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Lethal effects of short-wavelength visible
light on insects
Masatoshi Hori*, Kazuki Shibuya*, Mitsunari Sato & Yoshino Saito
Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
We investigated the lethal effects of visible light on insects by using light-emitting diodes (LEDs). The toxic
effects of ultraviolet (UV) light, particularly shortwave (i.e., UVB and UVC) light, on organisms are well
known. However, the effects of irradiation with visible light remain unclear, although shorter wavelengths
are known to be more lethal. Irradiation with visible light is not thought to cause mortality in complex
animals including insects. Here, however, we found that irradiation with short-wavelength visible (blue)
light killed eggs, larvae, pupae, and adults of
Drosophila melanogaster
. Blue light was also lethal to
mosquitoes and flour beetles, but the effective wavelength at which mortality occurred differed among the
insect species. Our findings suggest that highly toxic wavelengths of visible light are species-specific in
insects, and that shorter wavelengths are not always more toxic. For some animals, such as insects, blue light
is more harmful than UV light.
Understanding the influence of visible light (400–780 nm) on organisms is important for identifying novel
uses and examining hazards of exposure to visible light. However, little is known about the biological
toxicity of visible light. Although recent studies have described damage by short-wavelength visible light
(blue light, 400–500 nm) to the mammalian retina, called the ‘blue light hazard’
1–5
, there have been no reports on
the lethal effects of irradiation with visible light on complex animals, including insects. On the other hand, the
toxicity of shortwave UV light to organisms is well known. UVC (100–280 nm) and UVB (280–315 nm) induce
mutagenic and cytotoxic DNA lesions
6,7
, and UVC irradiation has lethal effects on insects
8
and microorganisms
9
.
The use of UVC irradiation for control of pests such as Tribolium castaneum,T. confusum,Cadra cautella, and
Trogoderma granarium, which infest stored grains, has been studied
10,11
. Lethal effects of UVC against larvae of
the silkworm Bombyx mori are also well known
12,13
. Lethal effects of UVB have been reported for spider mites
14
,in
which UVB irradiation strongly decreases survivorship and egg production. However, there are no reports that
describe lethal effects of UVB or UVA (315–400 nm) on insects, although UVA irradiation slightly decreases
adult longevity in the lepidopteran Helicoverpa armigera
15
. It is well known that shorter wavelengths of light are
more lethal
9,16,17
. In addition, positive effects of wavelengths ranging from UVA to green (500–560 nm) have been
reported for spider mites; irradiation with UVA, blue, and green light caused photoreactivation of mites damaged
by UVB irradiation
18
. Therefore, irradiation with visible light is not considered lethal to complex animals,
including insects. Here, in contrast, we show a strong lethal effect of blue light on insects. In this study, we found
that blue-light irradiation by a common LED can kill insect pests of various orders and that highly lethal blue-light
wavelengths are species-specific in insects.
Results
Lethal effects of irradiation with various wavelengths of light on D. melanogaster pupae.First, we investigated
the lethal effect of light (wavelengths from 378 to 732 nm) on D. melanogaster pupae using LEDs. Irradiation with
wavelengths of 378, 417, 440, 456, and 467 nm at 3.0 310
18
photons?m
22
?s
21
throughout the pupal stage
significantly increased the mortality of D. melanogaster pupae compared with their mortality under DD (24-h
dark) conditions (Fig. 1a, Supplementary Table 1). In particular, we identified two peak wavelengths (440 and
467 nm; Fig. 1a) that had strong lethal effects. More than 90% and 70% of pupae died before adult emergence after
irradiation with wavelengths of 467 and 440 nm, respectively; the lethal effects of these wavelengths were stronger
than those of UVA (378 nm). Wavelengths of 404 nm and $496 nm did not have a lethal effect on D.
melanogaster pupae (Fig. 1a, Supplementary Table 1). In wavelengths ranging from 378 to 508 nm, mortality
increased with increasing numbers of photons (Fig. 1b). Wavelengths of 440, 456, and 467 nm led to 100%
mortality at 4.0 310
18
photons?m
22
?s
21
; this number of photons did not have a lethal effect at wavelengths of 508,
657, and 732 nm. These results reveal, for the first time, that complex animals such as insects can be killed by
OPEN
SUBJECT AREAS:
ENTOMOLOGY
LASERS, LEDS AND LIGHT
SOURCES
Received
1 August 2014
Accepted
19 November 2014
Published
9 December 2014
Correspondence and
requests for materials
should be addressed to
M.H. (hori@bios.
tohoku.ac.jp)
*These authors
contributed equally to
this work.
