Insects

Eggs, final instar larvae, pupae and adults of Drosophila melanogaster; eggs and pupae of Culex pipiensmolestus; 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 = 50 mL) in a plastic box (72 × 72 × 100 mm). C. pipiensmolestus were supplied by Earth Chemical Co., Ltd. (Tokyo, Japan). The eggs, larvae and pupae were maintained in a plastic container (150 mm dia × 91 mm tall) containing 250 mL of water, with a constant supply of fishery feed (trout juveniles). Adults were maintained in a plastic cage (340 × 250 × 340 mm) containing two plastic cups (30 mm dia × 35 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 × 77 mm tall) on wheat flour containing 5% dry brewer's yeast. All insects were wild type and were maintained at 25 ± 1°C under a photoperiod of 16L:8D.

LED light radiation

LED lighting units (IS-mini®, ISL-150 × 150 Series; CCS Inc., Kyoto, Japan; light emission surface: 150 × 150 mm; 360 LEDs were equally arranged on a panel; LED type: φ 3-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−2·s−1) 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 (16L:8D photoperiod) conditions. The temperature data are summarized in Supplementary Tables 5 and 6.

Figure 4 Emission spectra of LED lighting units used for the experiments. Full size image

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 μL of water in a glass petri dish (60 mm dia × 20 mm tall). The petri dish was sealed with parafilm, placed in the incubator and irradiated with LED light for 7 d at 25 ± 1°C. 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 × 1018 photons·m−2·s−1 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 16L:8D 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 × 90 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 ± 1°C and the numbers of newly hatched larvae were counted under a stereomicroscope. The lethal effects of irradiation at 3.0 × 1018, 4.0 × 1018, 5.0 × 1018 and 10.0 × 1018 photons·m−2·s−1 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, L119) 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 × 15 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 ± 1°C. After irradiation, the petri dish was transferred to the thermostatic chamber (LP-1PH; Nippon Medical & Chemical Instruments Co., Ltd., Osaka, Japan) and maintained under 16L:8D (white fluorescent lamps were used during the light period) at 25 ± 1°C. The number of adults that emerged was counted after 10 d. Pupae that died before emergence were dissected under a stereomicroscope and their developmental stages were determined19. We investigated the lethal effects of irradiation at 5.0 × 1018, 7.0 × 1018, 10.0 × 1018 and 12.0 × 1018 photons·m−2·s−1. 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 emergence and released onto 10 mL of culture medium (same composition as for rearing stock cultures) in a glass petri dish (60 mm dia × 90 mm tall). The petri dish was irradiated with 467-nm LED light in the incubator at 25 ± 1°C. Flies were irradiated for 24 h d−1 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 × 49 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 ± 1°C. 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 × 1018 photons·m−2·s−1 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 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 × 38 mm tall), the opening of which was covered with a glass plate. The cup was placed in the incubator (25 ± 1°C) and irradiated with 417-nm LED light at 10.0 × 1018 photons·m−2·s−1 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 ± 1°C) 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) conditions. 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 × 15 mm tall). The petri dish was placed in the incubator (25 ± 1°C) and irradiated with LED light at 2.0 × 1018 photons·m−2·s−1 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.