JOURNAL OF MORPHOLOGY2t3:34t-347 (1992)

Influenceof Web-MonitoringTactics on the Densityof Mitochondriain LegMuscles of theSpider Family

BRENT D. OPELL ANo DAVID C, KONUR ni a Po t v t ec h n i c I ns t i t ut e qn d s tat e (rn iu e r si tv, Er:ff:w, {,i;: l:y; [; ;ri

ABSTRACT From electron micrographs we determined the ratio of mitochon- drial to myofibril cross sectional area in cells of the first leg anterior depressor muscles of adult females of four species, each from a different genus. Specieswith more active web-monitoring tactics and greater tracheal supplies to their first legs have muscle cells that are better supplied with mitochondria than those with less active tactics and less well-developed tracheal systems. These results demonstrate that, even in homologous tissues of closely related species,mitochondrial supply can change to accommodate changesin metabolic activity. o 1992Wiley-Liss, Inc.

Mitochondria are the sites of oxidative single monitoring line and shake the web '87a).In phosphorylation and are found in greater when a prey strikes it (Opell, both numbers in more active tissues.For example, orb-web and reduced-web , the first in insects mitochondrial densitv is low in legs play an important role in web monitor- small visceral muscles and inactive fat cells ing and manipulation. (Dean '85; '68; 'ZB) et al., Smith, Sohal, and As summarized in Table 1, previous stud- high in metabolically active tissues such as ies show that these differences in web-moni- flight muscles (Chapman,'54; Edwards and toring tactics are reflected in both the web- Ruska, '55; Levenbook '56; and Williams, monitoring force expressed by a species and Smith, '62), (Smith, '68; Malpighian tubules the degree of its tracheal development (Opell, Wigglesworth and Salpeter, '62), '87a,b). and anal Hyptiotes cauatus has the greatest papillae (Copeland,'64). The purpose of this absolute and relative resting forces (force study is to determine if changes in mitochon- exerted on a single, resting thread) and also drial density accompany diverging activity the greatest tracheal supply to its first legs levels in homologous muscles. We (i.e., the smallest volume of tissue served do this by comparing the mitochondrial sup- by each pm2 of tracheal cross plies of the leg muscles of spiders that have section). Uloboru,s glomosus has the next greatest different web-monitoring tactics. resting force and the next greatest The orb-web is the primitive web form in first leg tracheal sup- the family Uloboridae (Coddington, '86, 'g0; ply, followed by Miagrammopes animotus. '79). Opell, Members of orb-weaving genera The tracheal spiracle of uloborids is near the such as Uloborus and hang be- abdomen's posterior tip and the long abdo- neath the hubs of their horizontal webs while men of M. animolzs reduces the first leg's '86; waiting for prey (Lubin, Cushing and oxygen supply by increasing tracheal length '90; '88). Opell, Peaslee and Peck, In con- and, thereby, the distance over which oxygen trast, species that construct reduced webs must diffuse. The resting force expressed by more actively monitor and manipulate them has not been measured during prey capture. Members of the genus and attempts to do so for this study proved Hyptiotes spin taut, vertical triangle-webs unsuccessful, as spiders hung for only a few wtrich they monitor from the anchoring point secondson resting threads and failed to estab- of the web's apex thread (Lubin, '86; lish stable resting forces. However, as '82). Opell, this Members of the genus MiagratnnLopes species' tracheal system is restricted to its '90b) construct irregular webs that have one to abdomen (Opell, and its prosomal and eight prey capture lines that diverge at vary- legmuscles are supplied solelyby '86;Lubin hemolymph ingangles(Lubin, et al., '78; '90a). Opell, borne oxygen, it probably exerts less monitor- Like Hyptiotes, these spiders uJe a ing force than the other three species. o 1992 WILEY-LISS, INC. 342 B.D. OPELL AND D.C. KONUR

TABLE 1. Comparison of the mean leg uolumes, messes,tracheal areas, and resting forces of adult female uloborids Hyptiotes Uloborus Miagrammopes Octonoba cavatus glomosus animotus sinensis Leg I volume x 104pm3 1 27,438 48,822 104,188 Prosomal and leg weight mg3 L.49 1.91 2.38 g.as RestingforceN x 10-52 13.42 r0.72 7.09 Resting forcelleg I volume x 10 a/mm3 4.89 2.02 0.68 Resting force/prosomal and leg weight 9.01 5.61 2.89 Tracheal area serving leg I pm2 t 387 289 891 n Leg I volume/leg I tracheal arealsquare root distance 3,239 8,565 7,180 0 from spiracle to leg I1 rOpell,'87b. 2Opell,'87a. 3Opell,'90.

