Transmission of beneficial yeasts accompanies offspring production in Drosophila – an initial evolutionary stage of insect maternal care through manipulation of microbial load?

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.

Methods
Yeast Transmission by Individual Drosophila melanogaster 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 D. melanogaster eggs.
Larvae were individually placed in vials with sterile raspberry medium inoculated with specific yeast solutions.
Yeast isolates included Hanseniaspora uvarum, Pichia terricola, Wickerhamomyces pijperi, Candida californica, Starmerella bacilaris, and Saccharomyces cerevisiae.
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 H. uvarum.

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 H. uvarum 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.