DOI: 10.5061/dryad.9zw3r22fg
title: Evolution of sex-specific heat stress tolerance and larval Hsp70 expression in populations of Drosophila melanogaster adapted to larval crowding

description: The ability to tolerate temperature stress is an important component of
 adult fitness. In holometabolous insects like Drosophila
 melanogaster, adult stress resistance can be affected by growth conditions
 experienced during the larval stages. While evolution under crowded larval
 conditions is known to lead to the correlated evolution of many adult
 traits, its consequences on adult heat stress tolerance have not been
 investigated. Therefore, in the present study, we assessed the adult heat
 stress tolerance in populations of D. melanogaster adapted to a stressful
 larval crowding environment. We used replicate populations of D.
 melanogaster, selected for adaptation to larval crowding stress (MCUs),
 for more than 230 generations, and their respective controls (MBs). Larvae
 from selected and control populations were grown under crowded and
 uncrowded conditions and their adult heat shock resistance at two
 different temperatures was measured. Further, we compared Hsp70 expression
 in crowded and uncrowded larvae of both populations and also measured the
 Hsp70 expression after a mild-heat treatment in adults of selected and
 control populations. Our results showed that adaptation to larval crowding
 leads to the evolution of Hsp70 gene expression in larval stages and
 improves adult heat-stress tolerance ability in males, but not in
 females. 
description: This file ("Raw_data.xlsx") was generated in 2019 by
 Rohit Kapila. GENERAL INFORMATION 1.
 Title of Dataset: "Evolution of sex-specific heat stress tolerance
 and larval Hsp70 expression in populations of Drosophila melanogaster
 adapted to larval crowding" 2. Author Information
      A. Principal Investigator Contact Information
 Name: Prof. N. G. Prasad Institution: Indian Institute of Science
 Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge
 City, SAS Nagar, Punjab – 140306, India. Email: prasad@iisermohali.ac.in
      B. Associate or Co-investigator Contact
 Information Name: Rohit Kapila Institution: Indian Institute of Science
 Education and Research, Mohali Address: IISER Mohali, Sector 81, Knowledge
 City, SAS Nagar, Punjab - 140306, India. Email: rohit.kapila.24@gmail.com,
 mp14006@iisermohali.ac.in 3. Duration of data
 collection: 2018-2019 4. Geographic location of data
 collection: Mohali, Punjab, India 5. Information about
 funding sources that supported the collection of the data: IISER Mohali,
 Govt. of India. DATA & FILE OVERVIEW
 1. File List: "Raw_data.xlsx" Note: This file contains
 four separate data sheets (please see below for methodological details) in
 the following four tabs: Tab 1. " Survivorship at
 37 Degrees" (Data for assay done at 37 °C) Tab 2.
 " Survivorship at 37 Degrees" (Data for assay done at 38
 °C) Tab 3. "Larval Gene expression" (Data
 collected for estimating hsp70 expression in larvae)
 Tab 4. "Adult unshocked gene expression " (Data
 collected for estimating hsp70 expression in unshocked adults)
 Tab 5: “Adult shocked gene expression” (Data collected for
 estimating hsp70 expression in shocked adults)
 METHODOLOGICAL INFORMATION We used eight
 laboratory populations of D. melanogaster; four of which are selected for
 adaptation to larval crowding (MCU 1-4) and the other four are controls
 (MB 1-4). These populations- MB (Melanogaster Baseline controls) and MCU
 (Melanogaster Crowded as larvae, Uncrowded as adults) were maintained in
 our laboratory for 230 generations before beginning the current set of
 experiments at a 25°C and 90% RH in a 24-hour light regime.
 Each MCU population was derived from baseline control populations
 MB (i.e. MCU-1 was derived from MB-1 and so on). Each replicate of MCU was
 since then maintained as a separate population. Hence, the MCUs that are
 connected to MBs by the same replicate numbers are their direct
 descendants; therefore, they were treated as statistical blocks
 representing ancestry in analyses. MB populations were
 derived from four long-term laboratory populations of D. melanogaster
 called JB populations.  In the year 2006, all the four JB populations were
 mixed together to form a single large population called MB. After 10
 generations, the single MB population was split into 4 replicate
 populations called MB 1-4. The four populations of MB were then maintained
 as independent populations for 15 generations before MCU was derived from
 them. The detailed maintenance regime of control
 populations is as follows:   MB (Melanogaster Baseline)
 populations (a set of 4 independently maintained populations) are
 maintained on a 21-day discrete generation cycle on standard
 cornmeal-charcoal food. Eggs laid by ~12-day-old females are dispensed
 into glass vials (25 mm diameter x 90 mm height) containing 6–8 mL of
 cornmeal-charcoal food at a density of 60–80 eggs per vial. Forty such
 vials are set up for each of the four replicates. The vials are then
 incubated at 25 °C temperature, 90% RH, and constant light.  12 days
 post-egg collection, when almost all the adults have eclosed, flies are
 transferred into a Plexiglas cage (24x19x14 cm) containing a Petri plate
 of cornmeal-charcoal food and wet absorbent cotton for maintaining high RH
 levels. Thus, the adult number is approximately 2500 per population per
 generation. Fresh food plates are provided every alternate day. On day 18
 post-egg collection, the flies are provided with a fresh food plate
 supplemented with ad libitum live yeast paste. Two days later, the flies
 are provided with a fresh food plate and are allowed to oviposit for 18 h.
