1. Asia-Pacific Entomol. 9(3): 249-253 (2006) www.entomology.or.kr

A New Method for Preparing 51 ide Mounts of Whole Bodies of Microlepidoptera

Sangmi Lee* and Richard 1.. Brown

Mississippi Entomological Museum, Box 9775, Mississippi State, MS 39762-9775

*Corresponding author. E-mail: microlepi@hotmail.com Tel: +1-662-325-2085; Fax: + 1-662-325-8837

(Received May 30, 2006; Accepted September 19, 2006)

Vol. 9, No.3 (September 2006)

Journal of Asia-Pacific Entomology

J. Asia-Pacific Entomol. 9(3): 249-253 (2006) www.entomology.or.kr

SYSTEMATICS AND EVOLUTION

A New Method for Preparing Slide Mounts of Whole Bodies of Microlepidoptera

Sangmi Lee* and Richard 1.. Brown

Mississippi Entomological Museum, Box 9775, Mississippi State, MS 39762-9775

Abstract A new method is described for preparing slide mounts of whole bodies of microlepidoptera to facilitate comparative morphological studies. This me­thod conserves traditional characters of wing pattern while revealing wing venation and other morpholo­gical structures of the denuded body. Examples of new characters revealed on slide mounts of whole bodies and photographed with a Confocal Laser Scanning Microscope are given for selected species of Gelechioidea. Also, the historical use of morpholo­gical characters for defining taxa of Lepidoptera is briefly reviewed.

Key words Lepidoptera, Gelechiidae, Morphology, Systematics, History

Introduction

Morphological structures of the whole body including sclerites, tarsi, and other structures, have been used historically for defining species, genera, and family group taxa throughout the Insecta. Lepidoptera are unique in being the only order in which systematic studies historically have been based on a limited set of characters that does not include the whole body. Most of the characters used in classifications and identification of Lepidoptera have involved colors and patterns of wings, wing venation, mouthparts, antennae, and genitalia.

The color, pattern, venation, and shape of wings were the earliest characters used for differentiating and classifying taxa of Lepidoptera. These characters have been used. for differentiating species, genera, e.g., wing shape in Papilio L., as well as families, e.g., hindwing shape in Gelechiidae and the dominant white or yellow color of wings in most Pieridae.

*Corresponding author.

E-mail: microlepi@hotmail.com Tel: +1-662-325-2085; Fax: +1-662-325-8837

(Received May 30, 2006; Accepted September 19, 2006)

These characters have remained useful for identi­fication of many species and higher taxa. Harris (1766) was a pioneer in using wing venation for systematic purposes, but Herrich-Schaffer (1843­1856) was the first to use wing venation as the primary basis for developing a new classification for Lepi­doptera, including the microlepidoptera (Lindroth, 1973). The use of the venation for defining families and genera was promoted subsequently by Meyrick (1895) and others. Current identification keys to families in North America and elsewhere have em­phasized characters of venation, e.g., Triplehorn and Johnson (2005) and Common (1970).

Characters of mouthparts were used early by Savigny (1816), Burgess (1880), and Walter (1885), and many of these, e.g., scaled or naked proboscis and development of labial palpi, are used currently for defining taxa at generic and family levels. The taxonomic value of antennae in Lepidoptera was treated by Bodine (1896), and antennaI characters have been used since then to differentiate taxa at all levels from superfamily to species.

One of the earliest studies of Lepidoptera genitalia, at least in the United States, was that of Scudder and Burgess (1870). Smith was the first systematist in North America to make extensive use of genitalia in monographic treatments of Lepidoptera, specifically Noctuidae (e.g., Smith, 1891). Dampf (1909) and subsequent authors, e.g., Busck and Heinrich (1921) and Heinrich (1923, 1926), elaborated on descriptions of genitalia and amplified their use for defining genera and family group taxa of microlepidoptera. Other lepidopterists during this period rejected or ignored the use of genitalia for taxonomic purposes, notably Edward Meyrick, who is quoted by Clarke (1955) as stating "the genital organs of the imagos are likely to be of slight importance in the definition of groups; whilst neuration occupies a high position, except when directly influenced by an alteration in form of wing, which is seldom the case."

