JR. (1975) Distribution and feeding habits of a tropical Chaoborus population. Verh. lnt. Verrin. Theor. Anger. Limnol 19, 3 106-3 119. MALUEG K. W. and HASLER A. D. (1967) Echo-sounder studies in die1 vertical movements of Chaoborus larvae in Wisconsin (U.S.A.) lakes. Verb. Int. Verein. Theor. Anger. Limnol. 15, 1697-I 708. PARMA S. (1969a) The life cycle of Chaoborus crystallinus (DeGeer) (Diptera: Chaoboridae) in a Dutch pond. Vrrh. Int. Verein. Theor. Angers. Limnol. 17, 888-894. PARMA S. (1969b) Notes on the larval taxonomy. ecology. and distribution of the Dutch Chaoborus species (Diptera: Chaoboridae). Beaufortia 17, 21-50. ROTH J. C. (1968) Benthic and limnetic distribution of three Chaoborus species in a southern Michigan lake (Diptera: Chaoboridae). Limnol. Oceanogr. 13, 242-249. SWIFT M. C. and FORWARII R. B.. JR. (1980) Photoresponses of Chaoborus larvae. J. insect Physiol. 26, 365-371. TERAGU(‘HI M. and NORTHC‘OTF T. G. (1966) Vertical distribution and migration of Chaoborusflavicans larvae in Corbette Lake, British Columbia. Limnol. Oceanogr. 11, 164-176.
0022-1910/82/020183-xm03.00/0 Q 1982 Pergamon Press Ltd.
Primed in Greaf Britain.
PHOTORESPONSES OF SECOND-INSTAR CHAOBORUS LARVAE MICHAEL C. SWIFT and RICHARD B. FORWARD, JR. University of Maryland-CEES, Appalachian Environmental Laboratory, Frostburg State College Campus. Gunter Hall. Frostburg, MD 21532, U.S.A. and Duke University Marine Laboratory, Beaufort.
NC 28516. U.S.A.
(Received 9 June 198 I : revised 26 August 198 1)
die1 vertical migration of Chaoborus larvae varies with larval instar. Although light is involved in the control of vertical migration the contribution of larval photoresponses is unknown. In order to describe ontogenetic changes in larval photoresponses we measured photoresponses of second-instar Chaoborus puncfipennis larvae in the laboratory. The response spectrum of these larvae had peaks in sensitivity at 420 and 620 nm with a wide plateau of lower sensitivity from 460 to 600 and 640 to 680 nm. Dark adapted larvae were positively phototactic at intensities from IO-’ to 10’ Wme2 at 420 nm. The level of response decreased somewhat above 10m4 Wm-‘, and above IOmz Wm-* a small proportion of larvae shifted to a negative phototaxis. At 420 nm the threshold intensity was about 10m7 Wme2 for positive phototaxis and lo-? Wm-’ for negative phototaxis. Light adaptation increased the threshold intensity for positive phototaxis. The differences in larval photoresponses between second- and fourth-instar larvae suggests that the young instars are adapted to the photoenvironment of the water column and older larvae are adapted to avoid the water column except at very low light intensities. These predictions match the die1 distribution of these larvae. KeJ Word Index: Chaoboruspunctipennis, second instar, response spectrum, positive phototaxis, negative phototaxis. threshold intensity for phototaxis, ontogenetic changes in phototaxis Abstract-The
responses is not known. Thus, the object of this study was to measure photoresponses of ‘young’ larvae in the laboratory in order to determine the extent of ontogenetic changes in larval photoresponses.
