DOI: 10.5061/dryad.2z34tmpr1
title: Temperature during pupal development affects hoverfly developmental time, adult lifespan and wing length

description: Hoverflies (Diptera, Syrphidae) are cosmopolitan, generalist flower
 visitors and among the most important pollinators after bees and
 bumblebees. The dronefly Eristalis tenax can be found in temperate and
 continental climates across the globe, often synanthropically. Eristalis
 tenax pupae of different generations and different climate zones are thus
 exposed to vastly different temperatures. In many insects, the ambient
 temperature during the pupal stage affects development, adult size, and
 survival; however, the effect of developmental temperature on these traits
 in hoverflies is comparatively poorly understood. We here reared E. tenax
 pupae at different temperatures, from 10°C to 25°C, and quantified the
 effect on adult hoverflies. We found that pupal rearing at 17°C appeared
 to be optimal, with high eclosion rates, longer wings, and increased adult
 longevity. Rearing temperatures above or below this optimum led to
 decreased eclosion rates, wing size, and adult survival. Similar thermal
 dependence has been observed in other insects. We found that rearing
 temperature had no significant effect on locomotor activity, coloration or
 weight, despite evidence of strong sexual dimorphism for each of these
 traits. Our findings are important as hoverflies are key pollinators, and
 understanding the effects of developmental temperature could potentially
 be useful for horticulture.
description: <em><strong>Animals</strong></em> We collected egg batches from female <em>Eristalis tenax </em>hoverflies wild caught under permit in Wittunga Botanic Garden (Adelaide, South Australia) and reared the larvae in fresh rabbit dung (Nicholas et al. 2018) at room temperature (22.8 ± 1.0°C). Each batch contained around 200 eggs (Nicholas et al. 2018). Third instar larvae were moved to small containers for rearing in different temperature conditions until the adult hoverflies emerged from the pupae. All pupae were kept in small, transparent containers fitted with a mesh under a 16 hour light: 8 hour dark cycle, with light provided by Arlec LED lights (UC0168, 350 lumens, warm white, Arlec Electrical Services, Australia) controlled by a timer. We exposed the pupae to 5 different temperatures. The coldest was achieved using a fridge (Hisense, HR6AF243, Hisense Australia) set to 10°C (10.1 ± 0.47°C). The 12°C (12.1 ± 0.54°C) and 17°C (17.4 ± 0.37°C) pupae were kept in wine coolers (Kogan 8 Bottle Thermoelectric Wine Cooler, Kogan Australia Pty Ltd). The 23°C pupae (22.8 ± 1.0°C) developed at room temperature in insect rearing cages. The 25°C (25.4 ± 1.76°C) pupae were housed on a plant propagation heating mat controlled by a THD digital controller (Aldoheat Horticultural Products, Australia). In one case, 10 pupae reared at 25°C were moved to room temperature for the last 2 days of their pupation, but treated as part of the 25°C cohort. Upon eclosion, the adult flies were moved to insect rearing cages (BugDorms, Australian Entomological Supplies) with a 24.5 cm or 32.5 cm side for maintenance at room temperature under laboratory lights (Nicholas et al. 2018). When counting the eclosion rate, we treated the hoverflies that died before completely crawling out of the pupae as unsuccessful. Adult hoverflies were labelled with unique color marks (Semco acrylic paint) on the thoracic dorsum to enable tracking of individuals. We recorded the lifespan of adult hoverflies from the day of eclosion to their death. Hoverflies that died from unnatural causes (e. g. by freezing for experimental reasons) appear as censored datapoints, and 10 individuals were omitted from the survival analysis due to lacking data about their sex. <em><strong>Morphometrics</strong></em> For the hoverflies reared at 12°C, 17°C and 25°C, we assigned a group of individual hoverflies (30-40 per temperature, balanced sample from each sex and batch) that were kept in a separate cage and regularly weighed at an interval of 5-15 days. The first measurement took place within 1-2 weeks of emergence. Each hoverfly was weighed by placing it in a small tissue culture dish (35 x 10 mm, Sarstedt AG & Co. KG, Germany), using a Sauter AR 1014 electronic balance (Sauter GmbH, Germany). These measurements were done un-blinded. After the hoverflies died, we took high-resolution photographs of them using an Olympus SZX10 camera equipped with DF PLAPO 1X-4 JAPAN lens (130 hoverflies, done blinded), or Olympus E-M10 Mark II camera equipped with Olympus ED 12-50 mm f/3,5-6,3 EZ lens (57 hoverflies, unblinded). 