Aside from the genitalia and structures associated with the abdomen, the use of other structures of the whole bodies of Lepidoptera has been relatively

250 J. Asia-Pacific Entomol. Vol. 9 (2006)

ignored, except for a few taxa. Notable examples of taxonomic studies that have used characters of whole bodies include those of Ehrlich (1958) for butterflies, Davis (1975) for Neopseustidae, Davis (1978) for Eriocraniidae and Acanthopteroctetidae, Kristensen and Nielsen (1979) for Heterobathmiidae, ScobIe (1986) for Hedylidae, Dugdale (1994) for Hepialidae, Davis (1996) for Prototheoridae, and Nielsen and Kristensen (1996) for Lophocoronidae. Recent studies using characters of whole bodies include those of Kaila (2004) for Gelechioidea, Fibiger and Lafontaine (2004) for Noctuoidea, and Dacosta and Weller (2005) for Callimorphini (Arctiidae). Most of these authors do not provide details of the methodology for pre­paring the whole body preparations, but a few indicate that preparations are maintained in glycerin within microvials. Whereas slide mounts are generally used for genitalia preparations to facilitate comparisons between specimens, no method has been provided previously for preparing slide mounts of the whole body that would be comparable to those of genitalia.

The technique described here for the preparation of whole body mounts maximizes the use of tradi­tional characters, such as wing pattern, venation, and genitalia, and at the same time, reveals additional anatomical characters from the rest of the body. This method was developed originally for species of Tortricidae, and it has been modified recently for Gelechioidea and other microlepidoptera. Further modification would be needed for the larger species of Lepidoptera, recognizing that many large species cannot be mounted on standard microscope slides.

Methods

Specimen selection and labeling

Specimens are selected for whole body mounts that have all appendages and complete wing patterns, i.e., not specimens with worn wings. Poor quality speci­mens should never be used for either whole body mounts or genital dissections because intact wing patterns include diagnostic characters essential for identification of the species.

Data labels are removed from the selected specimen and placed on a second pin with an additional label providing a unique slide number. Each slide number label is prepared in duplicate with one copy affixed to the pin holding the specimen data and the other copy accompanying the whole body mount through the various reagents. The slide number along with selected specimen data, e.g., locality and collection date, sex of specimen, and collection deposition are recorded in both a written record book and database.

Specimen preparation

The left pair of wings is separated from the body for preservation of the color pattern and is set aside for future slide mounting (a distinctive labial palpus or leg can also be removed for dry mounting with this pair of wings). The right pair of wings for studying venation is also detached from the body and is set aside for future descaling, staining, and slide mounting.

The rest of the specimen, while still on the pin, is placed in 10% potassium hydroxide (KOH) for at least 8 hours (generally overnight) at room tempe­rature. Specimens that previously were stored in alcohol are not recommended for use in whole body mounts because appendages become distorted when placed in KOH. After the body is cleared overnight, the head, thorax, and abdomen are separated. An­tennae, labial palpi, and one half of the proboscis are detached from the head capsule, the thoracic segments are separated, and the genitalia are separated from the abdomen. These portions of the body are denuded in 20% ETOH and then stained. Stains that have been used successfully include Eosin, saffarin, and mercurochrome. Chlorazol black may be used as a second stain to differentiate membranous from scle­rotized structures. The time required for adequate staining varies depending on the stain as well as the taxon, but typically staining with chlorazol black requires less than ten seconds, saffarin and mercuro­chrome each require about ten minutes, and Eosin requires as much as four hours. After staining, the separated portions of the body are transferred to 70% ETOH and are further cleaned of scales and other non-scierotized tissue. Scales are more easily removed in 70% ETOH than 20%, but the body parts, especially antennae and tarsi, are more fragile and can be broken easily in higher concentrations of alcohol. A camel hair brush (size 000) is used to remove scales by lightly brushing the base of the appendage toward its apex. All body parts except wings are dehydrated about 8 hours in 100% ETOH before being mounted in Euparal on a microscope slide.

The right pair of wings that was removed earlier from the pinned specimen for studying wing venation is placed in 20% ETOH to be denuded. Alternatively, bleach can be used for clearing the scales. These wings are transferred to a watch glass and stained with Eosin overnight. The wings are further cleared of scales in 70% ETOH after staining. The wings are dehydrated in 100% ETOH before being mounted in Euparal on a microscope slide.

Slide mounting

Two slides are required for mounting the whole body.

Slide mounts of whole bodies of microlepidoptera 251

Slide "A" (labeled with the dissection number, e.g., MEM 2204A [personal or collection acronym and unique number]) includes the body parts except the wings (Fig. lA). The head and its appendages, pro­thorax, and mesothorax are mounted under one cover slip in the middle of the slide, and the metathorax, abdomen and genitalia are mounted under a second cover slip on the right side of the slide. A label with species identification, sex, selected collection data, mounting medium, and slide number is attached to the left side of the slide. Slide "B" includes the wings, e.g., MEM 2204B (Fig. IB). The left pair of wings that was removed earlier for study of the preserved wing pattern is dry mounted on the middle of the slide using clear nail polish around the edge of a cover slip. The right pair of wings is mounted in Euparal on the right side of the same slide. The cover slip is brushed with Euparal essence before being placed on top of the wings.