Becausethe first legs are important in web make them easy to identify, isolate, and han- monitoring, the activity level of their mus- dle and because their role as flexors of the cles, as predicted by a species' resting force patella is important for the leg movements and tracheal supply, should be reflected in associatedwith web monitoring and manipu- their mitochondrial supply. This study will lation. test the hypothesis that the density of mito- The ADDM were post-fixed for 30 min in chondria in first leg muscles is ordered, from 17oosmium tetroxide, washed with sodium gTeatest to least, as follows: Hyptiotes caua- cacodylate buffer for 5 min, and dehydrated tus, Uloborus glomosus, Miagrammopes ani- through a series of ethanol dilutions. Mus- motus, Octonobasinensis. cles were then infiltrated with Spurr's epoxy resin (hard formula) and polymerized for 24 MATERIAISAND METHODS hr at 65'C. Thin cross and longitudinal sec- Adult females of the following specieswere tions were stained with lead citrate and ura- used in this investigation: the introduced nyl acetate and examined and photographed Asian orb-weaver, Octonoba sinensis (Si- at x 1,600-3,300 with a transmission elec- mon), collected from free ranging popula- tron microscope. tions in greenhouses at Virginia Polytechnic Enlarged cross sectional photographs of Institute and State University (VPISU); the individual muscle cells (Fig. 1) were mea- eastern North American orb-weaver, Ul- sured with a digitizing tablet connected to a oborusglomosus (Walckenaer),collected from computer. We first determined the cell's total shrubbery on the VPISU campus; the east- surface area (TA), the combined surface ar- ern North American triangle-web-weaver, eas of its mitochondria (MA), the surface Hyptiotes cauatus (Hentz), collected in the area of its central clear region where a nu- forests of Montgomery and Craig Counties of cleus was often found (CA), and the com- Virginia; and the Puerto Rican "single-line- bined surface areas of the mitochondria found weaver," Miagramm,opes animotus Chicker- within the central clear region (CMA). '68, We ing, collected at the Center for Energy used the following formula to determine the and Environment Research's El Verde field area occupied by a cell's contractile elements station in the Luquillo National Forest. These (CEA): species were identified by reference to the CEA: TA - CA- (MA- taxonomic revisions of Chickering ('68), CMA) Muma and Gertsch ('64), Opell ('79), and However, in addition to myofibril bundles, Yoshida('80). this cortex region (: CEA) also contains sar- Specimens were anesthetized with carbon coplasmicreticula, T-system tubules, and in- dioxide, fixed at 20-26"C for 12-18 hr in 3Vo tracellular space(Figs. L, 2). We determined glutaraldehydel}o/o formaldehyde in 0.1 M the density of myofibrils within the CEA sodium cacodylatebuffer (pH 7.3), and rinsed (NIYD) by dividingthe total area of a represen- in 0.1 M sodium cacodylate buffer. First legs tative cortex region into the total area occu- were removed at the trochanter and their pied by this region's myofibril bundles. We retrolateral and prolateral anterior dorsal de- then multiplied each cell's CEA by its NIYD pressor muscles (ADDM; Whitehead and to determine its corrected total myofibril sur- '59) Rempel, were exposed.We chose these face area (CMYA). When CMYA is divided by muscles becausetheir large size and position MA it yields an index of how densely a cell's MITOCHONDRIAL DENSITY IN SPIDER LEG MUSCLES 343

