Seasonal changes in temperature and day length are distinct between rural and urban areas due to urban warming and the presence of artificial light at night. Many studies have focused on the impacts of these ubiquitous signatures on daily biological events, but empirical studies on their impacts on insect seasonality are limited. In the present study, we used the flesh fly Sarcophaga similis as a model insect to determine the impacts of urbanisation on the incidence and timing of diapause (dormancy), not only in the laboratory but also in rural and urban conditions. In the laboratory, diapause entry was affected by night-time light levels as low as 0.01 lux. We placed fly cages on outdoor shelves in urban and rural areas to determine the timing of diapause entry; it was retarded by approximately four weeks in urban areas relative to that in rural areas. Moreover, almost all flies in the site facing an urban residential area failed to enter diapause, even by late autumn. Although an autumnal low-temperature in the urban area would mitigate the negative effect of artificial light at night, strong light pollution seriously disrupts the flesh fly season adaptation.

Insects

Colonies of S. similis were obtained from females captured in Osaka City (34.59 °N, 135.50 °E) and Toyonaka City (34.80 °N, 135.45 °E), Japan, in 2014 and 2018, respectively. Stock cultures were maintained under diapause-averting long-day conditions (16-h light/8-h dark) at 25 ± 1 °C. Fluorescent light (FL15SW, FL20SW, FL30SW, or FL40SW; Panasonic, Kadoma, Japan) was used as the light source during the light phase (> 1,000 lux). Water, two sugar blocks, and a piece of beef or chicken liver were provisioned for adult flies in a plastic container (15 cm in diameter, 9 cm in depth) covered with nylon netting. A second, fresh piece of liver was provided as a larviposition site 12 days after the first provision. One day later, the livers with larvae were collected and transferred to an aluminium dish. The dish was placed on dry wood chips (30 mm to 50 mm in depth) as a pupariation/pupation substrate in a plastic container. Mature larvae ceased feeding and moved into the wood chips. A few days later, they pupariated and pupated. New adults emerged approximately 18 days after larviposition.

For experiments, newly emerged adults were kept under short-day conditions (12-h light/12-h dark) at 20 °C since the first provision of the liver. Twelve days after the provision of the liver, five to 10 flies were transferred to each experimental condition (see below). Three days later, that is 15 days after the first provision of the liver, pharate larvae in the uterus were collected by dissecting the female abdomen, to set the larviposition timing. The photoperiodic-sensitive stages of this species range from the embryonic stage in the uterus of the mother to the end of the larval stage and no maternal effect via the uterus induces diapause in offspring [35]. Thus, this larval collection does not affect diapause induction. Larvae collected were transferred to a piece of liver and maintained under the same conditions. Diapause status and sex were assessed 20 and 30 days after larval collection at 20 °C and 15 °C, respectively, in the laboratory diapause induction experiments, as described in the previous studies [49,50]. Diapause status and sex were assessed 20-30 days after larval collection in semi-natural conditions. The fly pupa is encased in a hardened larval cuticle (puparium). We opened the puparium and judged pupal mortality in the semi-natural experiments.

Laboratory diapause induction

Sarcophaga similis diapause incidence was measured under long-day, short-day, and light-at-night short-day (LAN; 12-h light/12-h dim light) conditions at 20 and 15 °C. In all conditions, flies were illuminated by fluorescent light (> 1,000 lux) during the light phase as described above. For the long-day and short-day conditions, no light was provided during the dark phase (0 lux). For the LAN condition, a fluorescent light (FL8WF, Panasonic, Osaka, Japan), covered with a black vinyl sheet to adjust the illuminance level to 0.01, 0.1, or 1 lux, was set in the incubator and left constantly on; thus, in the LAN conditions, the light phase was illuminated by both high and low illuminance, while the dark phase had a single fluorescent light with low illuminance. The spectrum of the fluorescent light measured by the colour rendering illuminometer CL-70F (Konica Minolta, Tokyo, Japan) is shown in Figure S1. The LAN conditions with 0.01, 0.1, and 1 lux dim light are abbreviated as LAN-0.01, -0.1, and -1, respectively. We chose these parameters as these illuminance levels are typical in urban areas [42] and the temperatures represented average temperatures of early October, when the flies begin to enter diapause, and early November, when most flies have entered diapause, in our field location in Osaka, Japan (Japan Meteorological Agency, www.jma.go.jp).

Semi-natural diapause induction

In the first experiment (Experiment 1), we compared diapause timing between rural and urban sites. In the second (Experiment 2), we compared diapause timing between two urban sites, one of which was less exposed to artificial light sources and the other was subject to severe light pollution. Illuminance and temperature at each site were recorded by data loggers (TR-74 Ui, T & D Corporation, Nagano, Japan) every two minutes during the experimental period.

In Experiment 1, we placed outdoor shelves (90.7 cm width, 45.7 cm depth, 90.0 cm height), covered by a clear vinyl sheet to shelter from rainfall, at rural and urban sites in 2015. We selected the Botanical Gardens of Osaka City University (BG; 34.77 °N, 135.68 °E) as the rural site, as no or few artificial light sources existed. We selected the campus of Osaka City University (OCU; 34.59 °N, 135.50 °E) as the urban site. This urban site faced the urban residential area but was covered by a tree branch; thus, it was partially protected from artificial light sources. Shelves at both sites faced north to prevent direct sunlight. Adult females that had been reared for 12 days under short-day conditions with the liver were placed on the outdoor shelves every week. Three days after the adult placement, larvae were collected and were continuously kept on the outdoor shelves. The data logger was placed on the shelf. In this experiment, we did not obtain data on mortality and diapause incidence on 2 November and 9 November 2015, as we were unable to check survival and diapause status within 30 days of the larval collection. No assessment of sex was conducted in this experiment.

In Experiment 2, we established two sites at OCU, approximately 130 m apart. At site 1, the outdoor shelf was established in the same manner as in 2015 but was placed in a new location due to operational unavailability. This new location was more exposed to residential artificial light sources than was the OCU urban site in Experiment 1. At site 2, we placed insect-rearing containers in a clear plastic container attached to the branch of a cherry blossom tree. This container faced north in a residential area near a 24-h convenience store; thus, it was exposed to light even during the night. The data logger was placed in the container to monitor temperature and illuminance. Adult females were placed at these locations every week and collected larvae were continuously kept at the same locations, as did in Experiment 1. The spectrum of the residential light sources in site 2 after sunset is shown in Figure S1. The residential light contained the various wavelengths of light and the pattern is similar to the spectrum of the fluorescent light, not that of the LED and the sodium light. The residential light in the site would be mainly from the fluorescent light.

Data analysis

All data were analysed using R version 3.1.2 [51]. In the laboratory experiment, diapause incidences under laboratory conditions were compared using Tukey-type multiple comparisons. Sexual differences were compared using Fisher’s exact test. In the semi-natural experiments, we calculated the length of the day when illuminance was continuously higher than 0.01, 0.1, and 1 lux from the illuminance data. Astronomical day length was calculated from sunrise and sunset of each day at BG and OCU obtained from the National Astronomical Observatory of Japan website (eco.mtk.nao.ac.jp). Differences between the astronomical day length and the length of the day at which illuminance was continuously higher than each set value were calculated as means ± standard deviations. The day of larval collection from the mother was regarded as the day for the experimental population. Two-way analysis of variance (ANOVA) was used for the diapause and mortality data after arc-sine transformation. No data on environmental parameters were available for 5 October and 6 October 2016 due to a typhoon.