Discussion

This method for preparing slide mounts of whole bodies has been adopted for an investigation of new characters that can be used for defining higher taxa in Gelechioidea. To date, whole body mounts have been made of males and females of 75 species in 48 genera of Gelechioidea. Examples of some of the characters revealed with whole body mounts have been photographed with a Confocal Laser Scanning Microscope (Figs. 2-6), which allows three-dimen­sional examination of images as well as capability for rotating the image to provide both lateral and frontal views.

Useful characters for differentiating taxa have been found by examining the internal and external areas of the head capsule (Fig. 2), including the form of the pi lifers (Fig. 3), shape of the clypeus, and the form of the platelike sitophore, which occurs at the bottom of the buccal cavity. The forms of the metascutellum and epinoturn (Fig. 4) also vary con­siderably among genera and families. The prothoracic spina (Fig. 5), which is the median process of the furcastemum, varies in the size and shape of its apex among genera. Denuded and slide mounted appen­dages, such as the labial palpus and hindleg (Fig. 6), facilitate meastirements of lengths and widths and comparison of setation.

The preceding examples are only a few of the characters that can be compared easily among genera and higher taxa with use of whole body mounts. Some of these characters will undoubtedly be found to be highly variable, but other characters will be important for defining higher taxa.

In conclusion, slide mounts of genitalia and whole bodies have advantages and disadvantages. The prin­cipal advantage is the ability to store, retrieve, and sort slide mounts quickly and easily, rather than having the preparation stored in a glycerin or other preservative inside a microvial. Slide mounts also facilitate comparison of differences between specimens because the slides can be examined with one on top of another after the mounting medium is dry. Slide mounted material can also be illustrated easily with confocal microscopy, as shown in Figs. 2-6. The only disadvantage of a slide mount is the inability to change the position of the body part to obtain a view from another angle, but this disadvantage is offset with use of confocal microscopy, as previously described.

Acknowledgment We thank William Monroe, Mississippi State University Electron Microscope Center, for assistance with the Confocal Laser Scanning Microscope, and Blake Layton, Department of Entomology and Plant Pathology, for the photography of the slides. Support for this research was provided by National Science Foundation grant DEB­0416078 and by Mississippi Agricultural and Forestry Experiment Station. Approved for publication as Journal Article No. J-l0940 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.

Literature Cited

Bodine, D. J896. The taxonomic value of the antennae of Lepidoptera. Trans. Amer. Entomo!. Soc. 23: I-56, pis. I-V.

Burgess, E. 1880. Contributions to the anatomy of the milk-weed butterfly, Danais archippus. Anniversary Mem. Boston Soc. Nat. Hist. 1880: 16 pp.

Busck, A. and C. Heinrich. 1921. On the male genitalia of the Microlepidoptera and their systematic importance. Proc. Entomo!. Soc. Wash. 23: 145-152.

Clarke, J.F.G. 1955. Catalogue of the type specimens of microlepidoptera in the British Museum (Natural History) described by Edward Meyrick. Vo!. I. 332 pp. Trustees of the British Museum (Natural History), London.

Common, I.F.B. 1970. Lepidoptera. pp. 765-866, in Insects of Australia, Ed. I.M. Mackerras. 1029 pp. Melbourne University Press, Melbourne.

Dacosta, M.A. and S.J. Weller. 2005. Phylogeny and classification of Callimorphini (Lepidoptera: Arctiidae: Arctiinae). Zootaxa 1025: 1-94.

Dampf, A. 1909. Ueber den Genialapparat von Rhopobota naevana Hb. (Lepid., Tortricid.). Dt. entomo!. Z. Iris 21: 304-329.

Davis, D.R. 1975. Systematics and zoogeography of the family Neopseustidae with the proposal of a new superfamily (Lepidoptera: Neopseustoidea). Smithson. Cont. Zoo!. 210: 1-45.

Davis, D.R. 1978. A revision of the North American moths of the superfamily Eriocranioidea with the proposal of a new family Acanthopteroctetidae (Lepidoptera). Smithson. Cont. Zoo!. 251: 1-131.

