Influence of Web-Monitoring Tactics on the Density ofMitochondria in Leg Muscles of the Spider Family Uloboridae

BRENT D. OPELL ANo DAVID C, KONUR

Er:ff:w, {,i;: l:y; [; ;ri n i a P o t v t e c h n i c I n s t i t u t e q n d s t at e (r n iu e r s i tv,

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

JOURNAL OF MORPHOLOGY 2t3:34t-347 (1992)

single monitoring line and shake the webwhen a prey strikes it (Opell, '87a).In bothorb-web and reduced-web spiders, the firstlegs play an important role in web monitor-ing and manipulation.

As summarized in Table 1, previous stud-ies show that these differences in web-moni-toring tactics are reflected in both the web-monitoring force expressed by a species andthe degree of its tracheal development (Opell,'87a,b). Hyptiotes cauatus has the greatestabsolute and relative resting forces (forceexerted on a single, resting thread) and alsothe greatest tracheal supply to its first legs(i.e., the smallest volume of tissue served byeach pm2 of tracheal cross section). Uloboru,sglomosus has the next greatest resting forceand the next greatest first leg tracheal sup-ply, followed by Miagrammopes animotus.The tracheal spiracle of uloborids is near theabdomen's posterior tip and the long abdo-men of M. animolzs reduces the first leg'soxygen supply by increasing tracheal lengthand, thereby, the distance over which oxygenmust diffuse. The resting force expressed byOctonoba sinensis has not been measuredand attempts to do so for this study provedunsuccessful, as spiders hung for only a fewseconds on resting threads and failed to estab-lish stable resting forces. However, as thisspecies' tracheal system is restricted to itsabdomen (Opell, '90b) and its prosomal andlegmuscles are supplied solelyby hemolymphborne oxygen, it probably exerts less monitor-ing force than the other three species.

Mitochondria are the sites of oxidativephosphorylation and are found in greaternumbers in more active tissues. For example,in insects mitochondrial densitv is low insmall visceral muscles and inactive fat cells(Dean et al., '85; Smith, '68; Sohal, 'ZB) andhigh in metabolically active tissues such asflight muscles (Chapman,'54; Edwards andRuska, '55; Levenbook and Williams, '56;

Smith, '62), Malpighian tubules (Smith, '68;

Wigglesworth and Salpeter, '62), and analpapillae (Copeland,'64). The purpose of thisstudy is to determine if changes in mitochon-drial density accompany diverging activitylevels in homologous arthropod muscles. Wedo this by comparing the mitochondrial sup-plies of the leg muscles of spiders that havedifferent web-monitoring tactics.

The orb-web is the primitive web form inthe family Uloboridae (Coddington, '86, 'g0;

Opell, '79). Members of orb-weaving generasuch as Uloborus and Octonoba hang be-neath the hubs of their horizontal webs whilewaiting for prey (Lubin, '86; Cushing andOpell, '90; Peaslee and Peck, '88). In con-trast, species that construct reduced websmore actively monitor and manipulate themduring prey capture. Members of the genusHyptiotes spin taut, vertical triangle-webswtrich they monitor from the anchoring pointof the web's apex thread (Lubin, '86; Opell,'82). Members of the genus MiagratnnLopesconstruct irregular webs that have one toeight prey capture lines that diverge at vary-ingangles (Lubin, '86;Lubin et al., '78; Opell,'90a). Like Hyptiotes, these spiders uJe a

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 Uloboruscavatus glomosus

Miagrammopes Octonobaanimotus sinensis

Leg I volume x 104 pm3 1 27,438Prosomal and leg weight mg3 L.49Rest ingforceN x 10-52 13.42Resting forcelleg I volume x 10 a/mm3 4.89Resting force/prosomal and leg weight 9.01Tracheal area serving leg I pm2 t 387Leg I volume/leg I tracheal arealsquare root distance 3,239

from spiracle to leg I1

48,8221 . 9 1

r0.722.025 .61

2898,565

104,1882 .387.090.682.89

8917 ,180

g.as

n

0

rOpe l l , ' 87b .2Opell , '87a.3Opell , '90.

Because the first legs are important in webmonitoring, the activity level of their mus-cles, as predicted by a species' resting forceand tracheal supply, should be reflected intheir mitochondrial supply. This study willtest the hypothesis that the density of mito-chondria in first leg muscles is ordered, fromgTeatest to least, as follows: Hyptiotes caua-tus, Uloborus glomosus, Miagrammopes ani-motus, Octonoba sinensis.

