Herbert W. Levi.
Techniques for the Study of Spider Genitalia.
Psyche 72(2):152-158, 1965.

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TECHNIQUES FOR THE STUDY OF
SPIDER GENITALIA
BY HERBERT W. LEVI *
Museum of Comparative Zoology
In response to many inquiries, I want to describe the techniques used for studying genitalia in the spider family Theridiidae. These procedures are not original, but have been adapted from methods used by several colleagues and students. One technique used in the family Araneidae is new and, I hope, helpful to students of that family. I would like to urge strongly against making permanent microscopic slides of genitalia. Most slides are not permanent; even balsam may crystallize after fifty years. In dehydration, sclerotized parts may warp and, as the slide dries, the cover glass may crush anatomical struc- tures. The slides often become separated from the parts of the specimen kept in alcohol and are lost. However, mounting the geni- talia of common species on slides often saves time in their study. The medium found most useful is Hoyer's fluid (Baker and Wharton). Luckily, most male spiders have two palpi. One, usually the left, is removed so that it can be turned to the desired angle. Palpi should always be examined completely submerged in 75 to 90% alcohol,
never dry. Drying shrinks and distorts softer parts; partial submer- sion produces undesirable reflections. To prevent the parts from floating away in convection currents caused by the heat of the micro- scope light,
the light is equipped with heat absorbing glass and the pedipalpus anchored in a piece of fibrous paper tissue at the bottom of the dish. Vaseline, sometimes recommended to' keep the palpus in position, may cling to it, smear over it, and is difficult to remove. Examination is by binocular dissecting microscope, at magni- fications of 150 to 240 times.
Small, translucent, weakly sclerotized palpi are common among theridiid spiders. A transfer to glycerine, after blotting off alcohol, may help to make visible the borders of transparent structures. A temporary slide mount in glycerine may be made for examination under a compound or phase microscope. The palpus should always be returned to alcohol in a microvial (4 X 10 mm) stoppered with cotton, to be kept with the spider in a larger vial (Fig. I). A per- manent mounting generally ruins the palpus for study, as sclerites become too transparent, may become distorted, and can not be turned * Manuscript received by the editor November 4, 1964 152




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19651, Levi - Spider Genitalia 153
Figures 1-5.
Techniques for the Study of Spider Genitalia. Fig. 1. Vial containing two smaller vials,
all stoppered with cotton. Fig. 2. Abdomen, showing place of incision.
Fig. 3. Abdomen with genital area bent over. Fig. 4. Abdomen positioned for drawing internal genitalia. Fig. 5. Part of
cephalothorax with pedicel and abdomen integument including genitalia. and placed conveniently. I have examined a few such slides of geni- talia of holotypes only twenty years old; they were almost useless. Expanding the palpus to examine the position of sclerites is of value in phylogenetic studies, but is rarely useful for descriptions in- tended to facilitate species recognition. After the alcohol is blotted off, the palpus is brought to boiling in 10% NaOH solution. Then the palpus is transferred directly to distilled water where the hema- todocha rapidly expands.
The palpus can then be transferred to
alcohol for storage. Unfortunately, boiling in NaOH warps, distorts and damages sclerites.
Full strength lactic acid can be substituted for sodium hydroxide.
Sclerotized palpi must be boiled longer than soft ones.




