DOI: 10.5061/dryad.fj6q573qx
title: Data from: Cold nights, city lights: artificial light at night reduces photoperiodically induced diapause in urban and rural populations of Aedes albopictus

description: 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.
description: We
 collected live <i>Ae. albopictus</i> 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 <i>Ae. albopictus</i> 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 24<sup>O</sup>C 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 Difco<sup>TM </sup>Nutrient
 Broth (Becton, Dickenson, and Company) in deionized (DI) water. After 48
 hours, larvae were sieved, identified as <i>Ae. albopictus</i>
 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 F<sub>1
 </sub>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
 9<sup>th</sup> at which time no more eggs were laid.
             For the
 experimental manipulation, we stimulated F<sub>1 </sub>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
 20<sup>th</sup> which corresponds with a photoperiod of 12:13
 hours of daylight at the latitude (38.3<sup>O</sup>N) of this
 study and is predicted to be short enough to produce diapause incidence in
 <i>Ae. albopictus</i> 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 6<sup>th</sup> the
 temperatures in the field were dipping below 10<sup>O</sup>C
 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 21<sup>O</sup>C 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
 0.3<sup>O</sup>C. 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,
 <i>Aedes triseriatus </i>and <i>Aedes
 japonicus</i>, which are not readily distinguishable as either eggs
 or pharate larvae, co-occur with <i>Ae. albopictus</i> in
 rural habitat. We are confident, however, that our urban collections were
 <i>Ae. albopictus</i> because all of the eggs collected at
 these sites in the previous weeks and identified for the colonies were
 <i>Ae. albopictus.</i> Moreover, we have two years of data
 from urban Saint Louis that contained no other <i>Aedes</i>
 species (Westby et al. unpublished).  We determined proportion diapause
 for two egg papers collected weekly from the three sites for the weeks of
 September 4<sup>th</sup> through October
 2<sup>nd</sup> 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.