Egg histology and the “invisible fraction”

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by Carlos Garriga (CLAS ’23 Biology)

Genetics has always fascinated me, especially how it is a crucial component of evolutionary studies of animal populations. To understand the full impact of genetics in populations, it is not enough to observe the genotypes and phenotypes of the living progeny; we must also consider offspring that may have died during prenatal development to understand the full scope of how inheritance affects survival. The population of unborn individuals not taken into consideration is known as the “invisible fraction.” In birds, it is easy to detect eggs that fail to yield live hatchlings. However, the naked eye often cannot determine whether a zygote was present in the egg. This means that without microscopic inspection, it is not possible to know if the egg did not hatch due to fertilization failure or because of prenatal mortality. A recent study by Hemmings and Evans (2020) presents data on prenatal mortality in populations of two species, great tits (Parus major) and blue tits (Cyanistes caeruleus), that underscores this principle. Here, I discuss the relevance of this study for work in the Curry Lab.

Hemmings and Evans studied great and blue tits in Wytham Woods in Oxfordshire, England, where scientists have studied the populations for over 50 years. The birds used nest boxes as their nest sites; Hemmings and Evans examined data from three years (2008 – 2010). The researchers collected unhatched eggs 14 days after the nests’ first hatching date to ensure that all eggs were given enough time to hatch. 

Hemmings and Evans first emptied contents of unhatched eggs into a phosphate-buffered solution to see whether they could detect a germinal disc or embryo. If either was present, the researchers conducted further examination under a stereomicroscope to identify the stage at which development ceased. If the yolk had disintegrated, Hemmings and Evans looked for signs of fertilization, also using a stereomicroscope. For eggs with no other evidence of fertilization, the researchers stained perivitelline layer of the yolk with a fluorescent DNA stain (Hoechst 33342). Hemmings and Evans then looked for presence of nuclei of embryonic tissue, sperm trapped in the perivitelline layer of the ovum, or penetration holes made by sperm that had entered the ovum. They determined fertilization primarily by the presence of nuclei of embryonic tissue, and unfertilized eggs by the absence of nuclei. 

Over the span of the 3 years studied, only 2.2% (9 of 416) of blue tit and 1.3% (5 of 375) of great tit eggs were unfertilized. This led Hemmings and Evans to conclude that the overwhelming majority of eggs contained a zygote that either survived to hatch or died during development. 

Importantly, only 50% of blue tits and 32% of great tits that died in the egg expired before the stage of development at which an embryo was discernable to the naked eye. This means that if Hemmings and Evans had not examined the egg contents microscopically, they would have incorrectly deemed roughly half of unhatched blue tit eggs and one third of unhatched great tit eggs as unfertilized. Thus, evolutionary studies done on birds without proper examination of unhatched eggs might contain skewed data regarding unfertilized eggs. Accordingly, the same studies might discard genetic data that could influence our understanding of evolutionary patterns. 

Hemmings and Evans (2020) provide key insight into methods for studying unhatched eggs and the importance of doing so. We know that reduced hatching success is a major cost of hybridization in mixed Carolina × black-capped chickadee populations (Driver et al. 2022), but no prior study of these species has examined fertilization rates in the level of detail proscribed by Hemmings and Evans. The 2020 paper has inspired me to conduct research on the “invisible fraction” in Carolina and black-capped chickadees for my senior thesis. This work may help us better understand prezygotic vs. postzygotic barriers to hybridization in chickadees. If most of the eggs produced by mixed pairs or hybrid parents are unfertilized, that would represent prezygotic barriers; if the eggs instead contain embryos that die during development, that would indicate postzygotic barriers. My primary goal, then, will be to clarify patterns of fertilization in our mixed Pennsylvania chickadee populations, at Hawk Mountain and at Tuscarora State Park.

Literature Cited

Driver, R., V. Ferretti, E. Burton, K. L. Cornell Duerr, M. McCoy, and R. L. Curry. 2022. Spatiotemporal variation in hatching success and nestling sex ratios track rapid movement of a songbird hybrid zone. American Naturalist

Hemmings, N., and S. Evans. 2020. Unhatched eggs represent the invisible fraction in two wild bird populations. Biology Letters 16:20190763.