SCIENTIFIC REPORTS | 4 : 7383 | DOI: 10.1038/srep07383 1
irradiation with certain wavelengths of visible light, and that visible
light is more harmful than UV light to some animals.
Lethal effects of irradiation with blue light on eggs, larvae, and
adults of D. melanogaster.Irradiation with a wavelength of 467 nm
had the strongest lethal effect on Drosophila pupae, although this
wavelength was also lethal to other developmental stages. The
mortality rate of eggs increased with increasing numbers of
photons (Fig. 2a); the majority of eggs died after 48-h irradiation
at $5.0 310
18
photons?m
22
?s
21
, whereas most eggs hatched under
dark conditions. Irradiation with a wavelength of 467 nm for 24 h
was lethal to final-instar larvae (L1–L2)
19
and showed a dose–
response relationship (Fig. 2b). Most flies died before adult
emergence after irradiation at 7.0 310
18
photons?m
22
?s
21
. Flies
died during earlier developmental stages as the number of photons
increased. Forty percent and 27% of flies died during the larval
stage following irradiation at 12.0 310
18
and 10.0 310
18
photons?m
22
?s
21
, respectively. Using these same irradiation levels,
more than 90% of flies died during the larval or prepupal stages (L1–
P4). With irradiation at 7.0 310
18
photons?m
22
?s
21
, the flies that
died before adult emergence were almost evenly divided among the
flies that died during the larval or prepupal stages (L1–P4) and those
that died during the pupal stage (P5–P15). Interestingly, none of the
irradiated flies died during the developmental stages of P5–P9. Adult
longevity decreased significantly as the number of photons increased
(Fig. 2c, Supplementary Table 2). In contrast, the longevity of adult
flies maintained under dark conditions was approximately 60 d.
Irradiation with a wavelength of 467 nm affected fly fecundity
(Fig. 2d); the mean number of eggs deposited by surviving females
decreased with increasing numbers of photons. These results show
that irradiation with blue light has a lethal effect on the pupal stage of
Drosophila, and also on other developmental stages of this insect—
including the adult stage, which is typically considered tolerant of
light irradiation.
Lethal effects of blue-light irradiation on C. pipiens molestus and
T. confusum.We also investigated the lethal effects of various blue-
light wavelengths (404–508 nm) on pupae of the mosquito Culex
pipiens molestus. Blue light irradiation was lethal to mosquito pu-
pae, although their tolerance was higher than that of D. melanogaster
pupae (Fig. 3a, b). Compared with DD conditions, irradiation with
wavelengths of 404, 417, and 456 nm at 10.0 310
18
photons?m
22
?s
21
throughout the pupal stage significantly increased the mortality of C.
pipiens molestus (Supplementary Table 3); the peak wavelength of
417 nm was highly lethal (Fig. 3a). Wavelengths of 404 and 417 nm
killed substantial proportions of pupae before adult emergence,
whereas wavelengths $440 nm were non- or negligibly lethal
(Fig. 3a). The lethal effect of 417 nm increased with increasing
numbers of photons; in contrast, the lethal effect of 404 nm was no-
minal, and the lethal effects of 440-, 456-, and 467-nm wavelengths
increased only slightly with increasing numbers of photons (Fig. 3b).
Irradiation with a wavelength of 417 nm was lethal to mosquito eggs,
and the mortality increased over time (Fig. 3c, Supplementary Table
4). Whereas only 34% of mosquitos died before hatching following
48 h of irradiation at 10.0 310
18
photons?m
22
?s
21
, approximately
90% of hatchlings from the irradiated eggs died within 72 h after
irradiation; this is compared with a 2% mortality rate of hatchlings
from the eggs maintained under dark conditions. Accordingly, even
if irradiated eggs hatched, most hatchlings died soon thereafter.
These results show that the lethal effect of blue light is not con-
fined to flies; however, the effective wavelength at which mortality
occurs is species-specific, and tolerance to blue-light irradiation
differs among insect species.