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Fig. 1. Cross section of the first leg anterior dorsal Fig. 2. Longitudinal section of the first leg anterior depressormuscle of Uloborus glomosus. CA, central clear dorsal depressor muscle of Uloborus glomosus. M, mito- area; M, mitochondria; MYO, myofibrils; N, nucleus; T, chondria; MYO, myofibrils. tracheole. 344 B.D. OPELL AND D.C. KONUR myofibrils are supplied by mitochondria. The M : Miagrammopes, lJ : Uloborus, and O : larger this index, the greater the cell's mito- Octonoba: chondrial supply. Hr : (H + M) + (H + (U + Ol2))+ (M + (U + o^12))16 This method assumes that the mitochon- Hz: (U + O)+ (U + (H + Mlz))+ (O+ (H +Ml2))16 dria of all species have similar shapes. If a species' mitochondria are greatly elongated He:Hr +Hrl2 in the longitudinal dimension (Fig. 2) this We inferred the resting force and tracheal method might underestimate that species' supply of the common ancestor of Hyptiotes mitochondrial surface area. We evaluated andMiagrarllrnopes from the mean values of each species'mitochondrial shapeby measur- Waitkera waitakerensis, an orb-weaver that ing the maximum and minimum dimensions is a primitive member of the outgroup of the of mitochondria in cross and longitudinal four species included in this study, and Ul- sectionsof muscle cells (Figs. 1, 2). We mea- oborus glornosus, the most primitive member sured either all of the mitochondria in a cell of the outgroup of the Hyptiotes-Miagram- (or, in the caseof longitudinal sections, all of nxopesclade (Coddington, '90). The values of the cell shown in a photograph) or a subset of U. glomoszs resting forces and first leg tra- mitochondria chosen by placing a numbered cheal supply are given in Table 1. Those for grid over a photograph and using a random W. waitakerensis are: relative resting forces, number table to select the mitochondria to be 1.93 x 10-a N/mma of leg I volume and measured. 5.58 x 10-5 N/mg ofprosomal and legweight; We determined the general shape of a spe- relative tracheal supply, 10,519 x 104pm3 of cies' mitochondria by comparing the ratio of leg I volume/p,mz of tracheal cross section/ its maximum to minimum mitochondrial di- square root ofdistance from tracheal spiracle mensions in cross section with that mea- to leg I basein pm (Opell, '87b,'90b,'92). sured in longitudinal section. If mitochon- dria are spherical then these ratios will be RESULTS equal. If mitochondria are elongated along the muscle's length then the ratio from longi- Results are presented in Table 2. Because tudinal sections will exceed that from cross data were not normally distributed for all sections. species, we used a Kruskal-Wallis k-sample The phylogenetic relationship of the gen- test to determine if there were interspecific era represented in this study is shown in differences in these values. There were no Figure 3. Hyptiotes and Miagramnxopes form significant differences (P > 0.145) in either a monophyletic group and Uloborus and Oc- the number of mitochondria per cell or the tonoba form a paraphyletic group (Codding- density of myofibrils in the cells' cortex. Mean '90). ton, We performed a transformational mitochondrial area differed significantly analysis of phylogenetic changes in the mito- among species,although one-tailed Wilcoxon chondrial supply of the ADDM by using the two-sample tests showed that the values of schemeof iterative averaging (Huey and Ben- Hyptiotes cauatus, Uloborus glomosus, and '87) nett, shown below, where H: Hyptiotes, Octonoba sinensis did not differ significantly (P > 0.333) but that Miagrammopes anirno- tushada smaller value than each of the other species(P < 0.033). Hyptiotes Miagrammopes Uloborus Octonoba Both total cell area and corrected myofibril 0.35 0.10 0.15 0.06 area per cell differed significantly between species, although there was no interspecific difference in corrected myofibril area per cell. As hypothesized, the ratio of mitochondrial area to corrected myofibril area differed sig- nificantly between species