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.

Fly Stocks

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.

Standard Lines

Drosophila melanogaster Oregon-R (O) and w¹¹¹⁸ (W) were taken as a representative of standard lines.

Wild-type Lines

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.

Inbred Lines

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.

Recombinant Inbred Lines

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 Drosophila lines were assessed. The description of developed lines with their representative symbols are outlined in Table 1.

Egg-to-Adult Viability

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:

Triacylglycerides (TAG) Assessment

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 Tennessen et al. (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.

Statistical Analyses

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 P-value of less than 0.05 was considered statistically significant in all cases.