MATERIAIS AND METHODS

Adult females of the following species wereused in this investigation: the introducedAsian orb-weaver, Octonoba sinensis (Si-mon), collected from free ranging popula-tions in greenhouses at Virginia PolytechnicInstitute and State University (VPISU); theeastern North American orb-weaver, Ul-oborus glomosus (Walckenaer), collected fromshrubbery on the VPISU campus; the east-ern North American triangle-web-weaver,Hyptiotes cauatus (Hentz), collected in theforests of Montgomery and Craig Counties ofVirginia; and the Puerto Rican "single-line-weaver," Miagramm,opes animotus Chicker-ing, '68, collected at the Center for Energyand Environment Research's El Verde fieldstation in the Luquillo National Forest. Thesespecies were identified by reference to thetaxonomic revisions of Chickering ('68),Muma and Gertsch ('64), Opell ('79), andYoshida ('80).

Specimens were anesthetized with carbondioxide, fixed at 20-26"C for 12-18 hr in 3Voglutaraldehydel}o/o formaldehyde in 0.1 Msodium cacodylate buffer (pH 7.3), and rinsedin 0.1 M sodium cacodylate buffer. First legswere removed at the trochanter and theirretrolateral and prolateral anterior dorsal de-pressor muscles (ADDM; Whitehead andRempel, '59) were exposed. We chose thesemuscles because their large size and position

make them easy to identify, isolate, and han-dle and because their role as flexors of thepatella is important for the leg movementsassociated with web monitoring and manipu-lation.

The ADDM were post-fixed for 30 min in17o osmium tetroxide, washed with sodiumcacodylate buffer for 5 min, and dehydratedthrough a series of ethanol dilutions. Mus-cles were then infiltrated with Spurr's epoxyresin (hard formula) and polymerized for 24hr at 65'C. Thin cross and longitudinal sec-tions were stained with lead citrate and ura-nyl acetate and examined and photographedat x 1,600-3,300 with a transmission elec-tron microscope.

Enlarged cross sectional photographs ofindividual muscle cells (Fig. 1) were mea-sured with a digitizing tablet connected to acomputer. We first determined the cell's totalsurface area (TA), the combined surface ar-eas of its mitochondria (MA), the surfacearea of its central clear region where a nu-cleus was often found (CA), and the com-bined surface areas of the mitochondria foundwithin the central clear region (CMA). Weused the following formula to determine thearea occupied by a cell's contractile elements(CEA):

CEA: TA - CA - (MA - CMA)

However, in addition to myofibril bundles,this cortex region (: CEA) also contains sar-coplasmic reticula, T-system tubules, and in-tracellular space (Figs. L, 2). We determinedthe density of myofibrils within the CEA(NIYD) by dividingthe total area of a represen-tative cortex region into the total area occu-pied by this region's myofibril bundles. Wethen multiplied each cell's CEA by its NIYDto determine its corrected total myofibril sur-face area (CMYA). When CMYA is divided byMA it yields an index of how densely a cell's

MITOCHONDRIAL DENSITY IN SPIDER LEG MUSCLES 343

.#-w''i:t&i*F',;

Fig. 1. Cross section of the first leg anterior dorsaldepressor muscle of Uloborus glomosus. CA, central cleararea; M, mitochondria; MYO, myofibrils; N, nucleus; T,tracheole.

Fig. 2. Longitudinal section of the first leg anteriordorsal depressor muscle of Uloborus glomosus. M, mito-chondria; MYO, myofibrils.

344 B.D. OPELL AND D.C. KONUR

myofibrils are supplied by mitochondria. Thelarger this index, the greater the cell's mito-chondrial supply.

This method assumes that the mitochon-dria of all species have similar shapes. If aspecies' mitochondria are greatly elongatedin the longitudinal dimension (Fig. 2) thismethod might underestimate that species'mitochondrial surface area. We evaluatedeach species' mitochondrial shape by measur-ing the maximum and minimum dimensionsof mitochondria in cross and longitudinalsections of muscle cells (Figs. 1, 2). We mea-sured either all of the mitochondria in a cell(or, in the case of longitudinal sections, all ofthe cell shown in a photograph) or a subset ofmitochondria chosen by placing a numberedgrid over a photograph and using a randomnumber table to select the mitochondria to bemeasured.

We determined the general shape of a spe-cies' mitochondria by comparing the ratio ofits maximum to minimum mitochondrial di-mensions in cross section with that mea-sured in longitudinal section. If mitochon-dria are spherical then these ratios will beequal. If mitochondria are elongated alongthe muscle's length then the ratio from longi-tudinal sections will exceed that from crosssections.

