Evaluating the role of reproductive constraints in ant social evolution

Abstract

The reproductive division of labour is a key feature of eusociality in ants, where queen and worker castes show dramatic differences in the development of their reproductive organs. To understand the developmental and genetic basis underlying this division of labour, we performed a molecular analysis of ovary function and germ cell development in queens and workers. We show that the processes of ovarian development in queens have been highly conserved relative to the fruitfly Drosophila melanogaster. We also identify specific steps during oogenesis and embryogenesis in which ovarian and germ cell development have been evolutionarily modified in the workers. These modifications, which we call 'reproductive constraints', are often assumed to represent neutral degenerations that are a consequence of social evolutionary forces. Based on our developmental and functional analysis of these constraints, however, we propose and discuss the alternative hypothesis that reproductive constraints represent adaptive proximate mechanisms or traits for maintaining social harmony in ants. We apply a multi-level selection framework to help understand the role of these constraints in ant social evolution. A complete understanding of how cooperation, conflict and developmental systems evolve in social groups requires a 'socio-evo-devo' approach that integrates social evolutionary and developmental biology.

Figure 1.

Germ cell development and oogenesis in ants. (a–e) Specification of the germ cells and formation of embryonic gonads. (a) The pole plasm, a specialized cytoplasm also known as germ plasm, is assembled in the posterior pole of the oocyte through the localization of multiple maternal determinants (here Vasa) to the posterior cortex. (b) The components of the germ plasm are inherited by the early embryo and incorporated in a cluster of cells, which thereby acquire a pole cell fate, at the cellularized blastoderm stage of embryogenesis. (c) Pole cells later on form the gonads (d), which split in two clusters (e) that later will give rise to either ovaries (females) or testes (males). (f) Typical reproductive organs of a queen of ants (here Acromyrmex). Queen reproductive organs contain two ovaries, each formed by several ovarioles connected to the uterus by oviducts. Note the presence of a prominent spermatheca, which is usually swollen in mated queens. (g,h) Focus on one ovariole captured by light microscopy (g) or stained with DAPI to reveal nuclei (h), showing follicles that grow in size as they approach the oviduct. As indicated, each follicle is formed by the oocyte and its cluster of nurse cells (both of germline origin) in addition to a layer of somatic follicle cells surrounding the oocyte. (i) At least two germline stem cells (GSC) can be found at the tip of the ovariole in close contact with the cells of the terminal filament (TF), in addition to the cystoblasts (Cb), which represent the immediate progeny of the GSC. Both GSC and Cb express high levels of the germline-specific protein Vasa (green colour). Note that the dividing Cb are connected with ring canals as revealed by actin staining in red colour (arrowhead); scale bar, 20 µm. (j,k) The cysts continue their incomplete divisions and the resulting daughter cells remain connected by microtubule (green colour) and cytoskeleton (red colour) material (arrowheads). (l) The cells forming the cysts are still indistinguishable from each other and continue expressing Vasa protein maintaining their germline identity; scale bar, 10 µm. (m) Two adjacent cysts where now one cell in each of the cysts appears larger than the rest and its nucleus shows signs that it has entered meiosis and therefore it has acquired oocyte fate. (n) The oocyte is now easily visible and has been encapsulated in a layer of follicle cells. (o) Initially, several cells of the cyst express the oocyte-determining marker Bicaudal-C (green colour), but the cell located posteriorly (arrowhead) express higher levels of the protein. (p) At this stage, only the oocyte expresses Bicaudal-C while the other cells of the cyst no longer express the protein. These cells have now adopted a nurse cell fate. Note the Bicaudal-C is localized in the anterior of the oocyte, indicating first signs of oocyte polarity. (q,r) The follicles are now growing in size, where the nurse cells actively synthesize maternal determinants that they provide to the oocyte. (s) A simplified schematic diagram of oogenesis (from i to r).

Figure 2.

Functions of female reproductive organs in ants. (a) Reproductive organs of a queen of A. rudis showing the high number of ovarioles in the ovaries. (b) Reproductive organs of a worker, from a queened colony, where each ovary contains only one short ovariole. (c) Reproductive organs of a worker from a colony without a queen, showing that the ovarioles are now longer, which indicates much higher activity. (d) Ovariole from an A. rudis worker showing the expression of tor mRNA in the early developing oocytes and in the nurse cells. tor mRNA is transported from the nurse cells to the oocyte in older follicles. (e) Ovariole of a worker from a queened colony, stained for nuclei (blue colour) and Vasa protein (green colour). Note in the mature oocyte the absence of Vasa localization at the posterior pole (e1), indicating that this oocyte is trophic. (f,g) Same ovariole as in (c), of a worker from a colony without a queen, stained for nuclei (blue colour) and Vasa protein (green colour). In addition to the higher number of follicles, many oocytes now localize Vasa to the posterior pole (f1 and g2) while other do not (g1). This indicates that the same ovariole is producing both trophic and reproductive oocytes in the absence of the queen. (h,i) Ovarioles of L. alienus workers from a colony without a queen, stained for nuclei (blue colour), actin (red colour) and Vasa (green colour). Note that these workers make oocytes with mis-localized Vasa (h2) and others with correct Vasa localization (h1, i1 and i2).

Figure 3.

The developmental basis of germ cell reduction or loss in the workers. (a–c) Worker-destined embryos of the ant M. pergandei stained for nanos mRNA to reveal the germ cells (GCs). (a) Embryo at early gastrulation, a stage where the germ cells are still developing properly. (b) A slightly older embryo showing that the germ cells are now degenerating. (c) A late embryo showing the remainder of germ cells, which have populated the gonads. (d–f) Embryos of the ant M. emersoni stained for Vasa protein to reveal the germ cells. (d) Early embryo showing the proper formation of the germ plasm at the posterior pole. (e) Embryo at gastrulation showing that the germ cells are undergoing degeneration. (f) A late embryo showing that the gonads are absent, and that Vasa now accumulates in the central nervous system (CNS).

Figure 4.

Summary of germ cell development and ovary function in ants. [a1–a3] Types of oocytes produced in ant ovaries. [a1] Viable with correct maternal determinant localization, [a2] trophic with no localization and [a3] failed where Vasa protein accumulates in the oocyte but its localization is not maintained at the posterior pole. Only oocytes with correct localization [a1] possess functional pole plasm and will undergo proper development. The pole plasm is inherited by the embryo [b1] and functions to specify the germ cells [b2]. Germ cells may develop fully [b3] and lead to the formation of complete gonads [b3 and c1] and therefore fully developed queen ovaries [d1]. Alternatively, germ cells may undergo degeneration during embryo development [b4] and lead to either reduced number of worker ovarioles [d2] or complete loss of worker ovarioles [d3]. Ovarioles of queens (or workers that are reproductively active) [d1a] are more active than normal worker ovarioles [d2a]. Queen ovarioles can produce both trophic and reproductive oocytes [a1–a2], whereas worker ovarioles are constrained at the molecular level and produce failed oocytes [a3] in addition to viable [a1] and trophic ones [a2]. Green, Vasa; blue, nuclei; red, actin.

Figure 5.

Crosstalk between reproductive constraints and multi-level selection solid lines with arrows indicate an increase, while truncated T-shaped lines indicate a decrease. The dashed line with arrow indicates that RC1 restricts ovaries to alternative functions. Reproductive constraints evolve as a consequence of social selective forces, but once they evolve, they positively feed back to decrease within-group competition and increase colony competitiveness.