Fish make C-starts to evade predator strikes. Double-bend (DB) C-starts consist of three stages: Stage 1, in which the fish rapidly bends into a C-shape; Stage 2, in which the fish bends in the opposite direction; and a variable Stage 3. In single-bend (SB) C-starts, the fish immediately straightens after Stage 1. Despite fish moving in three-dimensional (3D) space, fast-start responses of adult fish have mainly been studied in a horizontal plane. Using automated 3D tracking of multi-camera high-speed video sequences, we show that both SB and DB fast-starts by adult female least killifish () often contain a significant vertical velocity component, and large changes in pitch (DB up to 43 deg) and roll (DB up to 77 deg) angles. Upwards and downwards elevation changes are correlated with changes in pitch angle of the head; movement in the horizontal plane is correlated with changes in yaw angle of the head. With respect to the stimulus, escape heading correlates with the elevation of the fish at the onset of motion. Irrespective of the initial orientation, fish can escape in any horizontal direction. In many cases, the centre of mass barely accelerates during Stage 1. However, it does accelerate in the final direction of the escape in other instances, indicating that Stage 1 can serve a propulsive role in addition to its preparatory role for Stage 2. Our findings highlight the importance of large-scale 3D analyses of fast-start manoeuvres of adult fish in uncovering the versatility of fish escape repertoire.

Superfetation, the ability to simultaneously carry multiple litters of different developmental stages in utero , is a reproductive strategy that evolved repeatedly in viviparous animal lineages. The evolution of superfetation is hypothesized to reduce the reproductive burden and, consequently, improve the locomotor performance of the female during pregnancy. Here, we apply new computer-vision-based techniques to study changes in body shape and three-dimensional fast-start escape performance during pregnancy in three live-bearing fishes (family Poeciliidae) that exhibit different levels of superfetation. We found that superfetation correlates with a reduced abdominal distension and a more slender female body shape just before parturition. We further found that body slenderness positively correlates with maximal speeds, curvature amplitude and curvature rate, implying that superfetation improves the fast-start escape performance. Collectively, our study suggests that superfetation may have evolved in performance-demanding (e.g. high flow or high predation) environments to reduce the locomotor cost of pregnancy.

A live-bearing reproductive strategy can induce large morphological changes in the mother during pregnancy. The evolution of the placenta in swimming animals involves a shift in the timing of maternal provisioning from pre-fertilization (females supply their eggs with sufficient yolk reserves prior to fertilization) to post-fertilization (females provide all nutrients via a placenta during the pregnancy). It has been hypothesised that this shift, associated with the evolution of the placenta, should confer a morphological advantage to the females leading to a more slender body shape during the early stages of pregnancy. We tested this hypothesis by quantifying three-dimensional shape and volume changes during pregnancy and in full-grown virgin controls of two species within the live-bearing fish family Poeciliidae: Poeciliopsis gracilis (non-placental) and Poeciliopsis turneri (placental). We show that P. turneri is more slender than P. gracilis at the beginning of the interbrood interval and in virgins, and that these differences diminish towards the end of pregnancy. This study provides the first evidence for an adaptive morphological advantage of the placenta in live-bearing fish. A similar morphological benefit could drive the evolution of placentas in other live-bearing (swimming) animal lineages.

Swimming performance of pregnant live-bearing fish is presumably constrained by the additional drag associated with the reproductive burden. Yet, it is still unclear how and to what extent the reproductive investment affects body drag of the females. We examined the effect of different levels of reproductive investment on body drag. The biggest measured increase in body volume due to pregnancy was about 43%, linked to a wetted area increase of about 16% and 69% for the frontal area. We printed three-dimensional models of live-bearing fish in a straight body posture representing different reproductive allocation (RA) levels. We measured the drag and visualized the flow around these models in a flow tunnel at different speeds. Drag grew in a power fashion with speed and exponentially with the increase of RA, thus drag penalty for becoming thicker was relatively low for low speeds compared to high ones. We show that the drag increase with increasing RA was most probably due to bigger regions of flow separation behind the enlarged belly. We suggest that the rising drag penalty with an increasing RA, possibly together with pregnancy-related negative effects on muscle- and abdominal bending performance, will reduce the maximum swimming speed.

Historically, research on non-mammalian viviparity has suffered from anthropocentric views on reproduction, considering human (and mammalian) reproduction to be unique: live-bearing reproduction in non-mammalian species was considered a 'simple pattern' in which fertilized eggs developed and hatched inside the female [17,18]. Animals that lack 'true placentas', Figure 1-1. (next page) Phylogeny of the Poeciliidae indicating the level of matrotrophy and the presence of superfetation. Names depicted in bold indicate species used in this thesis. Boxes at the terminal ends indicate the presence (grey) or absence (white) of superfetation. Branch colour indicates a maximum likelihood reconstruction of maternal provisioning for natural log-transformed matrotrophy indices (MI), lnMI. Arrow indicates an MI of 1. The single egg-layer in the Poeciliidae family, Tomeurus gracilis, was excluded from this analysis. Adapted with permission from [67]. Figure 1-2. Overview of matrotrophy. A: Detail of an ovary of Phalloptychus januarius displaying matrotrophy. Arrowhead: maternal blood supply to a late-stage embryo. B: Frequency distribution of different levels of matrotrophy in Poeciliidae. Adapted from [28]. Inset: schematic overview of early-and late-stage embryos in lecithotrophic and matrotrophic species. C: The locomotor costs hypothesis predicts that the smaller oocytes at fertilization in matrotrophic fishes (B, inset) lead to a lower reproductive burden throughout pregnancy; the difference between the lecithotrophic and matrotrophic mode diminishes as pregnancy progresses.