Blue-light irradiation was lethal to pupae of the confused flour
beetle T. confusum (Fig. 3d). All beetles irradiated with wavelengths
ranging from 404 to 467 nm throughout the pupal stage at 2.0 310
18
photons?m
22
?s
21
died before adult emergence. However, irradiation
with the 532-nm wavelength did not have a lethal effect. These find-
ings show that blue-light irradiation can kill insects of various orders.
Discussion
In this study, we revealed for the first time that blue-light irradiation
can kill insect pests and that effective wavelengths of visible light are
species-specific. Our findings show that visible light is more harmful
than UV light to some animals. The insides of the containers and
media in which insects were housed did not register temperatures
that would have affected the survival of any of the developmental
stages in any of the irradiation treatments (Supplementary Tables 5
and 6). In addition, increases in lethal effects did not always corre-
spond to increases in temperature. In the irradiation treatments in
which increasing temperature corresponded to lethal effects, the
temperatures were not high enough to affect insect survival
20
.
Therefore, we concluded that temperature increases caused by
LED light did not cause the mortality. UVB and UVC directly
Figure 1
|
Comparison of the lethal effects of light irradiation on
Drosophila melanogaster
pupae using various wavelengths of LED light.
(a) Mortality of pupae irradiated with 3.0 310
18
photons?m
22
?s
21
. Data
are means 6standard error (SE). Different lowercase letters next to bars
indicate significant differences (Steel–Dwass test, P,0.05). LL, DD, and
LD indicate 24-h light, 24-h dark, and 16L58D photoperiod conditions,
respectively. (b) Dose–response relationships for lethal effects of
irradiation on pupae for each wavelength. Data are mean values.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 7383 | DOI: 10.1038/srep07383 2
damage DNA by inducing the formation of DNA lesions, notably cis-
syn cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimi-
done photoproducts
21
. The maximum absorption spectrum of
DNA ranges from 260 to 265 nm, and absorption rapidly declines
at longer wavelengths
22
. DNA damage induced by UVA is minimal
because UVA is not absorbed by native DNA
6,7
. However, UVA
indirectly damages lipids, proteins, and DNA by enhancing the pro-
duction of reactive oxygen species (ROS)
23–25
. Increases in oxidative
stress caused by UVA irradiation have also been shown in insects
such as the cotton bollworm Helicoverpa armigera
26
. In addition,
molecular-level responses to stress and damage by UVA irradiation
have been confirmed in insects
27,28
. However, lethal effects of UVA
irradiation on insects have not been shown
15,27
. Blue-light irradiation
injures organisms by stimulating the production of ROS. Many
microbial cells are highly sensitive to blue light as a result of the
accumulation of photosensitizers such as porphyrins and flavins
29
.
Mammalian retinas can also be severely damaged by ROS produced
by blue-light irradiation
4,5
. It is probable that the lethal effect of blue
light on insects is caused by the production of ROS, because the
effective wavelength is species-specific and not always associated
with the amount of photon energy delivered. In addition, light trans-
mission of D. melanogaster puparia was not wavelength-specific
(Supplementary Figure 1). These findings suggest that light absorp-
tion by certain inner tissues of the fly is wavelength-specific. That is,
species-specific chromophores or photosensitizers in insect tissues
absorb specific wavelengths of light, thereby generating free radicals.
Insects subsequently die from tissue damage caused by free-radical
formation.
We selected three insect species for the experiments presented
here. D. melanogaster is a major model animal species with a short
life cycle. Investigation of the lethal effects of blue-light irradiation on
D. melanogaster can be conducted with ease and can be useful for
Figure 2
|
Effects of irradiation with 467-nm blue light on eggs, larvae, and adults of
Drosophila melanogaster
.(a) Dose–response relationships for
lethal effects of irradiation with LED light on eggs. ‘‘0’’ photons represents the 24-h dark condition. Data are means 6standard error (SE).
(b) Relationship between light dose and developmental stage at which mortality occurred. Developmental stages of larvae and pupae were classified
according to Bainbridge and Bownes (1981)
19
. L1 and L2 are third-instar larvae, P1–P4 are prepupae, and P5–P15 are phanerocephalic pupae. No
irradiated flies died during the P5–P9 developmental stages. Data are mean values. Mortality (mean 6SE) of flies that could not emerge is indicated by the
black line. (c) Dose–response relationships for effects of irradiation with LED light on adult longevity. ‘‘0’’ photons represents the 24-h dark condition.
