Microscopic and Genetic Characterization of Bacterial Symbionts With Bioluminescent Potential in Pyrosoma atlanticum

The pelagic tunicate pyrosome,Pyrosoma atlanticum, is known for its brilliant bioluminescence, but the mechanism causing this bioluminescence has not been fully characterized. This study identifies the bacterial bioluminescent symbionts ofP. atlanticumcollected in the northern Gulf of Mexico using several methods such as light and electron microscopy, as well as molecular genetics. The bacteria are localized within the pyrosome light organs. Greater than 50% of the bacterial taxa present in the tunicate samples were the bioluminescent symbiotic bacteria Vibrionaceae as determined by utilizing current molecular genetics methodologies. A total of 396K MiSeq16S rRNA reads provided total pyrosome microbiome profiles to determine bacterial symbiont taxonomy. After comparing with the Silva rRNA database, aPhotobacteriumsp. r33-like bacterium (which we refer to as “PhotobacteriumPa-1”) matched at 99% sequence identity as the most abundant bacteria withinPyrosoma atlanticumsamples. Specifically designed 16S rRNA V4 probes for fluorescencein situhybridization (FISH) verified thePhotobacteriumPa-1 location as internally concentrated along the periphery of each dual pyrosome luminous organ. While searching for bacterialluxgenes in two tunicate samples, we also serendipitously generated a draft tunicate mitochondrial genome that can be used forPyrosoma atlanticumidentification. Scanning (SEM) and transmission (TEM) electron microscopy confirmed the presence of intracellular rod-like bacteria in the light organs. This intracellular localization of bacteria may represent bacteriocyte formation reminiscent of other invertebrates.

Sinopyrophorinae, a new subfamily of Elateridae (Coleoptera, Elateroidea) with the first record of a luminous click beetle in Asia and evidence for multiple origins of bioluminescence in Elateridae

The new subfamily Sinopyrophorinae within Elateridae is proposed to accommodate a bioluminescent species, Sinopyrophorusschimmeli Bi & Li, gen. et sp. nov., recently discovered in Yunnan, China. This lineage is morphologically distinguished from other click-beetle subfamilies by the strongly protruding frontoclypeal region, which is longitudinally carinate medially, the pretarsal claws without basal setae, the hind wing venation with a well-defined wedge cell, the abdomen with seven (male) or six (female) ventrites, the large luminous organ on the abdominal sternite II, and the male genitalia with median lobe much shorter than parameres, and parameres arcuate, with the inner margin near its apical third dentate. Molecular phylogeny based on the combined 14 mitochondrial and two nuclear genes supports the placement of this taxon far from other luminescent click-beetle groups, which provides additional evidence for the multiple origin of bioluminescence in Elateridae. Illustrations of habitus and main diagnostic features of S.schimmeli Bi & Li, gen. et sp. nov. are provided, as well as the brief description of its luminescent behavior.

A New Type of Luminescence in Fishes

A luminous organ of a hitherto undescribed type has been found in a Macrurid fish. This organ is described in the present paper. It consists essentially of an epithelium for the secretion of luminous substance, which has been thrown into long folds and wholly invaginated to form a gland. This gland is bound in connective tissue and has a compact appearance, and is furnished with supporting tissue internally. The duct is a flat and wide passage, continuous with the gland, which opens to the exterior about the anus in such a way as to surround the lower part of the rectum. The gland lies in the thickness of the body-wall forward of the rectum and between and behind the pelvic fins.The nature of the secretion is discussed and some experiments described. The luminescence appears to be due not to bacteria living as guests within the tissues of the fish, as has been shown for other species, but is essentially due to the well-known reaction wherein a substance luciferin is burnt to oxyluciferin in presence of the ferment luciferase, with emission of cold light.

STUDIES ON BIOLUMINESCENCE

1. Small dumps of the luminous cells of Mnemiopsis cannot readily be stimulated mechanically but will luminesce on treatment with saponin solution. Larger groups of luminous cells (such as are connected with two paddle plates) luminesce on mechanical stimulation. This suggests that mechanical stimulation to luminesce occurs chiefly through a nerve mechanism which has been broken up in the small dumps of luminous tissue. 2. The smallest bits of luminous tissue, even cells freed from the animal by agitation, that will pass through filter paper, lose their power to luminesce in daylight and regain it (at least partially) in the dark. 3. Luminescence of the whole animal and of individual cells is suppressed by near ultra-violet light (without visible light). 4. Inhibition in ultra-violet light is not due to stimulation (by the ultra-violet light) of the animal to luminesce, thereby using up the store of photogenic material. 5. Animals stimulated mechanically several times and placed in ultra-violet light show a luminescence along the meridians in the same positions as the luminescence that appears on stimulation. This luminescence in the ultra-violet or "tonic luminescence," is not obtained with light adapted ctenophores and is interpreted to be a fluorescence of the product of oxidation of the photogenic material. 6. Marked fluorescence of the luminous organ of the glowworm (Photuris) and of the luminous slime of Chatopterus may be observed in ultra-violet but no marked fluorescence of the luminous substances of Cypridina is apparent. 7. Evidence is accumulating to show a close relation between fluorescent and chemiluminescent substances in animals, similar to that described for unsaturated silicon compounds and the Grignard reagents.