DOI: 10.5061/dryad.4tmpg4fmb
title: Triacylglycerides and egg-to-adult viability in Drosophila melanogaster

description: Triacylglycerides (TAGs) are neutral lipids and are one of the major
 energy storage form in an insect aiding in normal physiology including
 reproduction of the organism. Importance of TAG metabolism in
 gametogenesis has been investigated in insects like mosquitoes, silkworm
 and Drosophila. However, a direct association between TAG levels and
 egg-to-adult viability has not been explored. In this study, we have
 assessed levels both stored and circulating TAG levels and egg-to-adult
 viability in Drosophila melanogaster lines with varying genetic
 backgrounds established through inbreeding and recombinant inbreeding
 strategies. We found noticeable variation in egg-to-adult viability and in
 both stored and circulating TAG levels in D. melanogaster lines.
 Apparently the D. melanogaster lines with higher levels of circulating TAG
 also had a higher egg-to-adult viability. To validate this, analysis of
 these variations through supervised correlation and unsupervised K-means
 clustering showed that levels of circulating TAG are positively associated
 with egg-to-adult viability irrespective of gender. The findings suggest
 that levels of circulating TAG promotes successful transition of egg to an
 adult. Taken together, the findings not only have a potential application
 in husbandry of ecologically and economically important insects but holds
 importance in translational research in fertility of vertebrates including
 humans.
description: <strong>Fly Stocks </strong> In
 the present study, four type of lines were used, namely: standard lines,
 native wild-type lines, strict inbred lines and Recombinant Inbred Lines
 (RILs), which represent different genetic backgrounds. The flies were
 reared on banana agar medium containing 50% banana, 1.667 % agar
 technical, 0.3% propionic acid, and 0.05% yeast at 25ºC, 70% relative
 humidity and 10-12hr day/night ratio.
 <strong><em>Standard
 Lines</em></strong> <em>Drosophila
 melanogaster</em> Oregon-R (O) and
 <em>w<sup>1118</sup></em> (W) were taken as a
 representative of standard lines.
 <strong><em>Wild-type
 Lines</em></strong> Two different wild-type
 lines namely wild-type old (L) and wild-type new (N) were used in the
 present study. The line L was developed from fruit fly isolates collected
 in Feburary’ 2019 (Amanullah et al., 2023). Line N was developed through
 isolates collected in July’ 2022. Both lines were exclusively maintained
 for over 200 generations in the lab. ANU-1 (A), a white eye isolated from
 L in September’ 2021, and since then it has been propagated and maintained
 under laboratory conditions.
 <strong><em>Inbred
 Lines</em></strong> The inbred crosses for
 the present study were an extension of a study previously conducted at our
 lab (Amanullah et al., 2023). From the set crosses using L population, two
 inbred crosses of Outbreed Sibling (OS) and Cousin Sibling (CS) with
 genomic homozygosity of 25% and 49.02% in the F1 generation, respectively,
 were taken. Nearly 500 lines were inbred for over 15 generations and 5
 lines were subsequently selected based on fecundity and equal
 male-to-female ratio for the present investigation.
 <strong><em>Recombinant Inbred
 Lines</em></strong> For setting up RILs,
 standard lines of O and W were crossed reciprocally with L, N and A. Six
 cross confirmations were set including four red-eyed lines (ON, NO, OL,
 LO) and two white-eyed lines (WA, AW) with 25 replicates of each in a
 single pair conformation. Out of 275 set crosses inbred for over 15
 generations, one representative from each cohort was selected for
 assessment in the present study. A total 16 different
 <em>Drosophila</em> lines were assessed. The description of
 developed lines with their representative symbols are outlined in
 <strong>Table 1</strong>.
 <strong>Egg-to-Adult Viability</strong>
 Assessment of egg-to-adult viability was carried out on standard
 cornmeal media containing 10.09% cornmeal, 6.07% dextrose, 3.04% sucrose,
 0.5% agar technical, 0.05% yeast, and 0.125% methyl 4-hydroxybenzoate.
 Five fly pairs (10 males and 10 females in each pair) of each strain were
 placed on 2.5% agar vials with yeast paste on top on day0. On day2 of pair
 placement, the flies from agar were removed and 1ml of 29% sucrose
 solution was added in the agar vials to help dissolve the yeast paste. The
 solution was then poured on a cell strainer (FALCON 70µm cell strainer).