Data are means 6SE. Different lowercase letters above bars indicate significant differences (Steel–Dwass test, P,0.05). (d) Dose–response relationships
for the effects of irradiation with LED light on fecundity. DD indicates the 24-h dark condition. Inset numbers (1.0, 5.0, and 10.0 310
18
) indicate light
dose in photons?m
22
?s
21
. Data are mean values.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 7383 | DOI: 10.1038/srep07383 3
studying damage caused by blue light or free radicals in animals. C.
pipiens molestus is a major mosquito species that is easily reared, and
thus is an appropriate model species for mosquito experiments.
Mosquitoes are one of the most medically important insect pests
and they transmit serious diseases, including malaria, dengue fever,
yellow fever, West Nile fever, and Japanese encephalitis. This study
showed lethal effects of blue light on mosquito pupae and eggs.
Reproduction of mosquitoes might be prevented by blue-light irra-
diation of water containing eggs, larvae, and pupae, and might
consequently prevent outbreaks of mosquito-borne diseases. T. con-
fusum is a globally important insect pest of stored grain. Our findings
showed the potential of blue-light irradiation for pest control in
stored products. That is, blue-light irradiation may be useful for pest
control in various situations including agriculture, sanitation, and
food storage. D. melanogaster and C. pipiens molestus belong to the
order Diptera, whereas T. confusum belongs to Coleoptera. This
implies that blue-light irradiation has lethal effects against multiple
insect orders. Current techniques in pest management utilize light to
influence insect behaviours, including attraction, repulsion, and light
adaptation in nocturnal species
30
. The present study suggests the
potential for a novel, clean, and safe pest-control technique that
can easily kill insect pests simply by radiating blue light (e.g., LED).
However, tolerance to blue light varied widely among the insect
species studied here. The order of tolerance was C. pipiens molestus
?D. melanogaster $T. confusum. The tolerance of C. pipiens
molestus to blue light was much higher than that of D. melanogaster,
although both species belong to the order Diptera. The habitats of
these three species differ. T. confusum inhabits stored foods in indoor
environments. D. melanogaster lives in both outdoor and indoor
habitats, but it occupies dark environments until adult emergence.
C. pipiens molestus usually lives in water in areas with low light until
adult emergence. Therefore, the quantity of light to which these
species are exposed is highest for C. pipiens molestus, followed (in
decreasing order) by D. melanogaster and T. confusum. Tolerance of
insects to blue-light irradiation is thought to be closely related to the
light exposure experienced in their natural habitats. The numbers of
photons of 470 nm and blue-light wavelengths (400–500 nm) in
direct sunlight in the field associated with our laboratory (Sendai,
Japan; 38uN, 140uE) were approximately 1.0–2.5 310
18
and 7.5–9.0
310
18
photons?m
22
?s
21
, respectively (1:00–2:00 PM in early sum-
mer). Therefore, we assume that D. melanogaster and T. confusum
cannot survive under direct sunlight because of the lethal effect of
blue light. Accordingly, eggs, larva, and pupae of D. melanogaster and
T. confusum require dark habitats. The relationships between insect
Figure 3
|
Lethal effects of blue-light irradiation on the mosquito
Culex pipiens molestus
and the confused flour beetle
Tribolium confusum
.
(a) Mortality of C. pipiens molestus pupae irradiated with various wavelengths of blue light at 10.0 310
18
photons?m
22
?s
21
during the pupal stage. Data
are means 6standard error (SE). Different lowercase letters next to bars indicate significant differences (Steel–Dwass test, P,0.05). DD indicates the
24-h dark condition. (b) Dose–response relationships for lethal effects of irradiation with each wavelength of light on pupae. Data are mean values.