226 hoverflies were photographed while still alive using the Olympus E-M10 Mark II camera, and to prevent their movement during photographing, we covered them with a Petri dish lid pressed against a soft cellulose square. Camera type and status during photographing (alive/dead) were used as predictors for statistical models to filter out potential systematic variability. Wings were cut from living or dead specimens using scissors and were stretched with a Petri dish for photographing. We used a ruler to calibrate the photographs, and then extracted wing length, measured between the point where the transversal h-vein joins the upper edge of the wing and the point where the R4+5 vein joins the tip of the wing (as in Ottenheim & Volmer 1999). We measured the length of the thorax, defined as the distance between the scutellum-metathorax border and the head-prothorax border, along the center of the metanotum. All measurements were conducted using ImageJ software (Schneider, Rasband & Eliceiri 2012). From the photographs, we defined the coloration of tergite 2 and tergite 3, which can be yellow, orange, brown, or black (Francuski et al. 2011). We used a color scale with 6 steps (pictograms, Fig. 5), with 3 lighter grades and 3 darker grades (as defined in Heal 1979). For this classification the size of the colored patches is more important than shade (Heal 1979). However, note that this is a simplified scale as other authors have used up to 22 different Eristalis tenax color morphs (Francuski et al. 2011). <em><strong>Activity</strong></em> To quantify locomotor activity we used the Locomotor Activity Monitoring system (LAM25, TriKinetics Inc, Waltham, MA, USA) with 25 mm diameter × 125 mm long Pyrex glass tubes (PGT25 × 125, TriKinetics Inc) positioned horizontally. Hoverflies were individually placed in each tube, where the ends were sealed with a cotton ball with water, some pollen, and honey (Thyselius & Nordström 2016). Additional water was added to the cotton balls twice daily (before 9:00 and after 16:00) by a Pasteur pipette to prevent drying out. Each time the hoverfly crossed the center of the tube, it would break an infrared beam, and this would be counted as an activity measurement. The hoverflies were kept in the Locomotor Activity Monitoring system (LAMS) for approximately 48 hours, starting in the morning, and the average activity between 10:00 and 16:00 on the second day of recording was used for quantification. We quantified the mean activity at 12°C, 17°C, or RT by placing the LAMS in wine cooler fridges or at room temperature, under a 16 hour light: 8 hour dark cycle. Sometimes a hoverfly would remain stationary in the middle of the tube for extended periods of time, continuously breaking the beam. To avoid giving erroneously high activity measurements, all measurements indicating more than 10 crossings per minute were replaced with a 1, and all non-null measurements immediately following it were replaced with a 0 (as in Thyselius & Nordström 2016). <strong><em>Statistical analysis</em></strong> Throughout the paper, N refers to the number of flies. In the text, all data given as mean ± stdev unless otherwise mentioned. Where the figures show boxplots, these indicate median and interquartile ranges, and the whiskers extend up to 1.5x of the interquartile range. Any data beyond this distance are considered as outliers, shown with individual points. All statistical analyses were performed using R 4.2.1 software. Where data were normally distributed, we conducted ANOVA analysis combined with Tukey’s HSD. In data with non-normal residuals, we used appropriate transformation. In non-normally distributed data, we used generalized linear model (GLM) of either quasi-poisson (over-dispersed count data) or quasi-binomial (proportional data) family. For ordinal response variable (coloration), we used cumulative link model (CLM). When dealing with repeated measurements (weight gain data), we checked the data using partial autocorrelation function (PACF) and found only weak temporal autocorrelation. Subsequently, we compared results and AIC of the weight gain model with and without autocorrelation structure, and as these two reported similar results, we implemented the model without it. For survival analysis, we used Cox proportional hazards model with time-splitting (tt) correction. 