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Psyche
(June
The epigynum of holotypes or rare species should not be removed from the body of the specimen. In many small spiders, especially if the epigynum is weakly sclerotized, the whole spider can be dropped into clove oil, after first blotting to remove excess alcohol. This procedure is satisfactory for routine examination of CZubiona, Dipoena and other small spiders having little pigment. After a few minutes' clearing, the ducts can be seen; longer clearing makes them too trans- parent. In these two genera, specific determination is based on charac- teristics of the duct that loops between the seminal receptacle and the external plate. In other genera the ducts are behind the seminal re- ceptacles. For description of new species, a more careful examination is necessary. After removal from clove oil, the spiders are again blotted and returned to alcohol for storage.** The procedure used for more careful examination is to make two cuts around the genital area (Fig. 2). The tools used are mounted, sharpened insect pins (minuten nadeln). The epigynal plate with seminal receptacles is folded back (Fig. 3) before or during submer- sion in clove oil. The specimen is then anchored on fibrous paper tissue for examination (Fig. 4). After examination the genitalia are bent back, like closing a door. In poorly preserved specimens; the structure sometimes breaks off. In that case it should be stored in a microvial. On very small spiders it is advantageous at times to sepa- rate cephalot1~orax and abdomen, but the epigynum and neighboring integument should remain attached to the pedicel (Fig. 5). Some- times a temporary slide can be made of the whole cephalothorax with genitalia.
If there are numerous specimens and difficulties in their study, it is best to make a temporary microscope slide of the epigynum with its accompanying structures. The epigynum is first cleared in clove oil and then, with a medicine dropper, it is transferred in a drop of clove oil to a microscope slide and covered with a coverglass. This preparation can be examined under a compound or phase microscope and the parts later returned to alcohol to be stored in a microvial. But there may be difficulties in this procedure. In Helvibis (Levi,
1964) it was possible to follow the course of the tortuous (but diag- nostic) epigynum ducts in only two species: one because the ducts were short and simple, the other because I could destroy one of the numerous specimens available and tease the ducts apart with needles. **Prof. M. E. Galiano informed me that examination under oil may damage iridescence or structural colors.




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19651 Levi - Spider Genitalia 155
Boiling the female genitalia in 10% NaOH may be necessary to clear heavily sclerotized parts, but this method invariably distorts and warps structures, swelling softer parts. It should only be used as an additional method if there are many specimens. Among the numerous difficulties of taxonomic research in the family Araneidae (= Argiopidae) is the problem of matching males and females. They are often collected separately and may be different in appearance. Abalos recently ( 1963) observed, in his study of reproductive behavior of spiders, that during the mating of many species the tip of the male palpal embolus, presumably carrying sperm, breaks off and remains in the female genital ducts. Though this had been noticed before in black widows, it appears to be widespread in theridiid spiders and orb weavers. The reason it has been overlooked is that the majority of males collected are virgin, in search of a female; the tip of the embolus is therefore still attached in most males preserved in collections. Because they die soon after mating, mated males are rarely collected. On the other hand, probably the majority of females collected have mated. In the genus Argyope now being studied, and presumably in other argiopids, the tip of the male em- bolus can readily be "fished out" of the epigynum by jiggling the projecting parts with a needle. Among related species of the genus Argyope, these tips differ strikingly in structure, athough the female genitalia are quite similar. An illustration of a broken off tip was supplied by Petrunkevitch (1930) with the hope of finding a male to match it. These tips not only permit matching males with females, but facilitate identification of females that have similar epigyna. Of course, the assumption is made that the male chose a mate of his own species. But the same method, applied to species in which the female is as easily determined as the male, may provide some data on the frequency of mating between species in nature. I would like to thank Dr. A. M. Chickering, Mr. Jon Reiskind and my wife for editing, and Miss Vida Kenk and Mr. F. Vuilleumier for translating the summary. The investigations were supported by Public Health Service Research Grant AI-01944. Resumen
Pre~araciones microsc6picas permanentes de 10s palpos y epiginios de arafias son desvantajosas ya que, a1 deshidratar las partes escle- rozadas se tuercen, se pueden destruir a1 secarse, y a menudo la preparaci6n es se~arada del resto del especimen guardado en alcohol y se pierde. Es mejor guardar 10s genitales en tubos pequefios (micro-