INTRODUCTION
THE DISTINCTIVEdie1 migration of Chaoborus larvae has been widely documented. Larvae are initially planktonic and the amplitude of the vertical migration usually increases as the larvae progress through the four larval instars (TERAGUCHIand NORTHCOTE, 1966; FEDORENKO and SWIFT. 1972; LEWIS, 1975). The typical migration pattern is fully developed in fourthinstar larvae, and consi$s of a rise from the sediments into the water column at dusk and return at dawn (JUDAY, 1921; EGGLETON, 1931; BERG, 1937; ROTH, 1968; PARMA, 1969a). Light appears to control the timing and pattern of larval migrations, as indicated by the correlation of migration pattern with underwater light intensity (TERAGUCHI and NORTHCOTE, 1966; MALUEG and HASLER, 1967; CHASTON, 1969; GOLDSPINK and SCOTT, 1971). Even though LAROW (1968, 1969) and SWIFT and FORWARD (I 980) have suggested mechanisms for the role of light in timing larval migrations, our knowledge of the interaction between larval photoresponses and underwater light is not sufficient to explain the ontogenetic changes in Chaoborus migration patterns. In general, ‘older’ instars are negatively phototactic (BERG, 1937; CCIOK and CONNORS,1964; LAROW, 1971; SWIFT and FORWARD, 1980) and ‘younger’ instars are positively phototactic (BERG, 1937; COOK and CONNORS, 1964; LAROW, 1971). However, detailed measurements of larval photoresponses have only been made for fourth-instar larvae (SWIFT and FORWARD, 1980). The extent to which ontogenetic changes in migration pattern are related to ontogenetic changes in larval photo-
MATERIALS
AND METHODS
Collection of larvae All experiments were done using second-instar Chaoboruspunctipennis (Say) larvae collected in Crane Pond, Newport, North Carolina. Larvae were collected in the late evening using a 240 pm mesh net hauled horizontally behind a rowing-boat. Samples were seived immediately through a 500 pm mesh strainer to remove third- and fourth-instar larvae and other large plankton. Bulk samples of larvae were returned to the laboratory and sorted under dim white light into plastic weighing pans containing about 4 ml of pond water which had been filtered to remove particles larger than 5 pm. Larvae were sorted individually by pipette; each pan received 20-25 larvae. The larvae were either dark or light adapted for at least 1 hr before being used in experiments. Larvae were collected about 2100 hr, sorted from about 2200 to 0100 hr, and tested from about 0100 to 0300 hr. Larvae were always used in experiments the night they were collected. Phototaxis Phototaxis was measured as movement towards (positive) or away (negative) from a directional light source. Details of the light source are given in SWIFT and FORWARD (1980). The stimulus light was obtained IX3
MICHAEL C. SWIFT AND RICHARD B. FORWARD. JR
184
by filtering light from a projector through heat, interference and neutral density filters. The experimental chamber was a lucite trough (13.8 x 2.4 x 3.9 cm) with 3.2 mm thick end walls. The chamber was divided into 5 equal sections. by lucite partitions which could be raised and lowered simultaneously. The protocol for all experiments was the following: (1) The trough was filled with about 60 ml of filtered pond water to a depth of 2 cm, the partitions were lowered, and the trough was positioned in front of the light source. (2) One pan of pre-sorted larvae was poured into the central section of the trough. (3) After 1 min in darkness, the partitions were gently raised, the stimulus light turned on for 30 set, and the partitions lowered. (4) The number of larvae in each section of the trough was recorded, the larvae and water were poured out, and the trough was re-filled for another trial. (5) Interference and/or neutral density filters were changed between trials when necessary. The protocol for the ‘dark control’ was the same except that the stimulus light was not turned on. The proportion of larvae showing positive phototaxis was defined as the number of larvae in the section of the trough closest to the light source divided by the total number of larvae in the trough. Similarly, larvae in the section furthest from the light were considered to show negative phototaxis. In view of the relatively large size of the trough with respect to the size of the larvae and the low number of larvae tested per trial, it is unlikely that the larvae caused much light scattering in the water or acted to substantially block the light when aggregated at the stimulus end of the trough. The mean (f S.D.) percentage positive and negative responses at each wavelength or intensity was calculated from the percentage data following arc sin square root transformation. These values were then plotted against wavelength or light intensity. Analysis of variance and multiple comparison techniques were used to examine the effect of wavelength or intensity on the level of response and to test for significant differences among treatment means. Light intensity was measured using a radiometer (EG & G, model 550-l) with the sensor located at the position where the end of the trough would be during experiments. Measurements were made in energy units (Wm -*). The quanta1 level was calculated using the energy level of a quantum at the wavelength of maximum transmission for each interference filter. In practice, the light intensity used in each
