Examining Trichoptera Genitalia with Confocal Laser Scanning Microscopy Christy Jo Geracy1 & Richard C
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Nectopsyche Neotropical Trichoptera Newsletter ISSN 1550 - 7971 Issue Number 3 June 2006 Examining Trichoptera Genitalia with Confocal Laser Scanning Microscopy Christy Jo Geracy1 & Richard C. Laughlin2 1Department of Entomology, Soils, and Plant Sciences, Clemson University, 2Department of Genetics and Biochemistry, Clemson University Corresponding Address: 114 Long Hall, Box 340315, Clemson University, Clemson, SC 29634 USA, [email protected] Introduction microscope and compiled into images by computer software. Table of Contents onfocal microscopy is a com- What makes confocal microscopy C Examining Trichoptera Genitalia mon tool that is based on the prin- potentially useful for taxonomy • with Confocal Scanning ciples of fluorescent microscopy. It and systematics is that it takes Microscopy.................................................1 has been around for decades, but image slices through the speci- • Editor’s Remarks..................................2 it has mainly been used by cellu- men at user-defined intervals • Colecta, cría y asociación en lar biologists to view intracellular and depths. This “digital dissec- Atopsyche (Collecting, rearing and structures labeled with fluorescent tion” process assures that only the associating Atopsyche, English and dyes. Recently, confocal imaging light that is in focus in any one Spanish)......................................................5 has been used for examining in- optical plane is captured. Out-of- • Featured photograph......................7 ternal morphology and the three- focus background noise, which • New Neotropical Trichoptera Lit- dimensional shape of the cuticle often obscures images taken with erature (2004-June 2006).....................8 in Blattodea (Periplaneta, Blaberus; conventional light microscopy, is • Being an Entomologist....................9 Larsen et al. 1997, Zill et al. 2000), eliminated. Each digital slice can • Featured illustration.......................10 and as well as muscle tissue and be examined individually, or the • E-mail List................................... ........11 internal organs in Diptera (Culex, slices can be compiled and ren- • Researchers, Addresses, Current Drosophila; Klaus et al. 2003). This dered into a three-dimensional Projects.............................................. .......12 technique takes advantage of the image that can be rotated about • Announcement 12th International fact that some types of insect tis- the X, Y or Z-axis. This allows the Symposium on Trichoptera...............13 sue, including most cuticular struc- investigator to see a three-dimen- • Call for Articles............ .....................14 tures, are autofluorescent under sional representation of the inter- • Call for Subscribers...................... ...14 red (543 nm) laser excitation. The nal connections within a specimen • On Choosing our Name.................14 fluorescence is detected by the without physically dissecting it. Page 1 This technique is potentially useful (plastic cover slips cannot be for working with rare specimens, used because they will melt dur- Editor’s Remarks voucher specimens, type mate- ing sample recovery). The mount- rial, or other material that cannot ed structures were allowed to dry It is my pleasure to bring be dissected practically or altered for approximately 30 minutes to you the third issue of Necto- permanently. until solidified. It was important psyche. I apologize for the de- In this report, we used an adult to position the genitalia as close lay in bringing you this issue. I male specimen of Polycentropus to the surface of one of the cover am pleased with the articles in- confusus Hagen as a test species to slips as possible to ensure they cluded herein contributed by measure the autofluorescence of were in the working range of CJ Geraci & Richard Laughlin the caddisfly genitalic structures, the objective. The farther away on confocal microscopy, Paola and to see if the three-dimension- the structure is from the cover Rueda on adult-larva associa- al images produced from confocal slip, the longer the distance the tion, and Ralph Holzenthal on laser scanning microscopy would laser has to penetrate; this can the latest Neotropical publica- contain information useful to tax- decrease resolution (some useful tions. I am very thankful for onomic and phylogenetic studies. tips: press the cover slips together their contributions. Specifically, we wanted to know only slightly -- too much pressure whether this technique can be can flatten and/or damage the ages through the Z-axis was col- used to view internal connections structures; using a label pen to lected (Fig. 2 A-D). The complete between membranous and sclero- circle the structures on the cover- series of image slices was then tized portions of the male phallus. slips makes it easeir to find them used to render a three-dimension- under the microscope). al image of the phallus (Fig. 3). Methods The protocols for mounting Imaging: The male genitalic struc- Specimen Recovery: To