The fundamental constraint shaping animal systems for internal gas transport is the slow pace of diffusion []. In response, most macroscopic animals have evolved systems for driving internal flows using muscular pumps or cilia. In arthropods, aside from terrestrial lineages that exchange gases via tracheal systems, most taxa have a dorsal heart that drives O-carrying hemolymph through peripheral vessels and an open hemocoel [], with Ooften bound to respiratory proteins. Here we show that pycnogonids (sea spiders), a basal group of marine arthropods [], use a previously undescribed mechanism of internal Otransport: flows of gut fluids and hemolymph driven by peristaltic contractions of a space-filling system of gut diverticula. This observation fundamentally expands the known range of gas-transport systems in extant arthropods.

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2 and release CO 2 directly across the cuticle. Diverticula of the midgut extend into the chelifores and almost to the end of every walking leg. Using video microscopy of tracers in the hemolymph and gut, together with experimental manipulation of gut contractility, we evaluated the relative contributions of several hypothesized mechanisms [ 4 Bogomolova E.V.

Malakhov V.V. Structure of the body cavity of the sea spider Nymphon brevirostre Hodge, 1863 (Arthropoda: Pycnogonida). 5 Dohrn A. Die Pantopoden des Golfes von Neapel. 6 Helfer H.

Schlottke E. Pantapoda. 7 Tjonneland A.

Kryvi H.

Ostnes J.P.

Okland S. The heart ultrastructure in two species of pycnogonids, and its phylogenetic implications. 2 transport: diffusion; heart-driven circulation; leg movements; and gut peristalsis. In all 12 species examined (see Figure 1 The physiology of oxygen transport by sea spiders. Show full caption 2 solution (N = 7). The bottom left two panels show an individual in the restricted movement group after injection and 180 min later, with fluorescein present throughout most of the first tibiae. The right panels show an individual adhered and paralyzed, and the fluorescein after 180 min still is confined centrally. By contrast, fluorescein moved significantly more slowly into legs of paralyzed sea spiders (statistics shown in 2 ) in the hemolymph of a set of Antarctic species (N = 26 individuals in 8 species) measured at –1 to +1°C. Antarctic species spanned body sizes from 0.03 to 12.64 g. (E) Effect of PO 2 on rate of peristalsis in Antarctic Colossendeis megalonyx (N = 11). (F, G) Effect of temperature on rate of peristalsis in (F) three temperate species (Achelia gracilipes (circles, N = 5), Achelia chelata (triangles, N = 1), Phoxichilidium femoratum (squares, N = 3) and (G) on Antarctic Nymphon australe (N = 13). (A) Micro-computed tomography reconstruction of Dodecalopoda mawsoni showing a dorsal view. This species is unusual in having six pairs of legs rather than the more typical four. Surface coloration indicates inferred cuticle thickness, with red being thinner and green thicker. (B) Time course of fluorescein moving into the legs of Antarctic Nymphon australe. Fluorescein was injected into the trunk via a cut coxa of one leg, and the mean penetration into each of the remaining seven legs was followed. Individuals were assigned to one of three groups: in mesh cups with free movement (N = 6); legs adhered to stiff mesh (restricted movement) (N = 6); and both adhered and paralyzed using MgClsolution (N = 7). The bottom left two panels show an individual in the restricted movement group after injection and 180 min later, with fluorescein present throughout most of the first tibiae. The right panels show an individual adhered and paralyzed, and the fluorescein after 180 min still is confined centrally. By contrast, fluorescein moved significantly more slowly into legs of paralyzed sea spiders (statistics shown in Table S1 C). These results confirm that neither diffusion nor gross leg movements drive significant transport of hemolymph. (C) Time series of a passing wave of gut peristalsis in the second tibia of Colossendeis megalonyx; also see Movie S2 . Approximately five waves passed in one direction before they reversed and traveled in the opposite direction. There was no coordinated timing of waves among legs. (D) Distribution of oxygen partial pressures (PO) in the hemolymph of a set of Antarctic species (N = 26 individuals in 8 species) measured at –1 to +1°C. Antarctic species spanned body sizes from 0.03 to 12.64 g. (E) Effect of POon rate of peristalsis in Antarctic Colossendeis megalonyx (N = 11). (F, G) Effect of temperature on rate of peristalsis in (F) three temperate species (Achelia gracilipes (circles, N = 5), Achelia chelata (triangles, N = 1), Phoxichilidium femoratum (squares, N = 3) and (G) on Antarctic Nymphon australe (N = 13). Relative to other arthropods, pycnogonids have small trunks and long legs, extremely reduced abdomens, and large proboscides ( Figure 1 A). They lack specialized gas exchange structures and instead take up Oand release COdirectly across the cuticle. Diverticula of the midgut extend into the chelifores and almost to the end of every walking leg. Using video microscopy of tracers in the hemolymph and gut, together with experimental manipulation of gut contractility, we evaluated the relative contributions of several hypothesized mechanisms [] of internal Otransport: diffusion; heart-driven circulation; leg movements; and gut peristalsis. In all 12 species examined (see Supplemental Information ), hemolymph was transported by the beating heart, but heart-driven flows were generally confined to the trunk and proximal segments of the legs (coxae; Movie S1 ). In a separate experiment using leg restriction and chemical paralysis, we confirmed that diffusion and leg movement play minor roles in longitudinal transport within the legs ( Figure 1 B).

