DOI: 10.5061/dryad.8pk0p2p0x
title: Impact of temperature and hypoxia on the size and survival of aquatic insects

description: It is crucial to understand impact of the globally rising temperatures on
 the functional traits of the insects. This is mostly due to the functional
 traits change, under temperature regime shift, can impact survivability of
 the individuals and population. Insects´ size is an important functional
 trait, affected by temperature. It was hypothesized, that many aquatic
 insects are getting smaller, with the temperature increase, probably due
 to the hypoxic-induced reduction of metabolic rate. This dataset contains
 data on the larvae and pupae size of non-biting midge (Chironomus
 riparius) and their change in the mesocosm setting, and their change under
 the impact of different combinations of temperature and hypoxia. We have
 compared the body size and survivability of C.
 riparius in six experimental treatments: three at 20°C, with
 high, medium and low oxygen concentration and three at 30°C, with the same
 three levels of oxygen concentrations. To achieve this comparison, we have
 collected and measured pupal exuviae and (at the very end of the
 experiment) larvae of the non-biting midge. This dataset contains digital
 measutments of the scaled images of larvae and pupal exuviae. All the
 exuviae photos are available at https://doi.org/10.5281/zenodo.14516968.
 Measurements were conducted on the digital images, using Fiji –ImageJ with
 calibrated scale (scales for the calibration are attached alongside the
 rest of the images at Zenodo) Our measurments
 https://zenodo.org/uploads/14516968 showed that C.
 riparius pupae were significantly smaller at 30°C treatments with
 medium and low oxygen levels than in the rest of the treatments. No
 significant reduction in pupal size was observed in the 30°C treatment
 with high oxygen content. We have found no significant differences in size
 of larvae between the treatments. However, we detected reduced larval
 survivability in the 30°C treatments with medium and low oxygen levels
 compared to the other treatments.
description: <strong>Experimental design</strong>
 Our experimental system utilized laboratory-reared populations of
 the non-biting midge <em>Chironomus riparius</em> (Meigen,
 1804). The mesocosm experiment was conducted from November 10, 2023, to
 March 23, 2024. Each experimental treatment included four replicate units,
 with each replicate consisting of a 2.7 L bucket placed inside an insect
 rearing cage. These cages were housed in a climate-controlled chamber
 maintained at 20 ± 2 °C, 60 % relative humidity, and an 8:16 hour
 light:dark cycle. Each bucket was filled with 100 g of clean sand and 2.7
 L of dechlorinated water. All replicates for a given treatment were placed
 within the same cage and climate chamber. Although larvae and pupae
 developed in isolated water-filled buckets with minimal inter-bucket
 interaction, replicates within the same cage must be regarded as
 pseudoreplicates. We established six treatment
 conditions: Treatments A, B, and C ("Lower" temperature group)
 were maintained at the chamber's background temperature, ranging from
 18.9 to 22 °C. Treatments D, E, and F ("Warmer" group) were
 exposed to elevated temperatures via submersible aquarium heaters set to
 30 °C, resulting in actual temperatures between 26.8 and
 28.5 °C. <strong>Specimens
 collection</strong> Pupal exuviae of emerging
 <em>C. riparius</em> adults were collected daily throughout
 the duration of the experiment. We focused exclusively on collecting
 exuviae to avoid disturbing the <em>C. riparius</em>
 populations within the experimental setups; no adult specimens were
 captured. At the conclusion of the experiment on March 23, 2024, all
 remaining live <em>C. riparius</em> larvae were collected from
 each treatment. No larval sampling was conducted during the experimental
 period in order to preserve the integrity of the developing populations.
 Pupal exuviae and larvae were preserved in 70 % ethanol and, within
 several weeks of collection, mounted on permanent slides using
 Hydromatrix™ mounting medium. Both life stages were mounted in a
 dorsal-ventral orientation.
