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Data from: Cold nights, city lights: artificial light at night reduces photoperiodically induced diapause in urban and rural populations of Aedes albopictus


Westby, Katie; Medley, Kim (2021), Data from: Cold nights, city lights: artificial light at night reduces photoperiodically induced diapause in urban and rural populations of Aedes albopictus, Dryad, Dataset,


As the planet becomes increasingly urbanized, it is imperative that we understand the ecological and evolutionary consequences of urbanization on species. One common attribute of urbanization that differs from rural areas is the prevalence of artificial light at night (ALAN). For many species, light is one of the most important and reliable environmental cues, largely governing the timing of daily and seasonal activity patterns. Recently, it has been shown that ALAN can alter behavioral, phenological, and physiological traits in diverse taxa. For temperate insects, diapause is an essential trait for winter survival and commences in response to declining daylight hours in the fall. Diapause is under strong selection pressure in the mosquito, Aedes albopictus; local adaptation and rapid evolution has been observed along a latitudinal cline. It is unknown how ALAN affects this photosensitive trait or if local adaptation has occurred along an urbanization gradient. Using a common garden experiment, we experimentally demonstrated that simulated ALAN reduces diapause incidence in this species by as much as 40%. There was no difference, however, between urban and rural demes. We also calculated diapause incidence from wild demes in urban areas to determine if wild populations exhibited lower than predicted incidence compared to estimates from total nocturnal darkness. In early fall, lower than predicted diapause incidence was recorded but all demes reached nearly 100% diapause before terminating egg laying. It is possible that nocturnal resting behavior in vegetation limits the amount of ALAN exposure this species experiences potentially limiting local adaptation.


We collected live Ae. albopictus at six sites (demes) during July and August, 2019; three locations were in back yards within the city of Saint Louis, MO and three locations were in oak-hickory forest habitat at Tyson Research Center (TRC), located approximately 38 kilometers outside of Saint Louis city (Fig. 1). TRC is a 2,000 acre, primarily forested, environmental field station bordered by state and county parks (38°31′N 90°33′W). To facilitate independence, collection sites were at least one kilometer apart which is the estimated maximum distance that adult Ae. albopictus will disperse naturally. At each site, four seven-litre black buckets were secured with ground stakes and provided initial inoculums of rain water and leaf detritus collected from the forest floor at TRC to attract oviposition; mosquitoes were allowed to colonize buckets naturally. We collected eggs weekly on seed germination paper that had been secured above the water line in each bucket. Egg papers were dried and stored in an environmental chamber set at 24OC and a 16:8 light:dark cycle. To establish colonies, eggs were stimulated to hatch in batches by submersing them in a solution of 0.35g/L of DifcoTM Nutrient Broth (Becton, Dickenson, and Company) in deionized (DI) water. After 48 hours, larvae were sieved, identified as Ae. albopictus and reared in group pans containing 10mL DI water and 0.0015g bovine liver powder per larva. Each of the six lab colonies were composed of mosquitoes collected from the six field sites during at minimum three collection events, providing at least 460 larvae per colony to produced F1 adults. F1 adults, in turn, produced the eggs used in the experimental manipulation to measure diapause incidence. We continued to collect eggs from the three city sites through the week of October 9th at which time no more eggs were laid.

            For the experimental manipulation, we stimulated F1 eggs to hatch using the afore described protocol. Four hundred first instar larvae were counted out for each deme and reared in group pans with four litres of water and 0.6g of bovine liver powder. The pans were then covered with mosquito netting to prevent oviposition and were placed in the field under the forest canopy at TRC. We had two sites at TRC, 1.5 kilometers apart, one we experimental illuminated and one we did not. Our experimentally illuminated site consisted of two high pressure sodium lights, one 75 Watt (Lithonia Lighting, OFL 70s 120 LP BZ M4) and the other 35 Watt (Eaton Lighting, W-35-H/PC) strapped to trees approximately 2.5 meters from the ground and positioned in a way that our larvae and adults received between 9 and 11 lux depending on their position on the table to which they were strapped. This amount of illuminance was chosen as it is commonly recorded in urban areas and was considered an intermediate level. No detectable light at night was measured at the non-ALAN treatment site (Dr. Meter LX1330B Digital Illuminance Meter). Caged larvae and adults were also strapped to a table at the non-illuminated site; the position of all cages was rotated every two days. Larvae were placed in the field on September 20th which corresponds with a photoperiod of 12:13 hours of daylight at the latitude (38.3ON) of this study and is predicted to be short enough to produce diapause incidence in Ae. albopictus nearing 100% (Urbanski et al., 2012 see supplemental material). Temperatures were logged (Onset HOBO MX2301) at both sites to ensure that there were no significant differences between sites, as temperature is also known to influence diapause rates in this species. By the week of October 6th the temperatures in the field were dipping below 10OC at night and adult mosquitoes were not taking blood meals, nor was there any observed mating, despite very frequent mating occurring in colony cages for this species. Thus, we moved the cages into the laboratory in environmental chambers set at 21OC with a 12:12 light:dark cycle. One chamber was set at complete darkness during the night cycle and the other chamber was manipulated but inserting two high pressure sodium lights, one at the top and the other at the bottom of the chamber. The lights were covered with aluminum foil to dim the lights to achieve illumination comparable to the field manipulation, and ranging between 8 and 12 lux. All cages were rotated inside chambers every two days. All of the eggs used to calculate diapause were laid after the mosquitoes had been moved into the chambers. Temperatures in the two environmental chambers varied by approximately 0.3OC. Adults were fed by placing definbrinated bovine blood (Hemostat Laboratories, Dixon, CA) into hog intestinal casing, warmed in a water bath, and placed on top of each cage. Eggs were collected in black cups lined with seed germination paper and filled with leaf infusion to stimulate oviposition. Egg papers were replaced every three to five days and stored in humidified bags in the chamber from which they were collected. After eggs were allowed to embryonate for two weeks, they were hatched with the aforementioned hatching protocol except that they were dried and hatched a second time. After all hatched larvae were counted (from both hatching events), the egg papers were bleached to determine embryonation [53]. Viable embryos that had not hatched were counted under a dissecting microscope and classified as being in diapause. We calculated the proportion of diapause eggs as the number of embryonated but unhatched eggs divided by the total number of embryos (hatched and unhatched). The effects of deme origin (urban or rural) on diapause incidence (proportion of eggs in diapause) was analysed using a GLMM using a quasi-binomial distribution with deme origin (urban or rural), light treatment (exposed to ALAN or not), and their interaction as predictors. Statistical analyses were performed in SAS 9.4.

            We also calculated the proportion of eggs in diapause laid by wild demes at the three urban sites from which we collected experimental mosquitoes. We were unable to do this at the three rural locations because two other species, Aedes triseriatus and Aedes japonicus, which are not readily distinguishable as either eggs or pharate larvae, co-occur with Ae. albopictus in rural habitat. We are confident, however, that our urban collections were Ae. albopictus because all of the eggs collected at these sites in the previous weeks and identified for the colonies were Ae. albopictus. Moreover, we have two years of data from urban Saint Louis that contained no other Aedes species (Westby et al. unpublished).  We determined proportion diapause for two egg papers collected weekly from the three sites for the weeks of September 4th through October 2nd after which no eggs were collected. Proportion diapause was determined using the same methods as for the experimental demes and we report the mean and standard error proportion for the two egg papers.


Tyson Research Center