(c) Mortality of C. pipiens molestus that were irradiated with 417-nm light for 48 h at 10.0 310
18
photons?m
22
?s
21
during the egg stage. Data are
means 6SE. Hours in parentheses show the elapsed time after discontinuation of irradiation. Different lowercase or capital letters next to bars indicate
significant differences among the three treatments for each time period (Steel–Dwass test, P,0.05). LL and DD indicate 24-h light and 24-h dark
conditions, respectively. (d) Mortality of T. confusum pupae irradiated with various wavelengths of light at 2.0 310
18
photons?m
22
?s
21
during the pupal
stage. Data are means 6SE. Different lowercase letters next to bars indicate significant differences (Steel–Dwass test, P,0.05). LD indicates 16L58D
photoperiod condition.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 7383 | DOI: 10.1038/srep07383 4
species (or habitats) and tolerance to blue light require further invest-
igation in order to utilize blue-light irradiation for pest control.
Blue-light irradiation may be useful for controlling various insect
pests. However, because the effective wavelengths of blue light are
species-specific, several wavelengths (or broad-spectrum blue light)
are needed for the simultaneous control of multiple species. In addi-
tion, genetic variation in resistance to UVC or ionizing irradiation
has been confirmed in D. melanogaster
31,32
. It is probable that there is
genetic variation in insect resistance to blue-light irradiation; this
variation should be investigated so that the use of blue-light irra-
diation for pest control can be realized in the near future.
The purpose of this study was to reveal the lethal effects of light;
the effects of low doses of blue-light irradiation on insects have not
yet been clarified. In mammals, low doses of UV exposure provide
health benefits including energy improvement, mood elevation, and
vitamin D production, although high rates of exposure can present
health risks such as increased susceptibility to cancer
33
. It is possible
that low doses of blue light can also have beneficial effects on insects.
Our findings facilitate the development of clean and safe pest-
control techniques, and provide important information on the
hazards of exposure to visible light.
Methods
Insects.Eggs, final instar larvae, pupae, and adults of Drosophila melanogaster; eggs
and pupae of Culex pipiens molestus; and pupae of Tribolium confusum were
maintained in our laboratory and used for the experiments. D. melanogaster was
purchased from Sumika Technoservice Co. (Takarazuka, Japan). The flies were
reared on culture medium consisting of glucose (2.5 g), dry brewer’s yeast (2.5 g),
agar (0.5 g), propionic acid (0.25 mL), 20% butyl p-hydroxybenzoate in 70% ethyl
alcohol (0.25 mL), and water (total medium volume 550 mL) in a plastic box (72 3
72 3100 mm). C. pipiens molestus were supplied by Earth Chemical Co., Ltd.
(Tokyo, Japan). The eggs, larvae, and pupae were maintained in a plastic container
(150 mm dia 391 mm tall) containing 250 mL of water, with a constant supply of
fishery feed (trout juveniles). Adults were maintained in a plastic cage (340 3250 3
340 mm) containing two plastic cups (30 mm dia 335 mm tall). Absorbent cotton
impregnated with 3% honey solution was placed in one of the cups as a food source,
and absorbent cotton soaked with water was placed in the other cup as an oviposition
substrate. T. confusum were provided by Fuji Flavor Co., Ltd. (Tokyo, Japan) and were
reared in a plastic container (130 mm dia 377 mm tall) on wheat flour containing
5% dry brewer’s yeast. All insects were wild type and were maintained at 25 61uC
under a photoperiod of 16L58D.
LED light radiation.LED lighting units (IS-miniH, ISL-150 3150 Series; CCS Inc.,
Kyoto, Japan; light emission surface: 150 3150 mm; 360 LEDs were equally arranged
on a panel; LED type: Q3-mm plastic mould) with power supply units (ISC-201-2;
CCS Inc.) were used for UV and visible light radiation. Insects were irradiated with
LED light in a multi-room incubator (LH-30CCFL-8CT; Nippon Medical &
Chemical Instruments Co., Ltd., Osaka, Japan). The emission spectrum was measured
using a high-resolution spectrometer (HSU-100S; Asahi Spectra Co., Ltd., Tokyo,
Japan; numerical aperture of the fibre: 0.2) Comparison of the emission spectra used
in the experiments is shown in Fig. 4. The number of photons (photons?m
22
?s
21
)was
measured using the spectrometer in a dark room and was adjusted using the power-
supply unit. The distance between the light source and the spectrometer sensor during
measurements was approximately the same as that between the insects and light
source in the incubator. Because the insects were irradiated through a glass lid,
polystyrene lid, or glass plate, the same lid or plate was placed between the light source
and sensor during measurement. The distances between the lid or plate and the light
source during measurements were approximately the same as those in the incubator.