 <strong>Imaging</strong> After the
 slides had dried, each specimen was photographed using a Leica Ivesta3
 stereomicroscope equipped with a Canon EOS 2000D camera. The resulting
 images were processed and analyzed in ImageJ software (version 1.54j)
 (Schindelin et al., 2012). Calibration and measurement of the images were
 performed in ImageJ using object micrometers photographed at the same
 magnification as the specimens. For larval specimens, head capsule length
 was measured from the posterior margin of the occipital sclerite to the
 ventral edge of the submentum (see Fig. 1A in associated paper). In pupal
 exuviae, total body length was measured from the anterior margin of the
 frontal apotome to the posterior edge of the anal lobes (see Fig. 1B in
 associated paper). <strong>Data
 Analysis</strong> All statistical analyses were
 performed using R version 4.2.3 (2023-03-15 ucrt) — "Shortstop
 Beagle". To assess within-group differences in body size across
 treatments, we applied one-way ANOVA followed by Tukey's post hoc
 test, as the size data met the assumptions of normality.
 To further explore pupal size variation, we developed two
 generalized linear mixed models (GLMMs) using a log-link function,
 implemented in the <code>glmmTMB</code> package. The first
 model included an interaction term between the two primary predictors:
 oxygen saturation and temperature:
 [1]<br><code>glmmTMB(Length of pupae ~ Temperature *
 Oxygen saturation + Date + (1 | Replicate bucket) + ar1(Date + 0 |
 Replicate bucket), family = Gamma(link =
 "log"))</code> In this model:
 <strong>Length of
 pupae</strong> denotes the measured total length of the pupal
 exuviae. 
 <strong>Temperature</strong> represents the average
 experimental temperature per treatment. 
 <strong>Oxygen saturation</strong> is the mean oxygen
 saturation for each treatment. 
 The <strong>Temperature * Oxygen saturation</strong>
 term captures the interaction between these two predictors.
 <strong>Date</strong> refers
 to the date of exuviae sampling. 
 <strong>Replicate bucket</strong> indicates the
 individual pseudoreplicate container within a treatment, specified as a
 random effect. An autoregressive
 (AR1) correlation structure was included for
 <strong>Date</strong> within each replicate bucket to account
 for temporal autocorrelation due to repeated measurements over
 time. A second, simplified
 model was also constructed, excluding the interaction term and including
 only the main effects:
 [2]<br><code>glmmTMB(Length of pupae ~ Temperature +
 Oxygen saturation + Date + (1 | Replicate bucket) + ar1(Date + 0 |
 Replicate bucket), family = Gamma(link =
 "log"))</code>
 <strong>References</strong>
 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V.,
 Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S.,
 Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K.,
 Tomancak, P. & Cardona, A. (2012). Fiji: an open-source platform
 for biological-image analysis. <em>Nature Methods</em>, 9(7),
 676-682. https://doi.org/10.1038/nmeth.2019
description: # Impact of temperature and hypoxia on the size and survival of aquatic
 insects --- We have submitted our raw data
 (**pupa.csv**,**HC.csv**,**D_treatment_21744379.csv**,**E_treatment_21744382.csv**,**F_tratment_21744381.csv**,**Climate_chamber_control_21744378.csv**, **c_ox.csv**), R project(**hypoxia_temp.Rproj**), R script (**chiro_code1.R**). ## Descriptions ### **pupa.csv** Raw measurements of the pupal exuviae of *Ch. riparius* derived from the photos at [https://doi.org/10.5281/zenodo.14516968](https://zenodo.org/uploads/14516968) by measuring specimens in Fiji * *index:* unique numeric index of each observation * *Treatment*: Experimental treatment group (e.g., temperature and oxygen level combination) assigned to the sample. * *Folder*: ID of the Folder containing treatment and the date of sampling * *Area*: The measured area (in pixels or calibrated units) of the specimen from the image. * *Mean*: The average grayscale pixel intensity within the measured area (used in image analysis). * *Min*: Minimum pixel intensity detected in