 The eggs and larvae were visualized under a stereomicroscope and a maximum
 of 50 eggs were placed in each vial. For each line, ten replicates were
 set at 25ºC, 70% relative humidity and 10-12hr day/night ratio. Total
 fecundity from each replicate was recorded and egg-to-adult viability was
 deduced using the following:
 <strong>Triacylglycerides (TAG)
 Assessment</strong> Body lysate of 18-24 hours
 old virgin male and female were made. For each gender of each strain, 3
 replicates of body lysates, each containing 5 flies were prepared. The
 lysate was used to measure free and stored form of TAG as described by
 <em>Tennessen et al.</em> (Tennessen et al., 2014). The flies
 were briefly anesthetized by cold treatment and washed thrice in ice cold
 1X Phosphate Buffered Saline (PBS). The flies were then dried on a filter
 paper and immediately transferred to a micro centrifuge tube containing
 100 µl PBS supplemented with 0.05% Tween 20 and Protease inhibitor
 (PBST-PI). The flies were homogenized for at least 1 min using a tissue
 homogenizer (Nippon Genetics Europe, Tissue grinder NG010). The micro
 centrifuge tubes were then heated at 70ºC for 10mins and centrifuged at
 14000rpm for 10mins at 4ºC. The supernatant was transferred to a fresh
 micro centrifuge tube. Pellet and supernatant were used to estimate stored
 and circulating TAG levels, respectively, using a Triacylglycerides
 Liquicolor kit (HUMAN) (CAT#10724). TAG standards with concentration
 ranging from 6.25mg/dl to 200mg/dl were prepared by serial dilution in
 PBST-PI. To measure circulating TAG levels, 20µl of supernatant was first
 diluted by a factor of 1:5 in 80 µl PBST-PI 20µl of diluted sample was
 loaded in 96 well plate. Subsequently, 100µl of TAG estimation kit reagent
 was added to each well and was incubated at 37ºC for 10mins. To measure
 stored form of TAG, pellet was first resuspended in 120µl of PBST-PI.
 Subsequently, 20µl of the suspension was mixed with 100µl of TAG
 estimation kit reagent in a micro centrifuge tube and incubated at 37ºC
 for 5mins. The tube was then centrifuged at 14000rpm for 10mins at 4ºC and
 100µl of the clear supernatant was transferred to the microplate.
 Absorbance was measured at 500nm in microplate reader (VARIOSKAN LUX) in
 duplicates. <strong>Statistical
 Analyses</strong> Statistical analyses were
 conducted using GraphPad Prism v8.01. Normality of the data was assessed
 using Shapiro-Wilk test. Statistical significance was determined using
 one-way ANOVA (Dunnett’s Post-hoc test) and Kruskal-Wallis (Dunn’s
 Post-hoc test). Linear regression was also carried out to assess
 correlation between free TAG values and egg-to-adult viability at 95%
 confidence interval. For K-means clustering analyses, the data for free
 TAG values and egg-to-adult viability was imported to DATAtab statistics
 calculator (https://datatab.net/)
 and unsupervised clustering was performed under default parameters. The
 allocated cluster values were exported and compared. A
 <em>P</em>-value of less than 0.05 was considered
 statistically significant in all cases.
description: # Triacylglycerides and Egg-to-Adult Viability in Drosophila melanogaster
 [https://doi.org/10.5061/dryad.4tmpg4fmb](https://doi.org/10.5061/dryad.4tmpg4fmb) ## Description of the data and file structure The data is about egg to adult viability and triacylglycerides levels in 16 different lines of Drosophila melanogaster  ### Files and variables #### File: Data\_Physiol\_Entomol.xlsx **Description:**  ##### Variables * Egg to Adult Viability (Row 1-15) * Triacylglycerides Free Form (TAG-S) in mg/dL (Row 16-26: Male ; Row 27-37: Female) * Triacylglycerides Stored Form (TAG-P) in mg/dL (Row 38-48: Male ; Row 49-59: Female) ## Code/software MS Excel  ## Access information Other publicly accessible locations of the data: * None  Data was derived from the following sources: * None