remove the structures and taking images with tures were imaged with a Zeiss genitalia from the gelatin solution the Carl Zeiss LSM 510 imaging LSM 510 Confocal Microscope the cover slip mount was placed in software were adapted from Klaus with either the 10X or 40X objec- a glass dish with distilled water and et al. (2003; see reference for de- tive by light or red laser (543nm). heated for approximately 20 min- tailed imaging procedures). To assess whether regions of the utes (low heat). When the solution cuticle were autofluoresent, im- had melted enough for the cover Mounting: The cleared male geni- ages using either the laser of slips to be separated, they were talia of Polycentropus confusus white light were taken (Fig 1, Pan- pulled apart carefully with forceps were mounted between two glass els 1 & 2). Light and laser images in order to avoid tearing the geni- cover slips in a solution of 1:17:17 were then digitally overlayed (Fig. talia. After the top cover slip was (w:v:v) gelatin:glycerine:water 1, Panel 3). Next, a serial set of im- removed the genitalia were con- Figure 1. Polycentropus confusus, tergum X and genitalia of male. Panel 1: autofluorescence under the red laser (543 nm); Panel 2: bright field image; Panel 3: red fluorescence overlaid onto brightfield image (Carl Zeiss Microscope LSM 510 software, 10X objective). Page 2 tained in a drop of media on the standing connections in any one py is useful for viewing arthropod bottom cover slip. The bottom cov- plane in the z-stack (Figure 2). cuticle and muscle tissue, but does er slip was put back into the dish of The three-dimensional render- not work well with darkened or distilled water and returned to the ing was most useful for view- solid cuticular structures. Confocal heat for approximately another 20 ing internal structures, and the images could be used in addition minutes (enough time to dissolve invagination of the endothecal to illustrations to show diagnostic the media). Finally, the genitalia membrane with its endothecal characters whose three-dimen- were removed from the cover slip spines. Deleting the proximal sional shape is important. The and placed in the distilled water slices exposed the invaginated internal connections between for an additional 30 minutes to endothecal membrane and se- muscle and cuticle that are shown fully dissolve the remaining gela- tae, which could then be rotated in the three-dimensional images tin. (Times are approximate, and about each axis to see where the can be used to help assess homol- may vary depending on the tem- membrane attached to a scleri- ogy for phylogenetic studies. The perature used and the size of the tized tube (Figure 3). most important factor in produc- mounted structures.) ing high quality images is properly Additional observations: We sur Observations and - mounting the structures between veyed various other taxa to test the cover slips. Damage to the Conclusions their levels of autofluorescence. structures may occur if care is not Polycentropus confusus: Carl Muscle tissue in the femur of a taken during the sample recovery Zeiss LSM 510 imaging software baetid mayfly, the peritrophic process, or if the structures are allowed us to view each optical matrix of a simuliid larva, and compressed too much between slice taken at the specified Z-axis the exocuticle of a tardigrade the cover slips. interval and overlay the fluores- all fluoresced brightly. However, cence onto the light image (Figure hardened and darkened cu- Free image analysis software: Carl 1). This showed any structures that ticle was not fluorescent, as has Zeiss LSM Image Browser Ver- were not fluorescent. It is not fully been reported previously (Zill sion 3.5 available for free down- understood why some some struc- et al. 2000). For example, Chi- load from the Carl Zeiss web site tures do not fluoresce while others ronomidae larval mouthparts (http://www.zeiss.com/) do. All portions of the male genita- were fluorescent except for the lia of P. confusus were fluorescent. mandibles, and the head capsule Software for converting LSM Figure 2 shows representative of Micrasema rickeri Ross & Un- files to QuickTime or .mpeg files: slices of the specimen at differ- zicker did not fluoresce. We also ImageJ (Image Process-ing and ent Z-axis coordinates. We found found that setae and other hol- Analysis in Java) available for free the limit for the z-stack was ~150 low structures fluoresced bright- download from the US National um, with a minimum interval size ly, but solid structures did not. Institutes of Health web site (http:// of ~0.5 um. Viewing the individual rsb.info.nih.gov/ij/) image slices was useful for under- Conclusions: Confocal microsco- Figure 2. Digital slices of the right lateral view of the phallus of Polycentropus confusus taken at sample intervals (40X objective; z-stack range: 0.0 - 118.4 um, 1.6 um interval). Page 3 References Klaus, A.V., V.L. Kulasekera, and Larsen, G.S., S.F. Frazier, and S.N. Zill, S., S.F. Frazier, D. Neff, L. Quimby, V. Schawaroch. 2003. Three-di- Zill. 1997. The tarso-pretarsal M. Carney, R. Dicaprio, J. Thuma, mensional visualization of insect chordotonal organ as an ele- and M.