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.

This file ("Raw_data.xlsx") was generated in 2020 by Rohit Kapila

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.
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.

Experimental design

Testis dissection
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
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.
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 
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’.

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
 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

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.

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)
Tab 2. "Testis size 9 day old males" (Data for Experiment 2)
Tab 3. "Accessory gland size 4 day old males" (Data for Experiment 3)
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
Testis size 12: Size of other half of testis measured during round one of measurement
Wing length 1: Length of wing measured during the first round of measurement
Testis size 21: Size of one half of testis measured during second round of measurement
Testis size 22: Size of other half of testis measured during second round of measurement
Wing length 2: Length of wing measured during the first round of measurement
Population: Selection regimes of flies (Two selection regimes: "MCU" and "MB")
Larval density: Density treatment in which larvae were grown (Two larval densities "HD" and "LD")
Block: (4 independent replicates of each selection regime; "1", "2", "3" or "4")
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.

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: 
Testis size 11: Size of one half of testis measured during round one of measurement
Testis size 12: Size of other half of testis measured during round one of measurement
Wing length 1: Length of wing measured during the first round of measurement
Testis size 21: Size of one half of testis measured during second round of measurement
Testis size 22: Size of other half of testis measured during second round of measurement
Wing length 2: Length of wing measured during the first round of measurement
Population: Selection regimes of flies (Two selection regimes: "MCU" and "MB")
Larval density: Density treatment in which larvae were grown (Two larval densities "HD" and "LD")
Block: (4 independent replicates of each selection regime; "1", "2", "3" or "4").

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
Accessory gland size 12: Size of other half of accessory glands measured during round one of measurement
Wing length 1: Length of wing measured during the first round of measurement
Accessory gland size 21: Size of one half of accessory glands measured during second round of measurement
Accessory gland size 22: Size of other half of accessory glands measured during second round of measurement
Wing length 2: Length of wing measured during the first round of measurement
Population: Selection regimes of flies (Two selection regimes: "MCU" and "MB")
Larval density: Density treatment in which larvae were grown (Two larval densities "HD" and "LD")
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
Accessory gland size 12: Size of other half of accessory glands measured during round one of measurement
Wing length 1: Length of wing measured during the first round of measurement
Accessory gland size 21: Size of one half of accessory glands measured during second round of measurement
Accessory gland size 22: Size of other half of accessory glands measured during second round of measurement
Wing length 2: Length of wing measured during the first round of measurement
Population: Selection regimes of flies (Two selection regimes: "MCU" and "MB")
Larval density: Density treatment in which larvae were grown (Two larval densities "HD" and "LD")
Block: (4 independent replicates of each selection regime; "1", "2", "3" or "4")