By contrast, peristalsis of the gut drove hemolymph and gut fluids throughout the body, except in parts lacking gut diverticula, i.e. palps, ovigers, and proboscis ( Figure 1 C, Table S1 and Movie S2 ). Gut peristalsis generated intermittent, periodic, countercurrent flows in the hemolymph and gut contents ( Movie S2 ). Countercurrent flow arises because each leg has a fixed volume defined by a semi-rigid cuticle. Because the volumes of the two main fluid compartments (hemolymph and gut) together must add up to approximately the total leg volume, gut liquids pushed distally by peristalsis displace distal hemolymph proximally toward the body (and vice versa when peristalsis pushes gut liquids proximally). The distance moved by gut particles in a single wave was as small as one quarter of a segment length in species with weak peristalsis (e.g. Nymphon australe) and more than one leg segment in species with vigorous peristalsis (e.g. Colossendeis megalonyx, and Nymphopsis spinosissima; Movie S2 ). Packets of hemolymph and gut fluid traversed similar distances but in opposite directions.

2 distributions in the body obtained using microelectrodes ( 2 for central regions (trunk, head, and proboscis). In temperate Achelia chelata, partial pressures of O 2 (PO 2 ) were highest in the first tibia (a distal leg segment; PO 2 < 3 kPa below ambient, air-saturated levels of 21 kPa) and declined in each segment moving proximally towards the trunk, which had the lowest PO 2 (7.5 kPa below ambient) ( 1,16 = 32.2, P < 0.0001). Antarctic species showed a similar pattern ( 1, 76 = 39.0, P < 0.0001), with an additional effect of body size; large-bodied individuals (>1 g body mass) had lower PO 2 (F 1, 17 = 21.1, P < 0.001, 2 status. Antarctic C. megalonyx exhibited <0.5 contractions min–1 in normoxia at seawater temperatures of −0.5–0°C but rates of contraction rose sixfold as PO 2 was depressed below 21 kPa ( 2 level F 1, 43 = 121.4, P < 0.001). Increasing temperature also increased the frequency of gut peristalsis up to around 20–25°C and 2–5°C in temperate and Antarctic species, respectively ( 2 transport to rates of consumption. distributions in the body obtained using microelectrodes ( Supplemental Information ) support the idea that legs can be a source of Ofor central regions (trunk, head, and proboscis). In temperate Achelia chelata, partial pressures of O(PO) were highest in the first tibia (a distal leg segment; PO< 3 kPa below ambient, air-saturated levels of 21 kPa) and declined in each segment moving proximally towards the trunk, which had the lowest PO(7.5 kPa below ambient) ( Figure S1 A, LME model: body section F= 32.2, P < 0.0001). Antarctic species showed a similar pattern ( Figure 1 D, LME model: body section F= 39.0, P < 0.0001), with an additional effect of body size; large-bodied individuals (>1 g body mass) had lower PO(F= 21.1, P < 0.001, Table S2 ). A respiratory role for gut peristalsis was also indicated by gut responses to changing Ostatus. Antarctic C. megalonyx exhibited <0.5 contractions minin normoxia at seawater temperatures of −0.5–0°C but rates of contraction rose sixfold as POwas depressed below 21 kPa ( Figure 1 E, LME model using linear part of increase: Olevel F= 121.4, P < 0.001). Increasing temperature also increased the frequency of gut peristalsis up to around 20–25°C and 2–5°C in temperate and Antarctic species, respectively ( Figure 1 F,G). Together, these data suggest that sea spiders alter rates of gut peristalsis in order to match rates of Otransport to rates of consumption.

2 transport. Countercurrent flows (as generated by gut peristalsis in pycnogonids) dramatically raise rates of transport of heat and gases between biological spaces separated by high-conductance barriers [ 8 Jackson D.C.

Schmidt-Nielsen K. Countercurrent heat exchange in the respiratory passages. 2 from distal parts of the legs to central areas, including the muscle-filled proboscis, where demand for O 2 is greatest ( We conclude that sea spiders use gut peristalsis for internal Otransport. Countercurrent flows (as generated by gut peristalsis in pycnogonids) dramatically raise rates of transport of heat and gases between biological spaces separated by high-conductance barriers [], such as gut walls. We propose that, in pycnogonids, this flow pattern transports Ofrom distal parts of the legs to central areas, including the muscle-filled proboscis, where demand for Ois greatest ( Figure S1 ). Legs function as the gills used by other arthropods, and the gut functions as a heart.

The gut plays a role in gas exchange in several other metazoan taxa, including aquatic larval Odonata and several groups of Echinodermata. In Cnidaria and Platyhelminthes, the digestive and circulatory functions are carried out jointly by a single system, the gastrovascular system. Shared digestive and respiratory functions may save energy. Gut peristalsis speeds digestion by agitating and moving liquids in tubes, and, if respiratory gases are easily co-transported and the gut is largely space-filling, O 2 may be adequately distributed without requiring other specialized systems for pumping and directing flows of blood.