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vials) (Fig. I ), con el espicimen. En especies comunes con numerosos ejemplares, las preparaciones pueden hacerse usando Hoyer o un medio de montaje similar para Acari (Baker and Wharton) . Para ilustrar un palpo, a veces es necesario separarlo de la arafia a objeto de orientarlo mejor. En tal caso deberia examinarse comple- tamente sumergido en etanol de 75 a 95%. A1 secarse las partes blandas se dafian. El palpo se fija en el fondo del recipiente con un pedazo de papel fibroso. El examen se hace con un microscopio binocular de diseccien entre 150 y 240 aumentos. Los palpos pequefios y transparentes pueden estudiarse transladiindolos a glicerina o, si se desea examinarlos con el microscopio compuesto o de fase, a un montaje temporal en glilcerina; per0 el palpo debe siempre volverse a guardar en alcohol.
Para dilatar el palpo, se hierve por unos minutos en NaOH a1 10% entonces se transfiere a agua destilada, y luego se guarda en alcohol. Sin embargo, 10s escleritos se tuercen y se dafian cuando se hierven en NaOH. Por el momento yo uso Acid0 lictico en vez de NaOH.
No se debe quitar el epiginio de 10s ejemplares raros. En especi-
menes pequefios puede estudiarse sumergiendo todo el animal en aceite de clavo. DOS incisiones (Fig. 2) permiten doblar la region de 10s genitales hacia atris
(Fig. 3) y, entonces, puede estudiarse
despuks de orientara cuidadosamente (Fig. 4). Mis tarde la 'puertecita" puede volver a cerrarse. En las arafias pequefias, el cefa- lotorax puede separarse a veces con el epiginio todavia fijo a1 pedicel0 ( Fig. 5 ) . Algunas veces, en ejemplares que no estin bien preservados, los genitales se desprenden o es necesario separarlos para examinarlos en detalle. En tal caso, el epiginio se guarda en un tub0 pequefio (Fig.1).
Cuando se trata de preparaciones transitorias, el cefaloterax entero y el epiginio
(Fig. 5) pueden montarse en aceite de clavo; o, una preparacibn temporal del epiginio, para su examen bajo un micro- scopio compuesto o de fase, puede hacerse con una gota de aceite de clavo cubierta con un cubre-objeto. Para el estudio detallado de especies comunes es necesario, algunas veces, sacrificar un ejemplar y separar con
aguias 10s conductos genitales. Luego, 10s genitales vuelven a guardarse en alcohol. Solo epiginios con estructuras muy esclerozadas pueden hervirse en NaOH a1 10%. Este mitodo deforma las estructuras e hincha las partes blandas. Los machos y las hembras de Araneidae son a menudo muy distintos en tamafio v apariencia. Abalos ( 1963), observe que en muchas especies el extreme del estilo del palpo del macho se desprende durante