The phylogenetic relationship of the gen-era represented in this study is shown inFigure 3. Hyptiotes and Miagramnxopes forma monophyletic group and Uloborus and Oc-tonoba form a paraphyletic group (Codding-ton, '90). We performed a transformationalanalysis of phylogenetic changes in the mito-chondrial supply of the ADDM by using thescheme of iterative averaging (Huey and Ben-nett, '87) shown below, where H: Hyptiotes,

Hyptiotes Miagrammopes Uloborus Octonoba

0 . 3 5 0 . 1 0 0 . 1 5 0 . 0 6

Fig. 3. Phylogenetic relationship of the four generarepresented in this study, with their mean mitochondrialarea to corrected myofibril area ratios and those for theirhypothetical ancestors Hr-Ha.

M : Miagrammopes, lJ : Uloborus, and O :Octonoba:

Hr : (H + M) + (H + (U + Ol2)) + (M + (U + o^12))16Hz : (U + O) + (U + (H + Mlz)) + (O + (H +Ml2))16H e : H r + H r l 2

We inferred the resting force and trachealsupply of the common ancestor of HyptiotesandMiagrarllrnopes from the mean values ofWaitkera waitakerensis, an orb-weaver thatis a primitive member of the outgroup of thefour species included in this study, and Ul-oborus glornosus, the most primitive memberof the outgroup of the Hyptiotes-Miagram-nxopes clade (Coddington, '90). The values ofU. glomoszs resting forces and first leg tra-cheal supply are given in Table 1. Those forW. waitakerensis are: relative resting forces,1.93 x 10-a N/mma of leg I volume and5.58 x 10-5 N/mg ofprosomal and legweight;relative tracheal supply, 10,519 x 104 pm3 ofleg I volume/p,mz of tracheal cross section/square root ofdistance from tracheal spiracleto leg I base in pm (Opell, '87b,'90b,'92).

RESULTS

Results are presented in Table 2. Becausedata were not normally distributed for allspecies, we used a Kruskal-Wallis k-sampletest to determine if there were interspecificdifferences in these values. There were nosignificant differences (P > 0.145) in eitherthe number of mitochondria per cell or thedensity of myofibrils in the cells' cortex. Meanmitochondrial area differed significantlyamong species, although one-tailed Wilcoxontwo-sample tests showed that the values ofHyptiotes cauatus, Uloborus glomosus, andOctonoba sinensis did not differ significantly(P > 0.333) but that Miagrammopes anirno-tushada smaller value than each of the otherspecies (P < 0.033).

Both total cell area and corrected myofibrilarea per cell differed significantly betweenspecies, although there was no interspecificdifference in corrected myofibril area per cell.As hypothesized, the ratio of mitochondrialarea to corrected myofibril area differed sig-nificantly between species and differs in thedirection predicted by the hypothesis. One-tailed Wilcoxon two-sample tests show thatthis ratio is significantly greater in 11. caua-tus thanin both U. glomosus (P : 0.013) andM. animotus (P: 0.007) and in U. glomosusthan in O. sinensis (P : 0.015). However, theratios 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 ofadult female Uloboridael

VariableHyptiotescavatus

Uloborusglomosus

Miagrammopesanimotus

Octonobasinensis P value

No. specimensNo. cells per specimenNo. mitochondria per cellArea per mitochondria pm2Total cell area pmzMyofibril density in cortexCorrected myofibril area pm2Corrected myofibril area/cell areaMitochondrial area/ corrected myo-

fibril areaMax. /min. mitochondria diameter

In cross sectionNo. specimensNo. mitochondria per cell

In longitudinal sectionNo. specimensNo. mitochondria per cell

64.8 -r 0.4

48.9-r 23. I0.90 ! 0.22

287.6 -+ 126.80 .60 * 0 .13

133.9 -F 69.20 . 4 5 - r 0 . 1 20.35 - f 0.10

1.28 + 0.094

20.0-+ 9.41 .50 + 0 .16

426.3 -r 2.2

43 .8 * 1 .0

37.3 -r 16.01 . 0 0 - + 0 . 5 1

460.8-+ 132.20.64 -r 0.06

242 .6 -+ 62 .80.54 r 0.090 .15 * 0 .08

1 . 4 2 - + 0 . 1 64

24.0 -r 10.61 . 3 4 - r 0 . I 2

250.0 r- 0.0

55 . 2 - + I . 6

34.2 -r 17.60 .40 + 0 .17

367.4 -+ 79.40.69 -r 0.12

236.8 * 86.40.62 -r 0.150 . 1 0 - r 0 . 1 1

1.21 -r 0.05

10.0 -r 0.01.40 - f 0.19

412.5 -r 5.6

4 -4 . 0 - r 2 . 5

19.1 - ' - 5.4 0.145I .L l -+ O.42 0.022

634.3 + 149.6 0.0130.65 - f 0.06 0.545

350.6 -r 85.9 0.0150.55 + 0.07 0.2530.06 * 0.02 0.006

I .24 -+ 0 . lO 0 .087/* -30.8 * 27.2I .32 -r 0.04 0.398

4 -5 2 . 5 - + 2 2 . 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)or M. animotus and O. sinensis (P : 0.357).