and differs in the direction predicted by the hypothesis. One- tailed Wilcoxon two-sample tests show that this ratio is significantly greater in 11. caua- tus thanin both U. glomosus (P : 0.013) and Fig. 3. Phylogenetic relationship of the four genera M. animotus (P: 0.007)and in U. glomosus represented in this study, with their mean mitochondrial : area to corrected myofibril area ratios and those for their than in O. sinensis (P 0.015).However, the hypothetical ancestors Hr-Ha. ratios do not differ significantly between ei- MITOCHONDRIAL DENSITY IN SPIDER LEG MUSCLES 345

TABLE 2. Comparison of mitochondrial densities in the first leg anterior dorsal depressor muscles of adult female Uloboridael Hyptiotes Uloborus Miagrammopes Octonoba Variable cavatus glomosus animotus sinensis P value No. specimens 6 4 5 4- No. cells per specimen 4.8 -r 0.4 3.8 * 1.0 5.2-+I.6 4.0-r 2.5 No. mitochondria per cell 48.9-r 23.I 37.3 -r 16.0 34.2 -r 17.6 19.1 -'-5.4 0.145 Area per mitochondria pm2 0.90 ! 0.22 1.00-+0.51 0.40 + 0.17 I.Ll-+ O.42 0.022 -+ Total cell area pmz 287.6 126.8 460.8-+ 132.2 367.4 -+ 79.4 634.3 + 149.6 0.013 Myofibril density in cortex 0.60 * 0.13 0.64 -r 0.06 0.69 -r 0.12 0.65 -f 0.06 0.545 -F Corrected myofibril area pm2 133.9 69.2 242.6-+62.8 236.8 * 86.4 350.6 -r 85.9 0.015 Corrected myofibril area/cell area 0.45-r 0.12 0.54 r 0.09 0.62 -r 0.15 0.55 + 0.07 0.253 -f Mitochondrial area/ corrected myo- 0.35 0.10 0.15 * 0.08 0.10-r0.11 0.06 * 0.02 0.006 fibril area Max. /min. mitochondria diameter In cross section 1.28 + 0.09 1.42-+0.16 1.21 -r 0.05 I.24-+ 0.lO 0.087 No. specimens 4 4 *-/ No. mitochondria per cell 20.0-+ 9.4 24.0 -r 10.6 10.0 -r 0.0 30.8 * 27.2 In longitudinal section 1.50+ 0.16 1.34-r0.I2 1.40 -f 0.19 I.32 -r 0.04 0.398 No. specimens 4 2 4 4- No. mitochondria per cell 26.3 -r 2.2 50.0 r- 0.0 12.5 -r 5.6 52.5-+22.3 rMean values are followed by standard deviations. P values are for Kruskal-Wallis k-sample tests of species effects on the variables.

ther U. glomosus andM. animotus (P: 0.196) for M. animotus to 0.11 -t 0.13. However, or M. animotus and O. sinensis (P : 0.357). these corrected values do not change the Kruskal-Wallis tests show no interspecific study's outcome. A Kurskal-Wallis test still differencesbetween the ratio of maximum-to- showed significant interspecific differences minimum mitochondrial dimensions in ei- (P : 0.006); H. cauatus still had a greater ther cross or longitudinal sections (Table 2). ratio than either U. glomosus (one-tailed When one-tailed Wilcoxon two-sample tests Wilcoxon test, P : 0.007) or M. animotus are used to compare the cross and longitudi- (P : 0.007); the ratios of U. glomosus and nal section ratios of each species,significant M. animotus do not differ significantly differencesare found inH. cauatus(P: 0.030) (P : 0.270): and the ratios of M. anitnotus and M. animotus (P : 0.019). but not in [/. and O. sinensis do not differ significantly glomosus (P: 0.409)orO. sinensis(P: 0.156). (P: 0.357). ln H. cauatus the longitudinal section ratio is The transformational analysis presented 1.17 times greater than the cross section in Figure 3 shows that the mitochondrial ratio and in M. animotus it is 1.16 times supply of the hypothetical ancestor of the greater. This indicates that in these species four speciesincluded in this study was most the mitochondria are not spherical but are similar to that of Uloborus.It also showsthat elongated along the myofibril axes and that this supply has increased in Hyptioles and mitochondrial cross sectional area underesti- decreased independently in Miagrammopes mates the total mitochondrial supply of their andOctonobo. Resting force and tracheal sup- muscle cells. ply values for the hypothetical ancestor of To account for this