DOI: 10.5061/dryad.1ns1rn8vt
title: Investment in adult reproductive tissues is affected by larval growth conditions but not by evolution under poor larval growth conditions in Drosophila melanogaster

description: In many insects, the larval environment is confined to the egg-laying
 site, which often leads to crowded larval conditions, exposing the
 developing larvae to poor resource availability and toxic metabolic
 wastes. Larval crowding imposes two opposing selection pressures. On one
 hand, due to poor nutritional resources during developmental stages,
 adults from the crowded larval environment have reduced investment in
 reproductive tissues. On the other hand, a crowded larval environment acts
 as a cue for future reproductive competition inducing increased investment
 in reproductive tissues. Both these selection pressures are likely
 affected by the level of crowding. The evolutionary consequence of
 adaptation to larval crowding environment on adult reproductive investment
 is bound to be a result of the interaction of these two opposing forces.
 In this study, we used experimentally evolved populations of Drosophila
 melanogaster adapted to larval crowding to investigate the effect of
 adaptation to larval crowding on investment in reproductive organs (testes
 and accessory glands) of males. Our results show that there is a strong
 effect of larval developmental environment on absolute sizes of testes and
 accessory glands. However, there was no effect of the developmental
 environment when testis size was scaled by body size. We also found that
 flies from crowded cultures had smaller accessory gland sizes relative to
 body size. Moreover, the sizes of the reproductive organs were not
 affected by the selection histories of the populations. This study
 highlights that adaptation to two extremely different developmental
 environments does not affect the patterns of reproductive investment. We
 discuss the possibility that differential investment in reproductive
 tissues could be influenced by the mating dynamics and/or investment in
 larval survival traits, rather than just the developmental environment of
 the populations.
description: This file ("Raw_data.xlsx") was generated in 2020 by
 Rohit Kapila <br> 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 165 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.<br> 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.<br> 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. Experimental
 design <br> Testis dissection<br> By the
 9th day post egg collection, in the high larval density treatment (HD),
 adults start to eclose. These adults were transferred into the Plexiglas
 cages (12 cm x 11 cm x 11 cm) with enough food and a non-crowded
 environment daily. Whereas flies from low larval density (LD) treatments
 were transferred into the cages on day 12 post egg collection. The
 protocol of transferring HD files in cages daily and transferring LD flies
 into cages 12-day post egg collection is a standard procedure used in
 larval crowding related studies to ensure that there is no crowding or
 resource limitation in adult stages. The maintenance of experimental flies
 was done mimicking the maintenance regime of our larval crowding selected
 (MCU) and larval crowding control (MB) populations. Until the day of
 assay, flies were maintained in the cages and were provided with a fresh
 food plate and moist cotton every alternate day. On the day of the
 experiment, in separate assays, 4-day old and 9-day old adult males were
 randomly sampled from the cages and were transferred into food vials.