description: ## Temperature during pupal development affects hoverfly developmental
 time, adult lifespan and wing length **Authors:** Klára Daňková Charles
 University, Prague, Czech Republic klara.dankova@atlas.cz Sarah Nicholas
 Flinders University, Adelaide, Australia sarah.nicholas@flinders.edu.au
 Karin Nordström\* Flinders University, Adelaide, Australia Uppsala
 University, Uppsala, Sweden karin.nordstrom@flinders.edu.au
 \*corresponding author ## Description: This ReadMe file describes the
 structure of the data folder that accompanies publication Daňková et al.
 2022 focusing on effect of the rearing temperature on activity, eclosion
 rate, pupal duration, morphometrics (body size, wing size, wing:thorax
 ratio, coloration), survival and weight gain of a cosmopolitan pollinator
 species, hoverfly Eristalis tenax (Diptera, Syrphidae). **Units and date
 format:** Temperature units: °C (Celsius) Unit system: metric Date format:
 DD/MM/YYYY ## Structure 1\_Raw\_data 1a) Raw\_data\_activity (1 folder
 with 10 text files) 1b) Raw\_data\_morphometry\_photos (1 folder with 3
 subfolders containing photos) 2\_Processed\_data 2a)
 "final\_data\_activity.csv" 2b)
 "final\_data\_pupal\_duration.csv" 2c)
 "final\_data\_eclosion\_rate.csv" 2d)
 "final\_data\_morphometry.csv" 2e)
 "final\_data\_survival.csv" 2f)
 "final\_data\_weight.csv" ### Detailed descriptions:
 **1\_Raw\_data** *1a) Name: Raw\_data\_activity* Description: We provide
 our data on hoverfly activity in two forms: raw (10 text files in
 "1\_Raw\_data" folder) and processed (spreadsheet called
 "final\_data\_activity.csv" in "2\_Processed\_data"
 folder). The "Raw\_data\_activity" folder contains raw data of
 hoverfly activity obtained right from Locomotor Activity Monitor LAM25
 (TriKinetics Inc, Waltham, MA USA). Files are in ".txt" format,
 named
 "activity\_TestingTemperature\_StartDate(DD.MM.)\_EndDate(DD.MM.)\_Year.txt". The "testing temperature" stands for an ideal temperature [°C] in the climabox during the experimental session (12 °C, 17 °C or 23 °C -- please note that "RT" stands for 23 °C and "18" stands for 17 °C). Each row of the ".txt" files contains following information: minute of measuring, date, time, 7x columns recording technical information (outputs of light sensors etc.), 24x columns recording activity of individual hoverflies in each of the 24 glass tubes [counts/minute], 8x empty columns (this corresponds to the bottom row of 8 glass tubes that was intentionally left empty due to higher error rate in measuring). LAM25 is a device in which the hoverflies were individually placed in horizontal glass tubes, sealed on both sides with a cotton ball with water, pollen and honey. Additional water was added to the cotton balls twice daily (before 9:00 and after 16:00) by a Pasteur pipette to prevent drying out. Each time the hoverfly crossed the center of the tube, it would break an infrared beam, and this would be counted as an activity measurement. The hoverflies were kept in the Locomotor Activity Monitoring system (LAMS) for approximately 48 hours, starting in the morning, and the average activity between 10:00 and 16:00 on the second day of recording was used for quantification. We quantified the mean activity at 12°C, 17°C or 23°C by placing the LAMS in wine cooler fridges or at room temperature, under a 16 hour light: 8 hour dark cycle. Sometimes a hoverfly would remain stationary in the middle of the tube for extended periods of time, continuously breaking the beam. To avoid this giving erroneously high activity measurements, all measurements indicating more than 10 crossings per minute were replaced with a 1 and all non-null measurements immediately following it were replaced with a 0 (as in Thyselius & Nordström 2016). For details on the processed data ("final\_data\_activity.csv"), see below. *1b) Name: Raw\_data\_morphometry\_photos* Description: Contains 3 subfolders of photos that were used for the analyses of thorax length, wing length, wing:thorax ratio and coloration. Subfolders: * 1\_Olympus\_SZX10\_photographed\_dead - These hoverflies were photographed after they died by Olympus SZX10 camera equipped with DF PLAPO 1X -4 JAPAN lens. Individuals could be identified