 These eggs are then used to start the next generation.
 The maintenance regime of MCUs is the same as MBs
 except: 1. Every generation, in MCUs, 800 eggs are
 collected in 1.5 ml of charcoal-cornmeal food as compared to 60 eggs in
 MBs in 6ml of food per vial. Because of crowding food runs out in MCUs
 vials in 3-4 days. Hence, there is larval competition because of crowding
 in MCUs. 2. Every generation 24 vials of 800 eggs are
 collected per population in MCUs (i.e 19,200 eggs per population) as
 compared to 40 vials of 60 eggs in MBs (2,400 eggs per population). In the
 larval stages, due to larval crowding conditions, there is high mortality
 in MCU populations and out of 19,200 eggs that are collected every
 generation, approximately 2400 individuals survive till the adult stages.
 Therefore, in the adult stages in both MCU and MB populations the adult
 population size is similar. 3. Because of crowding the
 eclosion pattern of adults changes and spreads over a span of 10 days,
 therefore to avoid adult crowding, from day 8 onwards (first fly eclosen
 day), daily eclosing adults of MCUs are transferred into a cage until day
 18. Whereas for MBs, since there is no crowding in vials, all the adults
 eclose by day 12 post egg collection, and they are transferred to cages on
 day 12 as described above. Standardization and
 generation of experimental flies: All the populations
 were standardized for one generation, where they were subjected to similar
 relaxed conditions, before using them for the experiments. This method is
 a standard practice to remove non-genetic parental effects. Egg collection
 for standardized flies was done at a density of 300 flies per bottle, and
 4 such bottles per population were collected. Adults emerging from these
 bottles were transferred to a Plexiglas cage (24x19x14 cm). 36 hours
 before experimental egg collection, a food plate with ad libitum yeast
 paste was provided followed by a 6-hour egg-laying window for experimental
 egg collection. For all the experiments discussed, each
 replicate of both selected and control populations had two
 treatments: 1. The high-density treatment had 600 eggs
 per vial containing 2 ml food. 2. The low-density
 treatment had 60 eggs per vial containing 6 ml food.  
 In our assays, we used 600 eggs per vial as the high-density
 treatment for both MCU and MB populations. During usual maintenance, MCU
 populations are held at a density of 800 eggs per vial. Using this density
 was necessary because the larvae of MB population do not survive to
 adulthood if grown at 800 eggs per vial density. Therefore, in the
 high-density assay conditions, the MCU’s were under slightly lower density
 compared to their normal maintenance. Larval crowding,
 as well as adaptation to crowding, affects development time in D.
 melanogaster population. Therefore, egg collection for different
 populations and treatments was done on different days, ensuring all the
 adults were of the same age on the day of the experiment.
 Adult heat shock treatment: From a pilot
 assay, we discovered that heat shock for an hour at 39 °C kills all the
 flies, whereas there was very little mortality below 36 °C. Therefore,
 flies were given a heat shock treatment for an hour at 37 °C and 38 °C.
 For high larval density treatments, newly eclosed flies were transferred
 daily to a Plexiglas cage (12 x 11 x 11 cm) containing a Petri plate of
 cornmeal-charcoal food and wet absorbent cotton for maintaining high
 humidity levels. For low larval density treatments, flies were transferred
 to cages on the 12th-day post egg collection. On the day of experiments
 (when adults from all the treatments were roughly 5 days old) flies were
 transferred into empty glass vials (25 mm diameter ? 90 mm height),
 covered with mildly moist cotton. 10 such vials, each with 5 flies of the
 same sex were set up for each treatment. Therefore, we assayed 50 males
 and 50 females per density treatment per selection regime for each block
 (i.e 400 flies per block). Assays for each replicate
 population were performed separately following the same protocol. The
 water bath was big enough to hold 100 vials at a given time. In each of
 our assays, we had a total of 80 vials ensuring each vial was subjected to
 the exact same temperature. Therefore, all the flies received the same
 heat-stress treatment. Soon after the flies were aspirated into empty
 vials, they were placed into a pre-heated, temperature-controlled, hot
 water bath for an hour. Vials from all the treatments were randomly
 shuffled and kept together in the water bath at the same time.  In
 separate assays, flies were subjected to heat stress at 37 °C and 38 °C
 for an hour. The temperature of the water bath was maintained and
 monitored throughout the experiment. After one hour of heat treatment,
 flies were transferred back into food vials, which were then kept at 25° C
 temperature-controlled incubator for 24 hours. Mortality was observed
 24-hour post-heat shock for each vial and the proportion of surviving
 individuals was calculated for each vial. Larval and
 adult Hsp70 Gene expression: For larval Hsp70 gene
 expression, egg collection was done from standardized flies. Third instar
 larvae, which were in their wandering stage were collected and stored at
 -80 °C till further use. Similarly, for adults, egg collection for the
 experiment was done from standardized flies. On the day of the experiment,
 flies from all four adult treatments were divided into two groups; shocked
 and un-shocked. Five flies of the shocked group were transferred into
 sex-separated empty glass vials. These vials were then kept in a water
 bath, which was maintained at 35 °C for an hour. This temperature does not
 normally induce any mortality within a one-hour duration in D.