 Shortly after that, a virgin female from an ancestrally related population
 (details of the population are described in the supplementary material)
 was introduced into the vial, and mating was observed for an hour. Since
 studies have shown that the mating status of males has an impact on the
 size of their testis and accessory glands in Drosophila, by observing
 single mating, we ensured that all the males were of the same ‘non-virgin’
 mating status in our assays. Throughout the experiment, just like the
 regular maintenance regime of the stock, all the flies were kept at 25 °C,
 60–80% RH, 24-hour light regime. Males that mated were frozen and kept at
 -20 °C until the dissections were done. Freezing the males does not affect
 the size of their reproductive tissues. From the frozen males, randomly
 chosen 20 males per population and density treatment were dissected for
 every block. Before dissection, all the frozen males were taken out from
 -20 °C to be thawed and brought to 25 °C. Dissections were done under a
 compound microscope (Leica MC120HD, Leica Microsystems GmbH, Wetzlar,
 Germany) and photographed at 40× using the attached digital camera
 connected with Leica Stereo Zoom Microscope (M 205C, Leica Microsystems
 GmbH, Wetzlar, Germany). From every dissected male, an image of the testes
 and the left-wing was taken. All the testes were dissected on a glass side
 in 1x PBS solution, and were uncoiled completely using fine forceps before
 imaging. Accessory gland dissection<br> Similar
 to testis dissection, in separate assays, 4-day old males and 9-day old
 males were randomly sampled from the cages and kept at -20 °C until the
 dissections were done. All the dissection was done in 1x PBS, on a glass
 slide under a compound microscope (Leica MC120HD, Leica Microsystems GmbH,
 Wetzlar, Germany) and was photographed at 40x zoom using the attached
 digital camera connected with Leica Stereo Zoom Microscope (M 205C, Leica
 Microsystems GmbH, Wetzlar, Germany). Thirty males per population and
 density treatment were dissected for every block.<br> Testes in
 Drosophila melanogaster are heavily coiled structures. To measure their
 area, it was necessary to uncoil them completely. The process of uncoiling
 the testes at times could lead to damage of accessory glands of the
 sample. Therefore, we dissected separate flies for testis and accessory
 gland dissections. The samples that were improperly dissected or were not
 imaged properly were not used for further analysis. A final count of the
 samples analyzed for each treatment per block is mentioned in the
 supplementary material. Measurement of the testis,
 accessory gland area, and wing size <br> NIH Image J version 1.50b
 was used to measure the area of testes and accessory glands, and length of
 the wing. An image of a standard stage micrometer (1 mm) glass slide was
 taken before imaging each organ mount at the same magnification (40×). The
 micrometer image served as a reference for the organ (for absolute
 length/pixels). Each image was analyzed manually by outlining the tissue
 using the ‘Polygon selections’ tool in image J. The area enclosed was then
 measured using the ‘Measure’ option provided under the ‘Analyze’ section
 of Image J. For wing size, we used the ‘Straight line’ tool in image J to
 join the anterior and posterior end of the longest vein of the wing. The
 selected length was measured using the 'Measure' option provided
 under the ‘Analyze’ section of Image J. The area of each organ was
 measured twice by an observer who was blind to the treatments. The average
 of the two measurements was taken and was used as the unit of analysis.
 For wing size, the length of second longitudinal vein was measured twice
 from the anterior crossvein to the end of the vein, and the average of
 both the readings was taken. For both accessory gland and testis, we
 calculated two types of area: 1) Absolute size, 2) Body normalized size.
 The values obtained from the average of two measurements of the area of
 organs was called the ‘Absolute size’ of that organ, whereas the area of
 an organ obtained by dividing the absolute size with the square of wing
 size for each sample was called the 'Scaled size’ of that
 organ’.
description: Note: In Tab 1, line 222, a value of 00 has been entered for wing
 size. This indicates that the wing measurement is missing. It does not
 mean that the value of wing size is 0. GENERAL
 INFORMATION 1. Title of Dataset: "Investment in
 adult reproductive tissues is affected by larval growth conditions but not
 by evolution under poor larval growth conditions in Drosophila
 melanogaster." 2. Author Information<br>  A.
 Principal Investigator Contact Information<br>  Name: Prof. N. G.
 Prasad<br>  Institution: Indian Institute of Science Education and
 Research, Mohali<br>  Address: IISER Mohali, Sector 81, Knowledge
 City, SAS Nagar, Punjab - 140306, India.<br>  Email:
 prasad@iisermohali.ac.in  B. Associate or
 Co-investigator Contact Information<br>  Name: Rohit
 Kapila<br>  Institution: Indian Institute of Science Education and
 Research, Mohali<br>  Address: IISER Mohali, Sector 81, Knowledge
 City, SAS Nagar, Punjab - 140306, India.<br>  Email:
 rohit.kapila.24@gmail.com <br> 3. Duration of
 data collection: 2017-2019 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. <br> 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. "Testis size 4 day
 old males" (Data for Experiment 1)<br> Tab 2. "Testis size
 9 day old males" (Data for Experiment 2)<br> Tab 3.