difference. we recalcu- Hyptiotes and Miagramn'Lopes are: relative lated mean total mitochondrial surface areas resting forces, 1.98 x 10-a N/mm3 of leg for H. cauatus andM. animotus muscle cells. volume and 5.60 x 10-5 N/mg of proso- We did this by treating each cell's total cross mal and leg weight; relative tracheal supply, sectional mitochondrial surface area as that 9,545 x 10a pm3lu,m2lpm. Like the mito- of a circle, determining its diameter, increas- chondrial density of this hypothetical ances- ing this diameter by 1.17 or 1.16, respec- tor (Fig. 3), these values are intermediate tively, determining the surface area of this between those of Hyptiotes and Miagram- larger circle, and then dividing this increased nlopes. mitochondrial surface area by the cell's cor- rected total myofibril area. DISCUSSION As a result of these recalculations, the mean The results of this study support the hy- mitochondrial area to corrected myofibril area pothesis that the mitochondrial supply of for H. cauatus increasedto 0.47 -t 0.15 and uloborid first leg muscles complements the 346 B.D. OPELL AND D.C. KONUR activity levels of this tissue. The density of LITERATURE CITED mitochondria is greatest in the first leg mus- Brosemer, R.W., W. Vogell, and T. Bucher (1963) Mor- cles of species that exert the most resting phologische und enzymatische Muster bei der Entwick- force and have the best developed tracheal lung indirekter Flugmuskeln von Locusta migratoria. Biochem. Z. 338:854-910. supplies to their first legs and is least in Chapman, G.B. (1954) Electron microscopyof ultra-thin speciesthat exert the least force and have the sections of insect flight muscles. J. Morphol. 95:237- poorest tracheal development. These conclu- 262. sions are not altered when the shape of each Chickering, A.M. (1968) The genus Miagrammopes (Ara- neae, Uloboridae) in Panama and the West Indies. species'mitochondria is taken into account. Mus. Comp . Zool. Breviora 28 9: l-28. These differences in mitochondrial density Coddington, J.A. (1986) The monophyletic origin of the may have been driven by changes in activity orb-web. In W.A. Shear (ed): Spiders: Webs, Behavior patterns and Evolution. Stanford: Stanford University Press, levels and tracheal or existing differ- pp.319-363. encesin mitochondrial densities and tracheal Coddington, J.A. (1990) Ontogeny and homology in the supplies may have permitted only certain male palus of orb-weaving spiders and their relatives, species to achieve greater levels of activity. with comments on phylogeny (Araneoclada: Arane- oidea,Deinopidea). Smithson. Contrib. Zool. 496:l-52. Support for the former hypothesis comes Copeland, E. (1964) A mitochondrial pump in the cells of from the contrasting mitochondrial densi- the anal papillae of mosquito larvae. J. Cell Biol.23:253- ties, resting forces, and tracheal supplies of 263. the sister genera Hyptiotes and Miagrdm- Cushing, P.E., and B.D. Opell (1990) Disturbance behav- (Tables iors in Uloborus glomosus (Araneae, Uloboridae): Pos- mopes L,2) and from the fact that the sible predator avoidance strategies. Can. J. Zool. 68: hypothetical ancestor of these genera ap- 1090-1097. pears to have had mitochondrial and tracheal Dean, R.L., M. Locke, and J.V. Collins (1985) Structure supplies that were intermediate for these of the fat body. In G.A. Kerkut and L.I. Gilbert (eds): Comprehensive Insect Physiolory, Biochemistry, and genera. Such a condition could not preadapt Pharmacolory. Vol. 3, Integument, Respiration, and this ancestor to the condition found in either Circulation. New York: Pergamon Press, pp. 155-210. extant genus. Instead, mitochondrial and tra- Edwards. G.A.. and H. Ruska (1955) The function and cheal supplies had to increase in Hyptiotes metabolism of certain insect muscles in relation to their structure. Q. FL Microsc. Sci.96;151-159. and decreasein Miagramnaopes to accommo- Huey, R.B., and A.F. Bennett (1987) Phylogeneticstud- date the diverging web-monitoring forces as- ies ofcoadaptation: Preferred temperatures versus op- sociated with changes in the web architec- timal performance temperatures in lizards. Evolution ture of these two genera. 