 melanogaster, but gives them a shock and induces Hsp70 expression (25).
 Un-shocked flies were kept in empty glass vials at 25 °C temperature for
 one hour. After an hour of heat shock treatment, flies were again shifted
 to 25 °C for an hour. After an hour, flies from both shocked and
 un-shocked treatments were stored at -80 °C until further use.
 RNA extraction and cDNA preparation: Flies
 were homogenized in Trizol (Sigma-Aldrich) using a motorized pestle. The
 standard RNA extraction protocol was followed to extract RNA (see
 supplement 1). Extracted RNA was suspended in 30 µl of DEPC treated water.
 Following this, to check the RNA quality and quantity, Absorbance260/280
 (A260/280) was calculated using a NanoDrop spectrophotometer (Thermo
 Scientific) and samples with A260/280&gt; 1.0 were further used. From
 the isolated RNA, cDNA was synthesized using a Verso-cDNA kit
 (Thermo-Fisher, USA) according to the manufacturer’s protocol. The
 obtained cDNA was further used in semi-quantitative real-time PCR
 (semi-qRT-PCR) for measuring the gene expression.
 Semi-quantitative real-time PCR (semi-qRT-PCR):
 Expression of the Hsp70 gene was measured from the cDNA
 synthesized from the extracted RNA. Expression of housekeeping gene actin
 was used as an internal control. The PCRs were performed using Maxima SYBR
 Green qPCR master mix (Thermo-Fisher, USA) and the Eppendorf Realplex
 master cycler. Primer sequences were designed using the gene sequence
 available at the NCBI server (available online at
 https://www.ncbi.nlm.nih.gov/pubmed) and were synthesized by Integrated
 DNA Technologies, USA.  The cycle threshold (CT) was obtained and the
 expression of each gene was normalized using the expression of internal
 control. The following calculations were done: delta CT (Normalized gene
 expression) = CT (Gene of Interest) - CT (Housekeeping gene) For adult
 Hsp70 gene expression, delta CT values under shocked and non-shocked
 conditions were obtained for high and low larval density treatment in both
 MCU and MB. Further, for all the samples, delta CT values of the samples
 from the shocked treatment were normalized with delta CT values of their
 respective un-shocked samples. The value thus obtained was denoted as
 delta delta CT. delta delta CT=delta CT (shocked
 sample) - delta CT (un-shocked samples). The delta
 delta CT of MCU and MB were compared to each other from their respective
 densities. RNA extraction protocol:
 In order to extract the RNA: * Two-step ethanol washing of all
 the samples was done. * Washed samples were then treated with 150 µl TRI
 reagent to homogenize the flies.   * To the homogenized mixture, 30 µl of
 chloroform was added. The whole mixture was then shaken vigorously and was
 allowed to stand undisturbed for 2-5 minutes. * The mixture was then
 centrifuged at 12,000 g for 15 minutes at 4 °C. * The supernatant obtained
 after the centrifugation was then transferred to a sterile
 micro-centrifuge tube. * 75 µl of isopropanol was then added to the
 supernatant obtained in the last step and the solution was mixed slowly. *
 The solution was then allowed to stand undisturbed for 10 minutes and was
 subjected to centrifugation for 10 minutes at 12,000 g at 4 °C. *
 Isopropanol was discarded and 1 ml of 75 % DEPC treated ethanol was added.
 * The solution was further vortexed and centrifuged at 7,500 g for 5 min
 at 4 °C. * The supernatant was then discarded. Excess ethanol was allowed
 to air dry for 5-10 minutes. * Finally, 30 µl of DEPC treated water was
 added to the RNA pellet. To re-suspend the RNA for further use.
 Primer sequences: Hsp 70
 Forward TCGATGGTACTGACCAAGATGAAGG Reverse
 GAGTCGTTGAAGTAGGCTGGAACTG Actin
 Forward: ATCGAGCACGGCATCAC Reverse:
 CACGCGCAGCTCGTTGTA