 "Accessory gland size 4 day old males" (Data for Experiment
 3)<br> Tab 4. "Accessory gland size 9 day old males" (Data
 for Experiment 4) DATA-SPECIFIC INFORMATION FOR
 "Raw data.xlsx" This file has four separate
 tabs. Below, we provide details separately for each tab.
 Tab 1. "Testis size 4 day old males" (Experiment
 1) 1. Number of variables: 9 2.
 Number of cases/rows: 261 3. Variable List: 
 Testis size 11: Size of one half of testis measured during round
 one of measurement<br> Testis size 12: Size of other half of testis
 measured during round one of measurement<br> Wing length 1: Length
 of wing measured during the first round of measurement<br> Testis
 size 21: Size of one half of testis measured during second round of
 measurement<br> Testis size 22: Size of other half of testis
 measured during second round of measurement<br> Wing length 2:
 Length of wing measured during the first round of measurement<br>
 Population: Selection regimes of flies (Two selection regimes:
 "MCU" and "MB")<br> Larval density: Density
 treatment in which larvae were grown (Two larval densities "HD"
 and "LD")<br> Block: (4 independent replicates of each
 selection regime; "1", "2", "3" or
 "4")<br> Note: line 222, a value of 00 has been entered
 for wing size. This indicates that the wing measurement is missing. It
 does not mean that the value of wing size is 0.
 <br> Tab 2. "Testis size 9 day old males" (Data
 for Experiment 2) 1. Number of variables: 9
 2. Number of cases/rows: 231 3. Variable
 List: <br> Testis size 11: Size of one half of testis measured
 during round one of measurement<br> Testis size 12: Size of other
 half of testis measured during round one of measurement<br> Wing
 length 1: Length of wing measured during the first round of
 measurement<br> Testis size 21: Size of one half of testis measured
 during second round of measurement<br> Testis size 22: Size of other
 half of testis measured during second round of measurement<br> Wing
 length 2: Length of wing measured during the first round of
 measurement<br> Population: Selection regimes of flies (Two
 selection regimes: "MCU" and "MB")<br> Larval
 density: Density treatment in which larvae were grown (Two larval
 densities "HD" and "LD")<br> Block: (4
 independent replicates of each selection regime; "1",
 "2", "3" or "4").
 <br> Tab 3. "Accessory gland size 4 day old
 males" (Data for Experiment 3) 1. Number of
 variables: 9 2. Number of cases/rows: 406
 3. Variable List:  Accessory gland size 11:
 Size of one half of accessory glands measured during round one of
 measurement<br> Accessory gland size 12: Size of other half of
 accessory glands measured during round one of measurement<br> Wing
 length 1: Length of wing measured during the first round of
 measurement<br> Accessory gland size 21: Size of one half of
 accessory glands measured during second round of measurement<br>
 Accessory gland size 22: Size of other half of accessory glands measured
 during second round of measurement<br> Wing length 2: Length of wing
 measured during the first round of measurement<br> Population:
 Selection regimes of flies (Two selection regimes: "MCU" and
 "MB")<br> Larval density: Density treatment in which
 larvae were grown (Two larval densities "HD" and
 "LD")<br> Block: (4 independent replicates of each
 selection regime; "1", "2", "3" or
 "4") Tab 4. "Accessory gland size 9 day
 old males" (Data of experiment 4) 1. Number of
 variables: 9 2. Number of cases/rows: 411
 3. Variable List:  Accessory gland size 11:
 Size of one half of accessory glands measured during round one of
 measurement<br> Accessory gland size 12: Size of other half of
 accessory glands measured during round one of measurement<br> Wing
 length 1: Length of wing measured during the first round of
 measurement<br> Accessory gland size 21: Size of one half of
 accessory glands measured during second round of measurement<br>
 Accessory gland size 22: Size of other half of accessory glands measured
 during second round of measurement<br> Wing length 2: Length of wing
 measured during the first round of measurement<br> Population:
 Selection regimes of flies (Two selection regimes: "MCU" and
 "MB")<br> Larval density: Density treatment in which
 larvae were grown (Two larval densities "HD" and
 "LD")<br> Block: (4 independent replicates of each
 selection regime; "1", "2", "3" or
 "4")