Autofluorescence in earthworm setae

AUTOFLUORESCENCE IN EARTHWORM SETAE Sarah A. McManus West Forsyth High School, 1735 Lewisville-Clemmons Rd, Clemmons, NC 27012 (e-mail: subversive.element@gmail.com) ABSTRACT When viewed under ultraviolet light, earthworm setae fluoresce yellow-green. The high contrast makes the setae more visible and could facilitate earthworm identification and structural studies using fluorescence microscopy. Because the setae are composed of sclerotized proteins similar to those found in arthropod cuticles, the mechanism of fluorescence is probably similar to that of other invertebrates such as scorpions and spiders. However, further research is needed to determine the specific proteins responsible for earthworm setae autofluorescence. Key words: Fluorescence, earthworms, setae. RESUMEN Cuando se observan las lombrices con luz ultravioleta, sus cerdas se ven amarillo-verde fluorescente. El claro contraste permite que las setas sean más visibles y se facilita identificar los diferentes tipos de lombrices así como estudiar su estructura con un microscopio de fluorescencia. Debido a que estas cerdas están compuestas de proteínas esclerotizadas como las encontradas en las cutículas de los artrópodos, el mecanismo de la fluorescencia es probablemente similar al de otros invertebrados como los escorpiones y las arañas. Sin embargo, se necesita seguir investigando para saber cuáles son las proteínas responsables de la autofluorescencia en las cerdas de las lombrices. Palabras clave: Fluorescencia, fluorescente, lombrices, cerdas, setas, sedas, Oligoqueta. To observe the fascinating fluorescent properties of earthworm setae, the only materials needed are a UV light (a blacklight), a worm, and a relatively dark area. As Fig. 1 shows, the position and grouping of the bristles are readily apparent due to their bright yellow-green glow, and one can watch the setae extend as the worm contracts and then retract as it stretches out (they even remain visible under the skin). For years, scientists searching for scorpions in the field have taken advantage of the fluorescent properties of similar cuticular structures by using handheld blacklights to spot glowing greenish scorpions against rocks and sand. Other invertebrates have developed their own uses for these markings, including a tiny jumping spider that waves green fluorescent pedipalps during courtship (Lim, Land & Li, 2007). Researchers have also used fluorescent proteins to study invertebrate physiology on a smaller scale. At Wake Forest University, Alex Jordan used a fluorescence microscope to view tiny pockets in the pheromone-emitting organs of a moth. His task was made much easier by the fact that the sacs contain the elastic protein resilin, which fluoresced and outlined the pouches in bright blue. Using resilin and the fluorescent amino acid dityrosine, other scientists were able to investigate molecular changes in tick cuticles as they swelled to accommodate a meal of blood (Andersen & Roepstorff, 2005). Like the scorpion exoskeleton, annelid setae are chitinous structures that originate in the cuticle and harden through sclerotization. Wankhede (2004) stated that, “It is widely accepted that chemical linking of cuticular proteins can lead to broadspectrum fluorescence,” mentioning several of these cross-linked proteins, including resilin and betacarboline. He concluded that a specific coumarin (7hydroxy-4-methylcoumarin) is largely responsible for scorpion fluorescence. Earthworm setae fluorescence could be caused by any one of these proteins or interactions among several; the coumarin seems to be a likely candidate based on the color of its fluorescence and its presence in such diverse groups as plants, some mollusks, scorpions and even beavers. (Wankhede, 2004) Fig. 1: Ventral view of Lumbricus sp. under UV light, showing rows of fluorescent setae. Inset: Ventral view of caudal region. The most immediate applications of UV imaging involve quick identification of setae position and grouping and improved ability to study and photograph fine setal structures. This technique can be used on living specimens without dyes or advance preparation. Additional research would be needed to determine the exact mechanisms causing the fluorescence and the specific proteins involved (possibly in a study similar to that of Wankhede, 2004). Other investigation could establish whether fluorescence varies with setal development, continued exposure to UV light, or preservation in various fixatives. It is not know if the effect occurs in all oligochaetes or if polychaete setae fluoresce as well; fluorescence has been observed in living Lumbricus and Eisenia, though not in one preserved earthworm or one preserved Nereis. (See Vukusic & Sambles, 2003 for a description of iridescence in polychaete setae.) Wankhede also mentions that some organisms use coumarins as sunblock; whether this is the case for earthworms such as Lumbricus and Eisenia could merit further research. Cautions about the use of UV light include potential damage to living earthworms and the need for basic eye protection. Do not look directly at the light, and wear coated glasses or safety goggles that block UV-A if warranted by the strength of the UV light source or the duration of use. Wankhede reported that scorpions preserved in ethanol leached fluorescent proteins into the fixative (causing the liquid to glow), which should be considered if comparing fluorescence in preserved specimens. LITERATURE CITED Andersen, Svend Olav and Peter Roepstorff. 2005. The extensible alloscutal cuticle of the tick, Ixodes ricinus. Insect Biochemistry & Molecular Biology, 35(10): 1181-1188. Jordan, A. and W. Conner. In Press. Dietary Basis for Developmental Plasticity of an Androconial Structure in the Salt Marsh Moth Estigmene acrea (Drury)(Lepidoptera: Arctiidae). Journal of the Lepidopterists’ Society. Lim, Matthew L. M., Michael F. Land, and Daiqun Li. 2007. Sex-Specific UV and Fluorescence Signals in Jumping Spiders. Science, 315(5811): 481. Vukusic, Pete and J. Roy Sambles. 2003. Photonic structures in biology. Nature, 424(6950): 852-855. Wankhede, Ravi A. 2004. Extraction, Isolation, Identification and Distribution of Soluble Fluorescent Compounds from the Cuticle of Scorpion (Hadrurus arizonensis). Retrieved 18 February 2007 from Marshall University Electronic Theses and Dissertations. Web site: http://www.marshall.edu/etd/masters/wankh ede-ravi-2005-ma.pdf