Crystalline Deposits Reveal Caste Identity of Late Embryos and Larvae of the Ant
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bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456267; this version posted August 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Crystalline deposits reveal caste identity of late embryos and larvae of the ant 2 Cardiocondyla obscurior 3 4 Tobias Wallner1 5 Eva Schultner1 6 Jan Oettler1* 7 8 1Zoology/Evolutionary Biology, University of Regensburg, Universitätsstrasse 31, 9 93053 Regensburg, Germany 10 11 *corresponding author 12 13 ORCID: 14 Tobias Wallner: 0000-0001-9135-6456 15 Eva Schultner: 0000-0002-5069-9732 16 Jan Oettler: 0000-0002-8539-6029 17 18 Keywords: 19 Caste; social insects, ant larvae; urate; ovarian development; eco-evo-devo bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456267; this version posted August 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Abstract 21 Social insects are interesting models for the study of anticipatory developmental 22 plasticity because of the striking differentiation into reproductive queens and 23 functionally sterile workers. A few ant genera, including Cardiocondyla, represent the 24 pinnacle of social evolution in the Hymenoptera, where workers have completely lost 25 their reproductive organs, minimizing reproductive conflicts between queens and 26 workers. Here we show that late embryos and larvae of queens of the ant C. obscurior 27 can be identified by the appearance of urate deposits around the forming ovaries. 28 The discovery of caste-specific urate patterns in C. obscurior and three additional 29 Cardiocondyla species will facilitate future studies of developmental plasticity in ants. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456267; this version posted August 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 30 Introduction 31 Alternative developmental trajectories leading to the division of labour between 32 reproductive queens and non-reproductive workers form the basis of 33 superorganismality, thereby permitting one of the major transitions in evolution [1]. 34 Research on caste development in ants has a long tradition (e.g. [2-5], and conceptual 35 discussions are ongoing (e.g. [6,7]. However, how the processes of caste 36 determination and differentiation are regulated at a proximate level is not well 37 understood. 38 Compared to honeybees, where caste fate is under strict nutritional control, the 39 factors underlying caste fate in ants are more diverse, ranging from genetic to socio- 40 environmental [8]. With this comes variation in the timing of developmental 41 divergence (e.g. [4,9], so that ants exhibit different degrees of “reproductive 42 constraints” [9]. In some species of Ponerine ants, workers retain full reproductive 43 potential, including the ability to mate and store sperm. In the majority of ant species 44 workers have lost the spermatheca but retain more or less functional ovaries capable 45 of producing haploid, male-destined eggs. Finally, workers from 11 genera 46 completely lack ovaries. These obligately sterile workers are an example of an 47 extended phenotype without any direct fitness, representing a highly derived form of 48 superorganismality. The biology of some myrmicine species with fully sterile workers 49 has been studied extensively (Cardiocondyla obscurior, Monomorium pharaonis, 50 Pheidole spec., Solenopsis invicta), but comparably little is known about their 51 development. Even less is known about development in the remaining seven genera 52 with workers lacking reproductive organs (to the best of our knowledge; Myrmicinae: 53 Wasmannia, Tetramorium, Pheidologetum; Dorylinae: Eciton; Ponerinae: Leptogenys, 54 Hypoponera, Anochetus). 55 Across the range of reproductive constraints, a diverse set of signals spanning nature 56 and nurture is likely to be involved in caste-specific development. Together with the 57 facts that ant larval mobility is variable, ant brood is reared in piles, and brood is often 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456267; this version posted August 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 58 relocated and can serve functional roles in the colony [10], this has rendered the study 59 of ant development a more complex problem compared to Drosophila research. In 60 particular, it has been difficult to study caste-specific developmental trajectories 61 because it is not possible to distinguish worker-destined larvae from queen-destined 62 and male-destined larvae (e.g. [11]. Here, we close this gap for the ant Cardiocondyla 63 obscurior by showing that queen- and worker-destined embryos and larvae can be 64 visually distinguished by crystalline deposits surrounding the developing ovaries of 65 queen-destined larvae. This discovery will facilitate the study of caste determination 66 and differentiation at the extreme end of the superorganismality spectrum, thus 67 bringing us closer to a general understanding of the mechanisms facilitating the 68 evolution of social insects. 69 70 Methods 71 Ants 72 C. obscurior is a cosmotropical tramp ant [12], with a very streamlined genome 73 (~193MB, [13]), the smallest ant genome known to date. Adult queens and workers 74 differ in size and morphology and workers lack ovaries. Larvae develop via three larval 75 instars which can be distinguished by the shape of the body and the degree of 76 sclerotization of the mandibles [14]. The ants used in this study were all from the Old 77 World lineage [13], maintained in the lab since 2010. Stock colonies were kept in a 78 climate chamber under a 12h/12h and 22°C/26°C night/day cycle at 70% humidity. 79 Experimental colonies were kept in round plaster-bottom nests with nest indentations 80 covered by dark foil under the same conditions. All colonies were provided with water 81 and fed three times a week with honey and pieces of cockroaches and fruit flies. 82 83 White crystalline spots 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456267; this version posted August 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 84 All larvae of C. obscurior exhibit white spots. After producing semi-thin sections (see 85 below), we used a polarization filter that revealed a crystalline reflection. These 86 crystalline structures are common in insect larvae and have been described as urate 87 crystals (see discussion). Hence, because it is a parsimonious explanation we use 88 “urate deposits” in the following when we refer to the white spots, even though we 89 are aware that this may not be correct and requires future verification. 90 91 Urate deposit localization 92 We characterized urate deposits in eggs and first instar (L1), second instar (L2) and 93 third instar (L3) larvae as unpaired (= worker-destined) or paired (= queen-destined) 94 by visually inspecting brood from stock colonies under a stereomicroscope. For better 95 detection of the patterns, eggs and L1 larvae were submerged in a dissection dish 96 containing PBT (0.3 %), after which they were mounted on a microscope slide and 97 sealed with nail polish. From each development stage, we selected and 98 photographed one representative individual with a paired pattern and one individual 99 with an unpaired pattern using a stereomicroscope connected to a camera (Keyence 100 VHX 500FD, Neu-Isenburg, Germany). 101 We additionally characterized urate patterns of third instar larvae from seven 102 Cardiocondyla species available in the lab. We further examined brood of six species 103 from four subfamilies available as live colonies. Lastly, we accessed Alex Wild’s photo 104 library for a broader overview of species 105 (https://www.alexanderwild.com/Ants/Natural-History/Metamorphosis-Ant-Brood/). 106 107 Caste fate and survival according to urate deposits 108 We tracked development of all stages to confirm that urate localization patterns are 109 associated with caste in C. obscurior. Brood was sampled from several stock colonies 110 and sorted by development stage and urate pattern as described above. Eggs and L1 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.13.456267; this version posted August 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 111 larvae were transferred to filter paper to remove excessive buffer. Eggs were then 112 transferred in groups of ~15 to experimental colonies containing 10 workers (eggs: 113 paired=192, unpaired=165). L1 larvae were transferred to experimental colonies 114 containing 10 workers; two colonies were setup with 17 queen-destined larvae each, 115 and two colonies with 19 and 4 for worker-destined larvae, respectively (L1: 116 paired=34, unpaired=23). L2 and L3 larvae were transferred in groups of ten to 117 experimental colonies containing 10-12 workers (L2: paired=50, unpaired=40, L3: 118 paired=50, unpaired=40).