Table 1. Quanta1 intensities
experiment was considere&obe the value at the centre of the trough. This value was calculated from the measured intensity at the entrance to the trough and from the attenuation coefficient of filtered pond water at each test wavelength. Measurements of the pond water were made with a spectrophotometer (Cary, Model 14) having a cell with a 10 cm path length. Spectral attenuation resulted from both scattering and absorption and increased with decreasing wavelength. Attenuation coefficients ranged from 0.008 (10 cm path length) at 660 nm to 0.108 at 380 nm. The light intensity of each wavelength to be tested on a given day was measured the night before the experiments were run. The actual optical densities of the neutral density filters were measured for each wavelength. Spectral sensitivity A response spectrum was produced by measuring the per cent positive phototactic response to stimulation with approximately equal quanta1 intensities of light at each test wavelength. Fifteen wavelengths were tested (400-680 nm in 20 nm steps). Positive phototaxis increases with stimulus intensity over a range of intensities between the lower threshold intensity and the intensity at which the maximum response occurs. Thus, the test intensity must be between these two extremes. Variations in response level among equal quanta1 intensities of different wavelengths indicates variations in sensitivity to those wavelengths; the greater the response the greater the sensitivity. Preliminary measurements indicated that a quanta1 intensity of about 5.0 x lOi quanta me2 see-i produced an appropriate level of response. Each wavelength was neutral density filtered to approximately this value. The mean quanta1 intensity was 4.6 x lOi quanta me2 set-i with a standard deviation of 0.5 x lOi quanta me2 sect’; actual values for each wavelength are given in Table 1. Five replicate sets of dark adapted larvae were tested at each wavelength, and 13 dark controls were tested. Intensity sensitivity To determine the pattern of phototaxis at different light levels we measured larval response to 12 intensities. All measurements were made at 420 nm except the two highest intensities. For these measurements a Corning 5-56 blue filter was used; the filter transmits light between 400 and about 555 nm. The 420 nm stimulus wavelength was chosen because of the peak in sensitivity at that wavelength.
used at each wavelength
in the
response
spectrum
measurements Wavelength bm) 400 420 440 460 480 500 520 540
Quanta 5.0 4.0 4.3 3.7 3.9 4.9 4.5 4.7
mm2 set-’ x x x x x x x x
10’4 IO” 10’” 10’4 10’4 10’4 10’4 IO’J
Wavelength bun) 560 580 600 620 640 660 680
Quanta 4.8 5.6 5.3 4.6 5.1 4.6 4.6
m x x x x x x x
* set _ 1 10’4 1OlJ 10’4 1OlJ 10’” IO” 10’f
Photoresponses
of second-instar
Chaohorus larvae
IX5
The experimental protocol was the same as in the response spectrum measurements except that stimulus intensity was changed between tests instead of wavelength. Dark controls were tested before and after each set of intensities was tested. Five replicate series of intensities were measured. Larvae were either dark adapted for at least 1 hr before being tested or light adapted at least 1 hour under GE cool white fluorescent lamps at a light intensity of about 1.54 Wme2. Light adapted larvae were held in the dark for the 30 set equilibration period before the partitions were raised and the stimulus lamp turned on. RESULTS The positive phototactic response of second-instar Chaoborus punctipennis larvae to equal quanta1 intensities of light is significantly affected (p < 0.001) by the wavelength of that light (Fig. 1). The response of these larvae is significantly greater at 400-440 nm and 620 nm than at all other wavelengths tested. Thus, sensitivity maxima occur at 420 and 620 nm. The response at all wavelengths was significantly greater than the dark controls. The sign of the phototaxis of dark-adapted larvae varied with light intensity. Positive phototaxis was significantly affected (p < 0.001) by changing light intensity (Fig. 2). The percentage of larvae showing a positive phototactic response was significantly greater than dark controls at all light intensities above 2.2 x 10 _ ’ Wm 2, the threshold intensity for positive phototaxis. The degree of response increased as light intensity increased to about 2 x 10e3 Wmm2 and then decreased somewhat as light intensity increased further. Negative phototaxis was also significantly affected by light intensity @ = 0.003) (Fig. 2). Negative phototaxis was significantly greater than the dark controls at intensities greater than 10m2 Wm-‘. Light-adapted larvae were less sensitive to light than were dark-adapted larvae (Fig. 2). There was a significant effect of intensity on the level of positive phototaxis @ = 0.013). The threshold intensity for positive phototaxis in these larvae was 10e3 Wm-? above this intensity the level of positive phototaxis was significantly greater than the dark controls. The level of negative phototaxis was not significantly affected by light intensity.