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19651 Levi - Spider Genitalia I57
la c6pula. El extremo del estilo palpal puede sepasarse del epiginio de la hembra para aparejar machos y hembras de la misma especie. Este mitodo se aplica con buen ixito en el ginero Argyope, en el cual la mayor parte de 10s machos colectados estin todavia virgenes, pues 10s machos mueren poco despues de la cbpula, y la mayor parte de las hembras estin fecundadas. Por lo tanto, es muy importante ilustrar cuidadosamente istos extremos de 10s estilos masculinos. Resumi f ran~ais
Les pripasations micro~scopiques permanentes sur lame de palpes et ipigynes d'araignies ne sont pas disirables. Les parties sclirifiies se diforment par dishydratation et peuvent Ctre icrasies par ass&he- ment et i1 arrive souvent que la lame soit siparie du reste du spicimen conserv6 en alcool et se perde. I1 est prifirable de garder palpes et ipigynes dans de petits tubes (4 X 10 mm) avec 1e spicimen (Fig. I). Pour les espkes communes dont on a de nombreux exemplaires on peut monter des lames en utilisant la solution de Hoyer ou bien des solutions semblables pour Acari (Baker et Wharton) . I1 se peut qu'on doive siparer un palpe de l'araignie si l'on veut llorienter correctement pour une illustration. Dans ce cas il faut l'examiner compl&ement immergi dans de l'ithanol 75 i 90%. Le desskhement ab?me les parties molles. On attache 1e palpe au fond de la cuvette au moyen d'un morceau de papier fibreux. On proc6de I1examen avec une loupe binoculaire grossissant de 150 i 240 fois. Les palpes de petite taille et transparents peuvent &re itudiis aprk &re transvasis dans de la glycirine ou bien apr& montage temporaire sur lame, en glycirine, pour examen au microscope optique ou au microscope 2 contraste de phase. Le palpe doit cependant &re remis en alcool pour conservation.
Pour itendre le palpe on le fait bouillir quelques minutes dans NaOH lo%, puis on le transpose dans de l'eau distillie, et enfin on le met dans l'alcool pour conservation. Cependant l'ibullition dans NaOH diforme et endolmmage les sclirites. J'emploie maintenant l'acide lactique i la place de NaOH.
L'6pigyne ne doit pas &re ditachi des spicimens rares. I1 peut &re itudii sur les petits exemplaires en submergeant l'araignie entiire dans de l'huile de girofle. Deux incisions (Fig. 2) permettent de courber en arri6re la region ginitale (Fig. 3)) qu'on peut itudier apris l'avoir soigneusement mise en place (Fig. 4). Par la suite on peut fermer la "porte" ainsi pratiquie. Chez les petites araignies on peut parfois ditacher le ciphalo~rhorax avec l'ipi~ne encore fix6 au pidicule ( Fig. 5 ) . Quelquefois l'ipigyne de spicimens ma1 conservis



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se casse, ou doit &re enlevi pour une itude ultirieure plus ditaillie. On conserve ensuite l'ipigyne dans un petit tube (Fig. I j. POUS des priparations microscopiques temporaires sur lame on peut monter le ckphalothorax et l'ipigyne entiers dans l'huile de girofle. On peut aussi priparer une lame temporaire en recouvrant l'ipigyne avec une goutte d'huile de girofle; une telle priparation protkie par une Iarnelle peut 6tse alors examinie au microscope optique ou i contraste de phase. Pour les espkes communes dont on fait une ktude ditaillie il se peut que l'on doive sacsifier un spicimen afin d'kcarter les conduits des ginitalia au moyen d'aiguilles. On remet ensuite Ies ginitalia en alcool pour conservation. On ne peut faire bouillir l'ipigyne dans NaOH 10% que si 1es structures sont fortement sclirifiies. Cette mithode abcme les structures et fait gonfler les parties molles.
Abalos ( 1963 j a observi que dans de nombreuses esp&es I'extrimitk de embolus du m5le se casse pendant l'accouplement. L'extrimiti de l'embolus palpal peut &re enlevi de l'ipigyne femelle pour assortir miles et fmelles (Chez les Araneidae les males et les femelles sont souvent tsk differents de taille et d'aspectj. Ceci se fait maintenant avec succ6s pour le genre Argyope, chez kquel la plupast des miles ricoltks son vierges, car les mXes meurent peu apr& l'accoupIement, et la plupart des femelles sont accouplies. Par conskquent il est important d'il1ustser ces extr6mitks de l'en~bolus mile. ABALOS, J. W. AND E. C. BAEZ
1963. On Spermatic Transmission in Spiders. Psyche 70: 197-207. BAKER, E. W., AND G. W. WHARTON
1952. An Introduction to Acarology. Macmillan, New York. LEVI, H. W.
1964. The Spider Genus Helvibis (Aradeae, Theridiidae). Trans. Amer. Microscop. SOC. 83 : 133-143.
PETRUNKEVITCH, A.
1930. The Spiders of Porto Rico, Trans. Connecticut Acad. Arts Sci, 30: 159-355.




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