using "First.photo" and "Wing.photo" variables from the "data\_morphometry.csv" file. For further information, see more detailed information about "data\_morphometry.csv" file below. * 2\_Olympus\_E-M10\_Mark\_II\_photographed\_alive - These hoverflies were photographed alive by Olympus E-M10 Mark II camera equipped with Olympus ED 12 50 mm f/3,5-6,3 EZ lens. When sorted by name, photos of hoverflies are preceded by photos that state their batch ID and rearing temperature (please note that "RT" stands for 23 °C and "18" stands for 17 °C). Individuals could be distinguished either by their individual marking (blue and white pattern on the thorax), or based on the fact that there are exactly 3 photos of each hoverfly (3 photos taken in a row). * 3\_Olympus\_E-M10\_Mark\_II\_photographed\_dead - These hoverflies were photographed after they died by Olympus E-M10 Mark II camera equipped with Olympus ED 12 50 mm f/3,5-6,3 EZ lens. When sorted by name, photos of hoverflies are preceded by photos that state their batch ID, rearing temperature and date of death. **2\_Processed\_data** *2a) Name: "final\_data\_activity.csv"* Description: Activity of hoverflies measured in the Locomotor Activity Monitor LAM25 (TriKinetics Inc, Waltham, MA USA). Variables: * Tube = identification number of the glass tube (= position) in the Locomotor Activity Monitor * Row = identification number of the row of tubes in the Locomotor Activity Monitor * Rearing.temperature = temperature at which the individual experienced the pupation period; from pupation to eclosion * Sex = sex of the individual (male/female) * Batch.ID = identification number of the batch of eggs; each batch was laid by a different female; "weight" stands for hoverflies from a group that was used for regular weight measurements and that has been initially established as a balanced sample of individuals from batches 3, 4, 5 and 7 * Testing.temperature = ideal temperature in the climabox during the experimental session * Actual.testing.temperature = actual temperature in the climabox during the experimental session measured with an additional thermometer; counted as an average of temperature at the beginning (first day) and at the end (third day) of the experimental session * Humidity = relative humidity in the climabox during the experimental session * Date = the second day of the experimental session * Notes = state of the individual at the end of the experimental session (alright = the individual was alive and well; dead = the individual was dead; ill = the individual had serious locomotory problems and died soon afterwards; one\_cotton\_fell = the individual was alive, well and was still in the experimental tube, but one of the food-provided cotton balls had fallen down, however, it most likely happened shortly before the end of the experimental session and therefore should not affect the results; got stuck = the individual got stuck with spilled water on the floor of the tube; added later = these individuals were accidentally added to the Locomotor Activity Monitor in the afternoon of the first day, not in the morning, but this should not affect the results as only data between 10:00 and 16:00 of the second day were used for the analysis * Activity.counts = counts of passing through the center of the glass tube per minute. As hoverflies sometimes stayed in the middle of the tube for prolonged periods of time, resulting in unrealistically high counts, the measurements indicating more than 10 crossing per minute were replaced with a 1 and all non-null measurements immediately following it were replaced with a 0. * Included.in.the.analysis.1 = dead and ill hoverflies were excluded; due to repeated measurements of several individuals, the dataset was split into two subsets that do not contain more than one measurement per individual; individuals with "yes" value in this column were part of the first dataset * Included.in.the.analysis.2 = dead and ill hoverflies were excluded; due to repeated measurements of several individuals, the dataset was split into two subsets that do not contain more than one measurement per individual; individuals with "yes" value in this column were part of the second dataset * Individual.ID = identity