Fig. 1. Response spectrum for second-instar C. punctipennis larvae. The per cent positive phototaxis stimulated by approximately equal quanta) intensities of each stimulus wavelength is plotted for each wavelength. Data points are means f S.D. C is the control response in darkness.
Fig. 2. Percentage phototactic response of dark-adapted light-adapted second-instar larvae as a function of intensity at a wavelength of 420 nm. Data points means + SD. for positive phototaxis (solid circles. lines), negative phototaxis (open circles, dashed lines), dark controls (points marked ‘Cf.
and light are solid and
DISCUSSION The response spectrum we measured for secondinstar C. punctipennis larvae is characterized by peaks in sensitivity at 420 and 620 nm and a lower but approximately constant sensitivity at wavelengths between these peaks. Our data differs a great deal from the only other measurements of spectral sensitivity of young larvae. LAROW (1971) showed that ‘immature’ C. punctipennis larvae were positively phototactic to 350 and 400 nm light and negatively phototactic to light at his test wavelengths above 440 nm. A response spectrum specifies the level of response stimulated by light of equal quanta1 intensity at each wavelength, and it will, therefore, vary as quanta1 intensity varies. LAROW (1971) apparently determined a response spectrum but didn’t report the quanta1 intensities used in his experiments. Because the sign of larval phototaxis varies with light intensity (SWIFT and FORWARD, 1980; this study), the differences between our measurements of spectral sensitivity and those of LAROW (1971) are probably due to differences in the light intensity used in our respective experiments. The spectral sensitivity of second-instar larvae differs from that of fourth-instar larvae. Fourth-instar larvae have a single peak of sensitivity at about 400 nm, a wide region of lower sensitivity (480420 nm), and a sharp decrease in sensitivity above 640 nm (SWIFT and FORWARD, 1980). Although the spectral sensitivity of fourth-instar larvae was determined from a true action spectrum and that of second-instar larvae from a response spectrum, there should be little qualitative difference between the results of these two methods (FORWARD and CRONIN, 1979). The consistent sensitivity to light in the violet part of the spectrum seen in both instars is probably an adaptation to the visual demands of the adult terrestrial environment. Since the attenuation coefficient of Crane Pond water increases dramatically
186
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AND
in the u.v.-violet region, the amount of violet light underwater is proportionately very low compared to other visible wavelengths. Several other insects with aquatic larvae and terrestrial adults exhibit the same pattern of larval sensitivity (HORRIDGE, 1969; AUTRUM and KOLB, 1978). The peak in sensitivity in the orange-red region of the spectrum found in second-instar larvae is not present in fourth-instar larvae. This difference may be related to differences in larval habitat. Second-instar larvae are typically found in the water column during the day while fourth-instar larvae are not (BERG, 1937; LEWIS, 1975). Because Crane Pond is highly coloured with humic material the spectral composition of underwater light is skewed towards the yellow-range part of the spectrum. Thus, the sensitivity maximum at 620 nm in second-instar larvae may be an adaptation to the light regime in the water column. Dark-adapted organisms generally exhibit a positive phototactic response to low light intensities which reverses to a negative phototaxis as the intensity increases (FORWARD, 1976). Dark-adapted secondinstar larvae in our experiments were positively phototactic at low light intensities, but there was no clear shift to a negative phototaxis as light intensity increased. At light intensities above about 10eJ Wrnm2 the percentage of larvae showing positive phototaxis decreased, and the percentage showing negative phototaxis increased. However, even at the highest intensity tested (50.0 Wme2) more larvae were positively phototactic than were negatively phototactic. The threshold intensity for positive phototaxis was about 2 x IO- ’ Wmm2 for a 30 set stimulus. This value is lower by about one log unit than the thresholds for planktonic crustaceans and fish larvae (FORWARD, 1976) and it is about the same as the threshold level for positive phototaxis in fourth-instar larvae when they are subjected to a 30 set stimulus. The response of dark-adapted second-instar larvae to increasing light intensity was very different from that of fourth-instar larvae. In second-instar larvae the percentage of larvae showing a positive phototaxis increased to a peak at about 10m4 Wm-* and declined somewhat as light intensity continued to increase. In fourth-instar larvae, however, there was a striking shift in the sign ofphototaxis from positive to negative at about 10m4 Wm-’ (SWIFT and FORWARD, 1980). Because of the difference in wavelength at which these two sets of experiments were run (420 and 540 nm) there is a difference in the number of quanta available at any given intensity between the two experiments. However, this difference is equivalent to only about one-half log unit in intensity. Thus, during larval development there is a striking shift in the sign of phototaxis at higher light intensities from positive to negative. Light adaptation generally decreases sensitivity so that the threshold intensity for phototaxis is raised to a higher value. Second-instar larvae responded as expected when light adapted. The percentage of the larvae showing a positive phototaxis increased as intensity increased but the threshold intensity was higher than for dark-adapted larvae. The light intensity in these experiments was apparently never high enough to stimulate a negative phototaxis.
RICHARD B. FORWARD,JR
The ontogenetic changes in photoresponses measured in the laboratory in our experiments parallel ontogenetic changes in eye morphology and the timing and pattern of vertical migration in Chaoborus larvae. Second-instar larvae appear to be adapted to the photoenvironment of the water column. They are sensitive to those wavelengths which can be expected to penetrate into the water column, and they exhibit little negative phototaxis--even at relatively high light-intensities. Fourth-instar larvae appear to be adapted to avoid the water column except at very low light intensities (SWIFT and FORWARD, 1980). They lack a peak in sensitivity to longer wavelengths of light, and they show a strong negative phototaxis even at relatively low light intensities. The role of the compound eye in light reception is not clearly known. It develops and becomes pigmented during the second, third and fourth instar. Visual observation indicated that the second-instar larvae used in our experiments had not developed pigmented ommatidia in the compound eye; they had a pigmented ocellus only. Fourth-instar larvae have fully developed compound eyes. This description of second-instar larvae adapted to the photoenvironment of the water column predicts that these larvae should be planktonic during the day. Second-instar larvae of several species have been described as being planktonic during the day and as undergoing a vertical migration of small amplitude (e.g. PARMA, 1969a, b; TERAGUCHIand NORTHCOTE, 1966; FED~RENKO and SWIFT, 1972; LEWIS, 1975). However, in Crane Pond second-instar larvae were never collected near the surface during daylight. Their exact die1 vertical migration pattern is unknown, but they were abundant near the surface at dusk or later. Perhaps the light intensity near the surface of Crane Pond during daylight was high enough to stimulate a negative phototaxis. It is clear that before we can fully understand the role of light in controlling vertical migration in Chaoborus it is necessary to measure underwater light at those wavelengths to which the larvae are sensitive and at a time when the larvae are responding to light. Acknowledgements-We would like to thank Mr. CARLETON GARNER for unlimited access to Crane Pond and for the use of his rowing-boat. This material is based upon work supported by the National Science Foundation under grant No. OCE 77-26838 to R. B FORWARD and Duke University Research Council Grant No. 453-5926-6634-2290 to M. C. SWIFT. Appalachian Environmental Laboratory Contribution No. 1254 (UM-CEES).