Insect containers were placed directly under the light source during irradiation. We
confirmed that the upper surfaces of the containers were irradiated homogeneously
by measuring the numbers of photons. In addition, we assumed that temperature
changes caused by the light source would not affect survival of the insects because
LED light emits little heat. To check this assumption, we measured the temperature
inside the containers using a button-type temperature logger (3650, Hioki E. E. Co.,
Ueda, Japan), of the insects and in the media except for water (filter paper, culture
medium, bottom of dish) using a radiation thermometer (IR-302, Custom Co.,
Tokyo, Japan). We measured water temperature using a digital thermometer (TP-
100MR, Thermo-port Co., Iruma, Japan). Temperatures that showed lethal effects in
several light treatments were measured in each experiment and under DD and LD
(16L58D photoperiod) conditions. The temperature data are summarized in
Supplementary Tables 5 and 6.
Lethal effects of irradiation with various wavelengths of light on D. melanogaster
pupae.Thirty pupae were collected from the rearing boxes within 24 h of pupation
and placed on a sheet of filter paper (Advantec, No. 1, 70 mm dia) impregnated with
700 mL of water in a glass petri dish (60 mm dia 320 mm tall). The petri dish was
sealed with parafilm, placed in the incubator, and irradiated with LED light for 7 d at
25 61uC. The numbers of emerging adults were counted 7 d after the start of
irradiation. Eight replications (petri dishes) were performed for each light dose and
wavelength. Initially, lethal effects at 3.0 310
18
photons?m
22
?s
21
were compared
among 12 wavelengths (378, 404, 417, 440, 456, 467, 496, 508, 532, 592, 657, and
732 nm). We investigated mortality of pupae under 24 h light (LL), 24 h dark (DD),
and 16L58D photoperiod (LD) conditions using white cold cathode fluorescent
lamps (CCFLs) in the light periods. The relationships between lethal effects and
numbers of photons were compared among the 12 wavelengths.
Lethal effects of irradiation with blue light on eggs, larvae, and adults of D.
melanogaster.1) Eggs.Five pairs of mated adults were released onto 10 mL of culture
medium (same as rearing stock culture) in a glass petri dish (60 mm dia 390 mm
tall) and allowed to lay 10 eggs on the medium within 6 h. The petri dish with eggs was
immediately sealed with parafilm and placed in the incubator. The eggs were then
irradiated with 467-nm LED light for 48 h at 25 61uC, and the numbers of newly
hatched larvae were counted under a stereomicroscope. The lethal effects of
irradiation at 3.0 310
18
, 4.0 310
18
, 5.0 310
18
, and 10.0 310
18
photons?m
22
?s
21
were
investigated. We also investigated egg mortality under DD conditions. Ten
replications (petri dishes) were performed for each light dose.
2) Larvae.Ten final-instar larvae (wandering third-instar stage, L1
19
) were collected
from the rearing boxes within 24 h of wandering out of the culture medium and
placed in a polystyrene petri dish (55 mm dia 315 mm tall). The petri dish was
sealed with parafilm, placed in the incubator, and irradiated with 467-nm LED light
for 24 h at 25 61uC. After irradiation, the petri dish was transferred to the ther-
mostatic chamber (LP-1PH; Nippon Medical & Chemical Instruments Co., Ltd.,
Osaka, Japan) and maintained under 16L58D (white fluorescent lamps were used
during the light period) at 25 61uC. The number of adults that emerged was counted
after 10 d. Pupae that died before emergence were dissected under a stereomicro-
scope, and their developmental stages were determined
19
. We investigated the lethal
effects of irradiation at 5.0 310
18
, 7.0 310
18
, 10.0 310
18
, and 12.0 310
18
photons?m
22
?s
21
. Ten replications (petri dishes) were performed for each light dose.
3) Adults.One pair of adults was collected from rearing boxes within 12 h of emer-
gence and released onto 10 mL of culture medium (same composition as for rearing
stock cultures) in a glass petri dish (60 mm dia 390 mm tall). The petri dish was
irradiated with 467-nm LED light in the incubator at 25 61uC. Flies were irradiated
for 24 h d
21
until both the male and female died. Every 24 h, we counted the number
of surviving adults and eggs deposited, and replaced the petri dish containing culture
medium with a fresh one. Ten replications (petri dishes) were performed for each
light dose.