code of individual hoverflies *2b) Name: "final\_data\_pupal\_duration.csv"* Description: length of the pupal period, measured as the time from pupation to eclosion Variables: * Batch.ID = identification number of the batch of eggs; each batch was laid by a different hoverfly female * Rearing.temperature = temperature at which the individual experienced the pupation period; from pupation to eclosion * Pupated = date of pupation * Eclosed = date of eclosion * Sex = sex of the individual (male/female) * Morning.or.afternoon = the part of the day at which the newly eclosed individual was found (0 = morning, 0.5 = afternoon). Note that the "afternoon" hoverflies might have also been eclosed in the late morning/noon. * Pupal.duration = number of days counted as difference between the day of eclosion and the day of pupation, plus 0.5 for hoverflies that eclosed in the afternoon * Notes = note that 10 pupae which were reared at 25 °C were moved to the 23 °C for the last 2 days of their pupation *2c) Name: "final\_data\_eclosion\_rate.csv"* Description: Eclosion success rate Variables: * Rearing.temperature = temperature at which the individual experienced the pupation period; from pupation to eclosion * Batch.ID = identification number of the batch of eggs; each batch was laid by a different female * Eclosed = number of individuals that eclosed successfully * Not.Eclosed = number of individuals that did not eclose successfully (did not eclose at all or died during crawling out of the pupa) * Total.pupae = sum of "Eclosed" and "Not.Eclosed" for each category *2d) Name: "final\_data\_morphometry.csv"* Description: Thorax length, wing length, wing:thorax ratio and coloration Variables: * Rearing.temperature = temperature at which the individual experienced the pupation period; from pupation to eclosion * Batch.ID = identification number of the batch of eggs; each batch was laid by a different female; "weight" stands for hoverflies from a group that was used for regular weight measurements and that has been initially established as a balanced sample from batches 3, 4, 5 and 7 * Sex = sex of the individual (male/female) * First.photo = name of the first photography portraying the individual hoverfly; usually followed by 1-3 additional photos that follow right afterwards (until another "first photo" occurs in the sequence of files); sometimes the wings were photographed separately, in such cases the name of the photo is in a column called "Wing.photo" * Wing.length = wing length (mm), measured between the point where transversal h-vein joins the upper edge of the wing and the point where R4+5 vein joins the tip of the wing (as in Ottenheim & Volmer 1999); empty cells indicate that the individual hoverfly lost/wore out its wings before we were able to measure them * Thorax.length = thorax length (mm), defined as the distance between the scutellum-metathorax border and the head-prothorax border, measured along the center of the metanotum * Wing.thorax.ratio = Wing.length/Thorax.length; empty cells indicate that the individual hoverfly lost/wore out its wings before we were able to measure them, thus it was impossible to calculate the Wing.thorax.ratio * Death = status at the moment of taking the photography of the body (died by natural causes/killed by freezing/alive) * Death.date = day of death; empty cells indicate that the date was not recorded for this particular individual in this dataset * Death.approx = approximated day of death; empty cells indicate that the date was not recorded for this particular individual in this dataset * Notes = note that the batch ID of one individual hoverfly was mislabelled in the photography * Wing.photo = name of photography of a wing of the individual hoverfly; empty cells indicate that the individual hoverfly lost/wore out its wings before we were able to photograph them, or that the photo's unique ID/name was lost (this happened to 6 data points) * Coloration = coloration on 6 step scale as defined in Heal 1979 (D = dark, MD = medium dark, M = medium, ML = medium light, L = light, UL = ultralight) * Alive.dead = state at the time of photographing (alive/dead) * Camera = camera used for photographing the specimen * Coloration.numeric = the previous coloration scale converted to numbers 1-6 (1 = the lightest, 6 = the darkest) * Body.photographed = date of taking photography of the body * Photo.age = age at the time of photographing (in days); empty cells indicate that date of death was missing for the particular individual in this dataset, and thus, we were unable to calculate the Photo.age * Phenotype = the previous coloration scale converted to a binary scale of either a light phenotype (UL/L/ML), or a dark phenotype (D/MD/M) *2e) Name: "final\_data\_survival.csv"* Description: Survival in time Variables: * Batch.ID = identification number of the batch of eggs; each batch was laid by a different female; "weight" stands for hoverflies from a group that was used for regular weight measurements and that has been initially established as a balanced sample from batches 3, 4, 5 and 7 * Rearing.temperature = temperature at which the individual experienced the pupation period; from pupation to eclosion * Eclosed = time interval in which the hoverfly has eclosed * Died = time interval in which the hoverfly has died * Sex = sex of the individual (male/female) * Eclosed.approximately = modus of the interval in which the hoverfly has eclosed * Died.approximately = modus of the interval in which the hoverfly has died * Status = cause of death of the individual (1 = died by unnatural causes, 2 = died naturally; individuals with status = 1 were included in the survival analysis as censored data) * Notes = more details on causes of death * Marking = individual colour marking; empty cells indicate that the individual hoverfly did not have a marking, or that the marking was lost/too worn out to be distinguished * Lifespan = difference between Eclosed.approximately and Died.approximately; 0.5 was added where the Eclosed.approximately was calculated based on an interval including an even number of days; individuals that died in the pupa or during the eclosion ("unsuccessfully eclosed") are not included in this dataset; an empty cell indicate one individual hoverfly that was seemingly severely damaged during the eclosion, and thus, it has fallen into our definition of the "unsuccessfully hatched" hoverflies, so we did not calculate its lifespan, despite this individual surviving for several days 2*f) Name: "final\_data\_weight.csv"* Description: Weight gaining in time Variables: * Rearing.temperature = temperature at which the individual experienced the pupation period; from pupation to eclosion * Eclosed = time interval in which the hoverfly has eclosed * Pupated = time interval in which the hoverfly has pupated * Sex = sex of the individual (male/female) * Weight = weight (g) * Measurement.number = number of weighting session of the certain temperature group (1 = first weighting session etc.) * Measurement.date = date of weighting * Measurement.time = time of the day of weighting * Notes = notes are mostly refering to individual color markings or state of the wings; in most of the individuals, the wings got very damaged as the hoverflies were aging, so we stopped recording the state of the wings at some point, as most of the hoverflies lost their wings (likely due to being kept in the relatively small insect cages) * Age.average = average age (in days) at the day of weighting, calculated as the difference between Measurement.date and Eclosed **References** * Ottenheim M., Volmer A. (1999) Wing length plasticity in Eristalis arbustorum (Diptera: Syrphidae). Netherlands Journal of Zoology 49:15-27. doi:10.1163/156854299X00029 * Thyselius, M., & Nordström, K. (2016). Hoverfly locomotor activity is resilient to external influence and intrinsic factors. Journal of Comparative Physiology A, 202(1), 45-54. **Acknowledgement** We thank the Botanic Gardens of Adelaide for their ongoing support, and Lukáš Janošík, Martin Weiser and Zdeněk Janovský for statistical analyses consultations. This research was funded by the US Air Force Office of Scientific Research (AFOSR, FA9550-19-1-0294 and FA9550-15-1-0188), the Australian Research Council (ARC, FT180100289 and DP210100740), Grant Agency of Charles University (464220/2020), the Faculty of Science Foundation of Charles University, and the Mobility Fund of Charles University.