REFERENCES AUTRUM H. and KOLB G. (1978) Spektrale Empfindlichkeit einzelner Sehzellen der Aeshniden. 2. vergl. Physiol. 60, 450-477. BERG K. (1937) Contributions to the biology of Corethru Meigen (Chaoborus Lichtenstein). Biof. Meddr. 13, l-101. CHASTON I. (1969) The light threshold controlling the vertical migration of Chaoborus puncripennis in a Georgia impoundment. Ecofogy 50,916-920. COOKS. F., JR. and CONNORS J. D. (1964) The direct response of Chaoborus astictopus to light (Diptera: Culicidae). Ann.
Photoresponses
of second-&tar
ent. Sot. Am. 57, 387-388. EGGLETON F. E. (193 1) A limnological study of the profundal bottom fauna ofcertain fresh-water lakes. Ecol. Monogr. I, 23 l-332. FEDORENKO A. Y. and SWIFT M. C. (1972) Comparative biology of Chaoborus americanus and Chaoborus trivittatus in Eunice Lake, British Columbia. Limnol. Oceanogr. 17, 721-730. FORWARD R. B., JR. (1976) Light and diurnal vertical migration: photobehavior and photophysiology of plankton. In Photochemical and Photobiological Reviews (Ed. by SMITH K. C.). Vol. 1. pp. 157-209. Plenum, New York. FORWARD R. B., JR. and CRONIN T. W. (1979) Spectral sensitivity of larvae from intertidal crustaceans. J. camp. Phvsiol. 133, 3 1 l-3 15. GOLDSPINK C. R. and SCOTT D. B. C. (1971) Vertical migration of Chaoborus flavicans in a Scottish loch. Freshwater Biol. 1, 41 l-421. HORRIDGE G. A. (1969) Unit studies on the retina of dragonflies. Z. vergl. Physiol. 62, l-37. JU~AY C. (1921) Observations on the larvae of Corethru punctipennis Say. Biol. Bul!. 40, 271-286. LAROW E. J. (1968) A persistent diurnal rhythm in Chaoborus larvae. I. The nature of the rhythmicity. Limnol. Oceanogr. 13, 250-256. LARo~ E. J. (1969) A persistent diurnal rhythm in
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Chaoborus larvae. II. Ecological significance. Limno/. Oceanogr. 14, 213-218. LAROW E. J. (1971) Response of Chaoborus (Dlptera: Chaoboridae) larvae to different wavelengths of light. Ann. ent. Sot. Am. 64, 461-464. LEWIS W. M.. JR. (1975) Distribution and feeding habits of a tropical Chaoborus population. Verh. lnt. Verrin. Theor. Anger. Limnol 19, 3 106-3 119. MALUEG K. W. and HASLER A. D. (1967) Echo-sounder studies in die1 vertical movements of Chaoborus larvae in Wisconsin (U.S.A.) lakes. Verb. Int. Verein. Theor. Anger. Limnol. 15, 1697-I 708. PARMA S. (1969a) The life cycle of Chaoborus crystallinus (DeGeer) (Diptera: Chaoboridae) in a Dutch pond. Vrrh. Int. Verein. Theor. Angers. Limnol. 17, 888-894. PARMA S. (1969b) Notes on the larval taxonomy. ecology. and distribution of the Dutch Chaoborus species (Diptera: Chaoboridae). Beaufortia 17, 21-50. ROTH J. C. (1968) Benthic and limnetic distribution of three Chaoborus species in a southern Michigan lake (Diptera: Chaoboridae). Limnol. Oceanogr. 13, 242-249. SWIFT M. C. and FORWARII R. B.. JR. (1980) Photoresponses of Chaoborus larvae. J. insect Physiol. 26, 365-371. TERAGU(‘HI M. and NORTHC‘OTF T. G. (1966) Vertical distribution and migration of Chaoborusflavicans larvae in Corbette Lake, British Columbia. Limnol. Oceanogr. 11, 164-176.