Vascular architecture in the bacteriogenic light organ of Euprymna tasmanica (Cephalopoda: Sepiolidae)

Abstract

Symbiosis between southern dumpling squid, Euprymna tasmanica (Cephalopoda: Sepiolidae), and its luminescent symbiont, the bacterium Vibrio fischeri, provides an experimentally tractable system to examine interactions between the eukaryotic host and its bacterial partner. Luminescence emitted by the symbiotic bacteria provides light for the squid in a behavior termed "counter-illumination," which allows the squid to mask its shadow amidst downwelling moonlight. Although this association is beneficial, light generated from the bacteria requires large quantities of oxygen to maintain this energy-consuming reaction. Therefore, we examined the vascular network within the light organ of juveniles of E. tasmanica with and without V. fischeri. Vessel type, diameter, and location of vessels were measured. Although differences between symbiotic and aposymbiotic squid demonstrated that the presence of V. fischeri does not significantly influence the extent of vascular branching at early stages of symbiotic development, these finding do provide an atlas of blood vessel distribution in the organ. Thus, these results provide a framework to understand how beneficial bacteria influence the development of a eukaryotic closed vascular network and provide insight to the evolutionary developmental dynamics that form during mutualistic interactions.

Keywords: aerobic; squid; symbiosis; vasculature.

Fig. 1.

Bright field imaging and emission spectra from the light organ in 1 d juveniles of E. tasmanica. A. Ventral view of an untreated juvenile light organ removed from the squid mantle cavity. Posterior portion of the animal is towards the top of the figure. B. Ventral view of a Scale-cleared light organ removed from the squid mantle cavity. Anterior portion of the animal is towards the top of the figure. C. Emission spectra controls for squid muscle tissue exhibiting normalized mean intensity (y-axis) over wavelength in nm (x-axis). The laser line used for the spectrum is in the top right corner of each graph. App, appendage. Scale bar=500 μm.

Fig. 2.

Region of interest (ROI) λ scans of blood vessels in the gills (A) and in the light organ (B) of a 14 d juvenile of E. tasmanica. A. Squid gill, XY optical section imaged with a 405-nm UV laser diode. The white box indicates the ROI scanned for the emission spectrum. B. Squid light organ, XY optical section of the central tissue in the light organ imaged with the 405-nm laser. The white box represents the ROI in which the emission spectrum was measured. C. Emission spectra for scans in A (black line) and B (grey line). Scale bar=100 μm.

Fig. 3.

A diagram proposing the orientation and hierarchy of blood vessels throughout the light organ, as described in this paper. Posterior is to the top. GL, gill; L, the largest vessel that bifurcates to each lobe of the light organ; LO, light organ; M, mantle; MA, the anterior branch of the second tier of vessels; MP, the posterior branch of the second tier of vessels; S, the third tier of vessels that sprawls throughout the organ; SH, systemic heart.

Fig. 4.

Optical sections of each light organ treatment observed. Light organs are oriented from a ventral view with posterior at the top. All images are a merger of laser lines 405 nm (blue), 488 nm (green), and 561 nm (red). The column labeled “Top” represents shallow optical sections of the surface of the light organ and large vessel; the “Deep” column represents the deep crypts and associated vessels. A–D. 1 d aposymbiotic (A,B) and symbiotic (C,D) samples exhibiting light organ surface and deep crypts. E–H. 4 d aposymbiotic (A,B) and symbiotic (C,D) samples exhibiting light organ surface and deep crypts. I–L. 14 d aposymbiotic (A,B) and symbiotic (C,D) samples exhibiting light organ surface and deep crypts. ap, anterior appendage; cc, crypt; po, pore. Scale bars=100 μm.

Fig. 5.

Diameter of vessels (in μm) in left and right lobes of the light organ of aposymbiotic and symbiotic animals. Animals from each treatment were sampled after 1 d, 4 d, or 14 d. Values are means ± standard error. A,F. L vessel. B,G. MP vessel. C,H. MA vessel. D,I. S vessel. E,J. Number of branch points (nodes) counted for each of the samples. Asterisk indicates significance at p<0.05.