Lethal effects of blue-light irradiation on C. pipiens molestus and T. confusum.1)
C. pipiens molestus pupae.Ten pupae were collected from the stock cultures within
1 h of pupation and released into water (100 mL) in a polyethylene terephthalate
(PET) ice-cream cup (101 mm dia 349 mm tall), the opening of which was covered
with a glass plate. The cup was placed in the incubator and irradiated with LED light
for 5 d at 25 61uC. The numbers of emerging adults were counted 5 d after the start
of irradiation. Ten replications (cups) were performed for each light dose and
wavelength. Initially, lethal effects at 10.0 310
18
photons?m
22
?s
21
were compared
among five wavelengths (404, 417, 440, 456, and 467 nm). We also investigated pupal
mortality rates under DD conditions. The relationships between lethality and number
Figure 4
|
Emission spectra of LED lighting units used for the
experiments.
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SCIENTIFIC REPORTS | 4 : 7383 | DOI: 10.1038/srep07383 5
of photons were then compared among seven wavelengths (404, 417, 440, 456, 467,
496, and 508 nm).
2) C. pipiens molestus eggs.Thirty eggs were collected from the stock cultures within
1 h of deposition and placed in water (50 mL) in a PET ice-cream cup (60 mm dia 3
38 mm tall), the opening of which was covered with a glass plate. The cup was placed
in the incubator (25 61uC) and irradiated with 417-nm LED light at 10.0 310
18
photons?m
22
?s
21
for 48 h. The number of newly hatched larvae was counted 48 h
after the start of irradiation. After checking hatchability, the cup with mosquitoes was
maintained under DD conditions for 72 h (25 61uC), and the mortality of newly
hatched larvae was then investigated. For comparison, hatchability and mortality
rates were investigated under LL (white CCFLs provided light for 48 h, after which
darkness was provided for 72 h) and DD (no irradiation, darkness for 120 h) con-
ditions. Ten replications (ice-cream cups) were performed for each light dose.
3) T. confusum pupae.Ten pupae were collected from the stock cultures within 24 h of
pupation and placed in a glass petri dish (30 mm dia 315 mm tall). The petri dish
was placed in the incubator (25 61uC) and irradiated with LED light at 2.0 310
18
photons?m
22
?s
21
for 14 d, after which we counted the number of adults that
emerged. The lethal effects of irradiation were compared among five wavelengths
(404, 417, 456, 467, and 532 nm). Ten replications (petri dishes) were performed for
each wavelength. We also investigated mortality of pupae under LD conditions (white
CCFLs were used).
Statistical analyses.Mortality and adult longevity were analysed using a generalized
linear model (GLM) followed by the Steel–Dwass test. Mortality of T. confusum pupae
was analysed by Steel–Dwass test without GLM, because 100% mortality occurred
under blue-light irradiation (404–467 nm) and 0% mortality occurred under LD
conditions. The lethal effects on C. pipiens molestus eggs were analyzed by using GLM
followed by the Steel–Dwass test among 417 nm irradiation, LL, and DD in each of 0
and 72 h after discontinuing irradiation.
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Acknowledgments
This study was supported by a grant entitled ‘‘Elucidation of biological mechanisms of
photoresponse and development of advanced technologies utilizing light’’ from the
Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan, and by JSPS KAKENHI
Grant Number 25660261. We wish to thank Earth Chemical Co., Ltd. and Fuji Flavor Co.,
Ltd. for kindly supplying insects for use in our study. We wish to thank for Dr. Yoshihara
(Graduate School of Agricultural Science, Tohoku University) for kindly advice on
statistical analyses.
Author contributions
M.H. designed the experiments. K.S., M.S. and Y.S. performed the experiments. M.H. and
K.S. analysed the data. M.H. wrote the manuscript. All authors reviewed the manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Hori, M., Shibuya, K., Sato, M. & Saito, Y. Lethal effects of
short-wavelength visible light on insects. Sci. Rep. 4, 7383; DOI:10.1038/srep07383 (2014).
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