DOI: 10.5061/dryad.ns1rn8pzp
title: Transmission of beneficial yeasts accompanies offspring production in Drosophila – an initial evolutionary stage of insect maternal care through manipulation of microbial load?

description: Parent-to-offspring transmission of beneficial microorganisms is
 intimately interwoven with the evolution of social behaviors. Ancestral
 stages of complex sociality-microbe vectoring interrelationships may be
 characterized by high costs of intensive parental care and hence only a
 weak link between the transmission of microbial symbionts and offspring
 production. We investigate the relationship between yeast symbiont
 transmission and egg-laying, as well as some general factors thought to
 drive the 'farming' of microscopic fungi by the fruit fly
 Drosophila melanogaster, an insect with no obvious parental care but which
 is highly dependent on dietary microbes during offspring development. The
 process of transmitting microbes involves flies ingesting microbes from
 their previous environment, storing and vectoring them, and finally
 depositing them in a new environment. This study revealed that fecal
 materials of adult flies play a significant role in this process, as they
 contain viable yeast cells that support larval development. During single
 patch visits, egg-laying female flies transmitted more yeast cells than
 non-egg-laying females, suggesting that dietary symbiont transmission is
 not random, but linked to offspring production. The crop, an extension of
 the foregut, was identified as an organ capable of storing viable yeast
 cells during travel between egg-laying sites. However, the amount of yeast
 in the crop reduced rapidly during periods of starvation. Although females
 starved for 24 hours deposited a smaller amount of yeast than those
 starved for 6 hours, the yeast inoculum produced still promoted the
 development of larval offspring. The results of these experiments suggest
 that female Drosophila fruit flies have the ability to store and regulate
 the transfer of microorganisms beneficial to their offspring via the
 shedding of fecal material. We argue that our observation may represent an
 initial evolutionary stage of maternal care through the manipulation of
 microbial load, from which more specialized feedbacks of sociality and
 microbe management may evolve.
description: Yeast Transmission by Individual <em>Drosophila
 melanogaster</em> Females: 750 µL of
 raspberry medium filled in plastic cups placed inside plastic
 vials. Observations made on mature female flies'
 residence time, body size, and yeast load. Patch
 residence time is calculated from entry and exit times.
 Yeast cells were collected from individual flies' bodies
 and patches and were quantified using a selective yeast medium and
 counting colony-forming units (CFU). 
 Transmitted Yeast Fungi as Dietary Symbionts:
 Axenic larvae prepared from freshly laid <em>D.
 melanogaster</em> eggs. Larvae were
 individually placed in vials with sterile raspberry medium inoculated with
 specific yeast solutions. Yeast isolates included
 <em>Hanseniaspora uvarum, Pichia terricola, Wickerhamomyces pijperi,
 Candida californica, Starmerella bacilaris, </em>and<em>
 Saccharomyces cerevisiae</em>. Survival of
 larvae to adulthood was observed for each treatment.
 The Crop as a Potential Storage Organ of Symbiotic
 Yeast Fungi: Upon emergence, the flies were
 sorted by sex and placed in separate sterilized plastic cages with a
 sterile raspberry medium inoculated with <em>H. uvarum.</em>
 Sterile water-agar was provided to facilitate the
 association of flies with the yeast and the maturation of eggs in
 ovaries. After three days, 28 females were
 individually placed in vials, and out of those, 14 females were introduced
 to males. The following day, females were transferred
 to fresh vials with sterile raspberry medium for oviposition for five
 hours and then removed. The number of eggs laid into
 the raspberry medium was counted to differentiate egg-laying activity
 between mated and virgin females. All females were
 dissected, and mature oocytes at stage 14 were counted from both
 ovaries. Crop and body size measurements were taken
 for each female. Each crop was crushed in 500 µl
 saline, and the entire suspension was spread-plated on a selective yeast
 medium. Plates were incubated at 25 °C for two days,
 and the number of viable yeast colonies was counted and quantified as
 CFU/mL. In the second
 experiment: Four-day-old females
 mono-associated with <em>H. uvarum</em> were used.
 Females were subjected to either 6 or 24 hours of
 fasting. After fasting, females (30 per treatment)
 were placed individually in vials with a sterilized raspberry
 medium. Each female was given two hours to oviposit,
 and then the eggs were counted. The surface of the
 patches was washed with a saline solution, and the washed patches were
 used for yeast plating. Yeast washing and plating
 were performed using the track dilution technique.
 Additionally, a separate experiment with the same setup was
 conducted, but this time the patches were not washed, and eggs were
 allowed to develop into the adult stage. Days to
 emergence and survival of the emerged larvae were recorded.
 Feces as a Source of Heritable Symbiont
 Fungi: Assessment of viable fungal
 symbionts in adult fecal material. Observations made
 on mated and virgin flies. Fecal droplets were
 collected and their area was calculated. Yeast cell
 population growth was quantified using a microplate reader.
description: 1. Yeast transmission by individual <em>Drosophila
 melanogaster</em> females 
 Investigated the relationship between yeast symbiont transmission
 and egg laying in <em>Drosophila melanogaster</em>.
 Found that egg-laying females transmitted
 more yeast cells, suggesting a connection between symbiont transmission
 and offspring production. Data
 Description: Excel file with raw
 data including sample details, patch residence time, egg count, yeast
 counts from the patch and fly, and wing length as a proxy for fly
 size. 2. Transmitted yeast
 fungi as dietary symbionts Tested
 whether yeast fungi transmitted by individual <em>Drosophila
 melanogaster</em> females serve as dietary symbionts.
 Examined the survival of larvae in
 different treatments (control and various yeast species).
 Data Description: 
 Data indicating the survival of larvae in different
 treatments, including control and specific yeast species.
 3. The crop as a potential storage organ
 of symbiotic yeast fungi Explored
 the potential of the crop in <em>Drosophila melanogaster</em>
 females to store viable yeast cells and transfer them to subsequent
 breeding patches. Manipulated
 mating and hunger status to examine the relationship between crop size,
 yeast storage, and fasting. 
 Assessed yeast retention in the crop after fasting and its impact
 on offspring development. Data
 Description: 3.1 Data: 
 Includes oocyte count, egg count, wing size, crop size, yeast
 counts from the crop, and mating status of individual flies.
 3.2 Data: 
 Examined yeast counts, egg count, and yeast viability under
 different fasting durations (6 hours and 24 hours). 
 3.3 Data: 
 Recorded egg count, pupae count on day 6 and day 7, total pupae
 count, adult count on day 9 and day 10, and overall adult count.
 Calculated proportions of pupae and adults
 relative to eggs. 4. Feces as a
 source of heritable symbiont fungi 
 Investigated the presence of viable fungal symbionts in adult
 fecal material, considering coprophagy as the primary vertical transfer
 pathway in <em>Drosophila</em>. 
 Assessed fecal droplet size, lag time, and mating
 status of flies to determine the initial cell number in fecal
 solutions. Data
 Description: Includes fecal
 droplet size, lag time, mating status, and calculated initial cell numbers
 in fecal solutions. 
 Supplementary material for Experiment 2. 
 Provides additional information on the identification
 of yeast isolates collected from the raspberry system using blast hit
 analysis against the NCBI nucleotide collection database.