the measured area. * *Max*: Maximum pixel intensity detected in the measured area. * *Length*: The calibrated length measurement (in mm ) of the specimen, derived from image analysis. * *Date*: R-date format converted date ### **HC.csv** Raw measurements of the larval head capsules of *Ch. riparius* derived from the photos at [https://doi.org/10.5281/zenodo.14516968](https://zenodo.org/uploads/14516968) by measuring specimens in Fiji * *Folder*: Identifier for the image batch or directory containing the photo files. * *Photo names*: File name or unique identifier of each image used for analysis. * *Area*: The total measured area of the object in the image, typically in calibrated units (e.g., mm²) or pixels. * *Mean*: Average pixel intensity (grayscale value) within the measured area, useful for texture or brightness analysis. * *Min*: Minimum pixel intensity detected in the measured region of interest. * *Max*: Maximum pixel intensity detected in the measured region of interest. * *Angle*: Orientation angle of the object in the image, measured during image analysis (unit typically in degrees). * *Length*: The calibrated length measurement (in mm ) of the specimen, derived from image analysis. ### **D\_treatment\_21744379.csv**, **E\_treatment\_21744382.csv**,**F\_tratment\_21744381.csv**,**Climate\_chamber\_control\_21744378.csv** Temperature readings from the Onset UA-001-64 HOBO 64K Pendant Temperature/Alarm (Waterproof) for the treatments D, E, F and climate chamber control, respectively * *#*: Row index or entry number in the data log; typically assigned automatically by the logging system. * *Date Time, GMT+02:00*: Timestamp of the recorded measurement, provided in local time (GMT+02:00). * *Temp, °C (LGR S/N: 21744379, SEN S/N: 21744379, LBL: D)*: Temperature reading in degrees Celsius, recorded by the specific logger and sensor units identified by their serial numbers. * *Coupler Attached (LGR S/N: 21744379)*: Boolean status (e.g., TRUE/FALSE or 1/0) indicating whether the data logger’s coupler was attached at the time of recording. * *Host Connected (LGR S/N: 21744379)*: Indicates whether the logger was connected to a host device (e.g., computer or base station) during the measurement. * *Stopped (LGR S/N: 21744379)*: A flag indicating whether data logging had been stopped at the time of this entry. * *End Of File (LGR S/N: 21744379)*: Marker showing whether this row represents the end of a logged data file; may be used for parsing or troubleshooting. ### **c\_ox.csv** Temperature readings from the Onset UA-001-64 HOBO 64K Pendant Temperature/Alarm (Waterproof) for the treatments D, E, F and climate chamber control, respectively \#24 hours oxygen dynamic after application of N2 to the low-oxygen treatment, oximeter readings * *A*: Placeholder for the index. * *Datetime*: Timestamp for each recorded measurement, typically in ISO format or local time. * *battery*: Battery level of the logging device at the time of recording, usually expressed as a percentage or voltage. * *id1*: Identifier for the logging device or sensor used to collect the data. * *user*: User-defined label or note, potentially identifying the person collecting the data or the treatment condition. * *Oxygen*: Measured oxygen concentration or saturation level, with units indicated in the adjacent *unit* column. * *unit*: Unit of measurement for the oxygen data (e.g., mg/L, % saturation). * *temperature*: Temperature reading at the time of measurement, corresponding to the unit in *unit_t*. * *unit_t*: Unit of temperature (°C). * *pressure*: Atmospheric or water pressure measurement, depending on context. * *unit_p*: Unit of pressure (e.g., hPa, kPa, mmHg). * *mode*: Operational mode or status of the logger (e.g., logging, standby, calibration), if recorded by the device. ## R Code R is necessary to run the code **chiro_code1.R**; it can be loaded using R project - **hypoxia_temp.Rproj**, we have produced the code using R version 4.2.3 (2023-03-15 ucrt) -- "Shortstop Beagle". Code contains list of all the necessary packages (for loading), some of them might require installation.