4l:1098-1 115. Levenbook. L.. and C.M. Williams (1956) Mitochondria Mitochondrial supply is known to increase in the flight muscles of insects. III. Mitochondrial during development to meet the increased c;rbochrome c in relation to the aging and wing beat metabolic demands of insect flight muscles frequency of flies. J. Gen. Physiol. 39:497-512. (1986) (Brosemer et al., '63) and fat bodies (Sohal, Lubin, Y.D. Web building and prey capture in '73). Uloboridae. In W.A. Shear (ed): Spiders: Webs, Behav- However, to our knowledge, this is the ior, and Evolution. Stanford: Stanford University Press, first study to demonstrate a phylogenetic pp. 132-171. changein the mitochondrial density of homol- Lubin, Y.D., W.G. Eberhard, and G.G. Montgomery (1978) (Araneae: ogous arthropod muscles to accommodatethe Webs of Miagrammopes Uloboridae) in the Neotropics. Psyche 85:I-23. changing activity patterns of adults. A lim- Muma, M.H., and W.J. Gertsch (1964) The spider family ited phylogenetic analysis (FiS. 3) suggests Uloboridae in North America north of Mexico. Am. that mitochondrial density may be quite plas- Mus. Novit. 2196:143. (1979) tic. In the four genera of Uloboridae that we Opell, B.D. Revision of the genera and tropical American speciesof the spider family Uloboridae. Bull. studied, one showed an increase and two Mus. Comp . Zool. 148: 433-549. showed independent decreasesin mitochon- Opell, B.D. (1982) Post-hatching development and web drial density from the group's hypothetical production of Hyptiotes cuuatus (Hentz) (Araneae: Ul- oboridae).J. Arachnol. 1O;185-191. ancestor. Opell, B.D. (1987a) Changes in web-monitoring forces associated with web reduction rn the spider family ACKNOWLEDGMENTS Uloboridae. Can. J. Zool. 65: lO28-1034. Opell,B.D. (1987b)The influenceof web monitoring Joseph Uknalis prepared thin sections of iactics on the trachealsystems of spidersin the fami$ muscles. National SCienCeFoundation gTants Uloboridae(Arachnida, Araneida). Zoomorphology 107: BSR-840?979 and BSR-8917935 supiorted ^255-25e' fieldwork and provided some th"";il;;i"t' "i"1;i"";jt"t"?ffi'"Tf.ft:f:frU?T:1T"il:#Yffn and equipment used in this study."f Soiiobtoi.26:B7s-88r. MITOCHONDRIAL DENSITY IN SPIDER LEG MUSCLES 347

Opell, B.D. (1990b) The relationship of book lung and Sohal, R.S. (1973) Fine structural alterations with age in tracheal systems in the spider family Uloboridae. J. the fat body of the adult male housefly, Musca domes- Morphol. 206:2LI-216. tica. Z. Zellforsch. Mikrosk. Anat. 140:169-175. Opell, B.D. (1992) Web-monitoring force exerted by the Whitehead, W.F., and J.G. Rempel (1959) A study of the spider Waitkera waitakerensis. J. Arachnol. (in press). musculature of the black widow spider, Latrodectus Peaslee,J.E., and W.B. Peck (1983) The biolory of Oc- mactans (Fabr.). Can. J. Zool.37:83I-870. tonobq octonaria (Muma) (Araneae, Uloboridae). J. Wigglesworth, V.B., and M.M. Salpeter (1962) Histolory Arachnol. 11:51-67. of the malpighian tubules in Rhodnius prolixus Stol Smith, D.S. (1962) C1'tological studies of some insect (Hemiptera). J. Insect Physiol. 8:299-307. muscles. Rev. Can. Biol. 2 1:27 9-301. Yoshida, H. (1980) Six Japanese species of the genera Smith, D.S. (1968) Insect Cells: Their Structure and Oct o nob a and P hi lop one I I a (Ar aneae: Uloboridae). Acta Function. Edinburgh: Oliver and Boyd. Arachnol. 29:57-64.