Kruskal-Wallis tests show no interspecificdifferences between the ratio of maximum-to-minimum mitochondrial dimensions in ei-ther cross or longitudinal sections (Table 2).When one-tailed Wilcoxon two-sample testsare used to compare the cross and longitudi-nal section ratios of each species, significantdifferences are found inH. cauatus (P: 0.030)and M. animotus (P : 0.019). but not in [/.glomosus (P: 0.409) orO. sinensis (P: 0.156).ln H. cauatus the longitudinal section ratio is1.17 times greater than the cross sectionratio and in M. animotus it is 1.16 timesgreater. This indicates that in these speciesthe mitochondria are not spherical but areelongated along the myofibril axes and thatmitochondrial cross sectional area underesti-mates the total mitochondrial supply of theirmuscle cells.

To account for this difference. we recalcu-lated mean total mitochondrial surface areasfor H. cauatus andM. animotus muscle cells.We did this by treating each cell's total crosssectional mitochondrial surface area as thatof a circle, determining its diameter, increas-ing this diameter by 1.17 or 1.16, respec-tively, determining the surface area of thislarger circle, and then dividing this increasedmitochondrial surface area by the cell's cor-rected total myofibril area.

As a result of these recalculations, the meanmitochondrial area to corrected myofibril areafor H. cauatus increased to 0.47 -t 0.15 and

for M. animotus to 0.11 -t 0.13. However,these corrected values do not change thestudy's outcome. A Kurskal-Wallis test stillshowed significant interspecific differences(P : 0.006); H. cauatus still had a greaterratio than either U. glomosus (one-tailedWilcoxon test, P : 0.007) or M. animotus(P : 0.007); the ratios of U. glomosus andM. animotus do not differ significantly(P : 0.270): and the ratios of M. anitnotusand O. sinensis do not differ significantly( P : 0 . 3 5 7 ) .

The transformational analysis presentedin Figure 3 shows that the mitochondrialsupply of the hypothetical ancestor of thefour species included in this study was mostsimilar to that of Uloborus.It also shows thatthis supply has increased in Hyptioles anddecreased independently in MiagrammopesandOctonobo. Resting force and tracheal sup-ply values for the hypothetical ancestor ofHyptiotes and Miagramn'Lopes are: relativeresting forces, 1.98 x 10-a N/mm3 of legvolume and 5.60 x 10-5 N/mg of proso-mal and leg weight; relative tracheal supply,9,545 x 10a pm3lu,m2lpm. Like the mito-chondrial density of this hypothetical ances-tor (Fig. 3), these values are intermediatebetween those of Hyptiotes and Miagram-nlopes.

DISCUSSION

The results of this study support the hy-pothesis that the mitochondrial supply ofuloborid first leg muscles complements the

346 B.D. OPELL AND D.C. KONUR

activity levels of this tissue. The density ofmitochondria is greatest in the first leg mus-cles of species that exert the most restingforce and have the best developed trachealsupplies to their first legs and is least inspecies that exert the least force and have thepoorest tracheal development. These conclu-sions are not altered when the shape of eachspecies' mitochondria is taken into account.

These differences in mitochondrial densitymay have been driven by changes in activitylevels and tracheal patterns or existing differ-ences in mitochondrial densities and trachealsupplies may have permitted only certainspecies to achieve greater levels of activity.Support for the former hypothesis comesfrom the contrasting mitochondrial densi-ties, resting forces, and tracheal supplies ofthe sister genera Hyptiotes and Miagrdm-mopes (Tables L,2) and from the fact that thehypothetical ancestor of these genera ap-pears to have had mitochondrial and trachealsupplies that were intermediate for thesegenera. Such a condition could not preadaptthis ancestor to the condition found in eitherextant genus. Instead, mitochondrial and tra-cheal supplies had to increase in Hyptiotesand decrease in Miagramnaopes to accommo-date the diverging web-monitoring forces as-sociated with changes in the web architec-ture of these two genera.

Mitochondrial supply is known to increaseduring development to meet the increasedmetabolic demands of insect flight muscles(Brosemer et al., '63) and fat bodies (Sohal,'73). However, to our knowledge, this is thefirst study to demonstrate a phylogeneticchange in the mitochondrial density of homol-ogous arthropod muscles to accommodate thechanging activity patterns of adults. A lim-ited phylogenetic analysis (FiS. 3) suggeststhat mitochondrial density may be quite plas-tic. In the four genera of Uloboridae that westudied, one showed an increase and twoshowed independent decreases in mitochon-drial density from the group's hypotheticalancestor.

ACKNOWLEDGMENTS

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