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Figs. 1-6. Whole body mounts of Gelechioidea. I, Position of body parts of Machimia tentoriferella Clem. (Amphisbatidae) on slides A and B: A, head, prothorax, and mesothorax (left coverslip), metathorax and abdomen (right coverslip), Ab =

abdomen, An = antenna, G = genitalia, H = head, L = labial palp, P = proboscis, T1 = prothorax, T2 = mesothorax, T3 = metathorax; B, intact (left coverslip) and denuded (right coverslip) wings. 2, Head capsule with sitophore (arrow) of Gnorimoschema gallaesolidaginum (Cham.) (Gelechiidae). 3, Setose pilifers and base of proboscis of Carpatolechia sp. nov. (Gelechiidae). 4, Metascutellum and epinotum (arrow) of Glyphidocera juniperella (Glyphidoceridae). 5, Prothoracic furcasternum and spinae (arrow) of Aroga composilella (Wlk.) (Gelechiidae). 6, First tarsomere of hind tarsus of Aroga composilella (Wlk.).

Davis, D.R. 1996. A revision of the southern African family Prototheoridae (Lepidoptera: Hepialoidea). Entomol. Scand. 27: 393-439.

Dugdale, l.S. 1994. Female genital configuration in the classification of Lepidoptera. N. Z. l. Zool. I: 127-146.

Ehrlich, P.R. 1958. The comparative morphology, phylogeny and higher classification of the butterflies (Lepidoptera, Papilionoidea). Kansas Univ. Sci. Bull. 39: 305-370.

Fibiger, M. and J.D. Lafontaine. 2004. A review of the higher classification of the Noctuoidea (Lepidoptera) with special reference to the Holarctic fauna. Esperiana II: 7-75.

Harris, M. 1766. The Aurelian or Natural History of English Insects, Namely Moths and Butterflies, together with the Plants on which They Feed. 77 pp. London.

Heinrich, C. 1923. Revision of the North American moths of the subfamily Eucosminae of the family Olethreutidae. Bull. U.S. Natl. Mus. 123: i-iv, 1-298, pis. 1-59.

Heinrich, C. 1926. Revision of the North American moths of the subfamilies Laspeyresiinae and Olethreutinae. Bull. U.S. Natl. Mus. 132: i-v, 1-216, pIs. 1-76.

Herrich-Schaffer, G.A.W. 1843-1856. Systematische Bear­beitung der Schmetterlinge von Europa, zugleich als Text, Revision und Supplement zu Jakob Huebners Sammlung Europaischer Schmetterlinge, vols 1-6. 1885 pp. GJ. Manz, Regensburg.

Kaila, L. 2004. Phylogeny of the superfamily Gelechioidea (Lepidoptera: Ditrysia): an exemplar approach. Cladistics 20: 303-340.

Kristensen, N.P. and E.S. Nielsen. 1979. A new subfamily of micropterigid moths from South America. A con­tribution to the morphology and phylogeny of the

Slide mounts of whole bodies of microlepidoptera 253

Micropterigidae, with a generic catalogue of the family (Lepidopera: Zeugloptera). Steenstrupia 5: 69-147.

Lindroth, C.H. 1973. Systematics specializes between Fabricius and Darwin: 1800-1859. pp. 119-154, in History of Entomology, Eds. R.F. Smith, T.E. Mittler and C.N. Smith. 517 pp. Annual Reviews Inc., Palo Alto, California.

Meyrick, E. 1895. A Handbook of British Lepidoptera. vi + 843 pp. Macmillan, London.

Nielsen, E.S. and N.P. Kristensen. 1996. The Australian moth family Lophocoronidae and the basal phylogeny of the Lepidoptera-Glossata. Invert. Taxon. 10: 1199-1302.

Savigny, J. 1816. Memoires sur les animaux sans vertebre. 1. fascicule: Theorie des organs de la bouche des Crustaces et des Insectes. 117 pp., 12 tt. Paris.

Scobie, M. J. 1986. The structure and affinities of the Hedyloidea: a new concept of the butterflies. Bull. Brit. Mus. (Nat. Hist.) Ent. 53: 251-286.

Scudder, S.H. and E. Burgess. 1870. On asymmetry in the appendages of hexapod insects, especially as illustrated in the Lepidopterous genus Nisoniades. Proc. Boston Soc. Nat. Hist. 13: 282-306, figs. 1-17.

Smith, J.B. 1891. Contributions toward a monograph of the Noctuidae of temperate North America. Revision of the species of Mamestra. Proc. U.S. Nat!' Mus. 14: 197-276.

Triplehorn, C.A. and N.F. Johnson. 2005. Borror and DeLong's Introdution to the Study of Insects. 7'h Edition. 864 pp. Thompson Brooks/Cole, Belmont, California.

Walter, A. 1885. Beitrage zur Morphologie der Lepidoptera. I. Zur Morphologie der Schmetterlingsmundteile. lena Zeitschr. Naturwiss. 18: 751-807. 2 tt.