the effect of remating on sperm number in the spermatophores of teleogryllus commodus (gryllidae)

The effect of remating on sperm number in the spermatophoresof Teleogryllus commodus (Gryllidae)

Robert Sturma

Brunnleitenweg 41, Salzburg, Austria

Abstract. Factors controlling sperm production in members of the Orthoptera have notbeen fully elucidated. In this study, the influence of intermating interval and ad libidummating on sperm number was investigated in black field crickets (Teleogryllus commodus).Remating at a variety of time intervals was not characterized by a significant change insperm number compared with the first mating. Ad libitum mating (i.e., continuous availabil-ity of unmated females) had two main effects on spermatophore production and spermnumber: first, there was a trend toward increased time between copulations with eachsuccessive remating, and second, the number of spermatozoa encapsulated in the transferredspermatophore declined after most rematings, with 61.8% of the initial sperm number beingproduced for the second spermatophore and 51.3% of the initial sperm number beingproduced for the third. The decrease in mean sperm number was accompanied by increasedvariance in sperm number in later rematings. This study suggests that males are willing tosuffer a decrease in sperm number if a mating opportunity occurs before the completion ofsperm production.

Additional key words: black field cricket, mating, nuptial gifts

Contrary to the limited number of eggs producedby females, the number of sperm produced by malesis remarkably high, which often results in an almostinfinite mating capacity of the male participants(Dewsbury 1982; Mann 1984; Reinhold & vonHelversen 1997). Although energetic investment ofmales in their gametes is relatively low with respectto that of females (Bateman 1948; Trivers 1972), thecosts which males incur in the production andpackaging of sperm may be manifest in several waysin repeated matings (Sakaluk 1985), includingextension of the intercopulatory interval, increase incopulation times, and decrease of ejaculate volumesprovided in subsequent matings. In many insects,the produced sperm mass is transferred to thefemale in a spermatophore that may consist of twoparts, the smaller sperm-containing ampulla and thelarger jelly–like spermatophylax (Greek: “spermguard”), which is devoid of sperm. In Lepidoptera,the quantity of sperm contained within subsequentspermatophores decreases if the male is presentedwith a female immediately after or within 24 h of aprevious mating (George & Howard 1968; Outram1971; Rutkowski 1979; Sims 1979); however, in

some male katydids (Orthoptera, Tettigoniidae)investment in successive copulations is held constantby increasing the time span between two spermato-phore transfers. In the acalypterate flies, Anastrephasuspensa LOEW 1862 and Drosophila melanogasterMEIGEN 1830, ejaculate volumes are influenced byintercopulatory intervals, which in turn are partlydetermined by female choice insofar as virginfemales prefer virgin males over freshly mated males(Markow et al. 1978; Sivinsiki 1984).

In the Orthoptera (and especially the crickets),those species providing a bipartite spermatophorecommonly remate after production of both the ejac-ulate and a large nuptial gift, the latter distractingthe female’s attention from the sperm during themating process. In some bushcrickets (e.g., Poecili-mon veluchianus RAMME 1933), the nuptial meal pro-duced by the male may amount to about 30% ofthe male’s weight, driving the energetic costs of mat-ing to high levels (Gwynne 1990; Simmons 1990;Simmons & Bailey 1990; Heller & Reinhold 1994).In exceptional cases, males of this species may alsoremate quickly, and deliver a spermatophore withreduced weight and fewer sperm. This, however,leads to the conclusion that available spermatophorematerial rather than available sperm numberaE-mail: [email protected]

Invertebrate Biology 130(4): 362–367.© 2011, The American Microscopical Society, Inc.DOI: 10.1111/j.1744-7410.2011.00240.x

influences the time point of spermatophore delivery(Reinhold & von Helversen 1997). Males of theblack field cricket Teleogryllus commodus (WALKER

1869) and other field crickets distributed all over theworld (e.g., Gryllus assimilis (FABRICIUS 1775), G.bimaculatus DE GEER 1773, Acheta domesticus(LINNAEUS 1758)) do not deliver a spermatophylaxas nuptial gift during the mating process, andinstead apply alternative strategies (guarding thefemales, supply of food from external sources) todistract females’ attention from the ampulla. Asreported in a previous study (Sturm 2003), thespermatophores produced by males of T. commodusare characterized by constant size from first to lastmating process. The number of sperm, on the otherhand, is subject to significant fluctuations amongthe collected spermatophores. Possible relationshipsbetween sperm production and intermating intervalhave not previously been investigated in detail.Likewise, the effect of ad libidum matings on inter-copulatory interval and sperm number is unknown.Descriptions of these relationships should help toanswer the essential question of whether males arewilling to suffer a decrease in the number of spermtransferred to females at mating if a mating oppor-tunity presents itself before the male can make acomplete recovery of his sperm supply.

Methods

Study animal and rearing conditions

The cricket, Teleogryllus commodus, is a medium-sized, hemimetabolous, exopterygote insect endemicto Australia and New Zealand that can be easilyreared under laboratory conditions. To exclude envi-ronmental effects on sperm quantity as far as possi-ble, crickets used for this study, especially allgenerations of the sub-adult life stage, were rearedunder identical conditions. Individuals were kept ata constant temperature of 25 ± 1°C, 60% relativehumidity, and on a 12 h light/12 h dark photope-riod (Sturm & Pohlhammer 2000; Sturm 2002).Nymphs were collected in plastic boxes(L 9 W 9 H: 50 9 40 9 30 cm) filled with a 2-cmlayer of dry peat soil and increased in surface by theaddition of egg cartons, and adults were separatedby gender and kept in 5-L glass vessels filled withwrinkled paper. Nymphs and adults were providedwith food ad libitum in the form of a special diet forlaboratory animals (Altromin® 1222, AltrominSpezialfutter GmbH & Co. KG, Lage, Germany),lettuce leaves, and water contained in small dishesplugged with cotton wicks.

Mating and spermatophore collection

An initial experiment was designed to investigateeffects of intermating interval on sperm number.Each male appropriate for the study (weight greaterthan average, adult for at least 8 d) was used twice,first in an initial mating and again for remating.After the first mating, with a virgin female, a freshvirgin female was offered to each male at a predeter-mined intermating interval (2, 4, 6, or 8 h, withn=20 males per treatment).

For the investigation of the effect of ad libidummating on sperm quantity, selected males (n=20)were placed together with virgin females in isolatedmating vessels, and mated females were replaced bynew virgin females of an equal size after eachspermatophore transfer. The process was observedover a time period of 12 h (light photoperiod),thereby counting the number of copulations andquantifying the sperm mass enclosed in each deliv-ered spermatophore.

Immediately after spermatophore transfer, thesperm-containing capsules were removed from thefemales using a pair of soft forceps. Subsequent totheir removal, spermatophores were submerged in10 mL of insect Ringer’s solution (Sturm &Pohlhammer 2000), liberated from the opaque masssurrounding the ampulla using a pair of modified,pincer-like forceps, and examined with a stereomi-croscope. The maximal diameter of the sperm mass(dsm) was measured for each spermatophore exam-ined.

Sperm counting

To obtain initial values of sperm number con-tained in the spermatophores, ten sperm capsules ofvirgin males of similar weights and ages (8 d aftereclosion) were subjected to an electron microscopicquantification procedure. To do this, spermato-phores isolated as described above were fixed in aparaformaldehyde–glutaraldehyde mixture (Karnov-sky 1965) for 3 h, washed in sodium-cacodylatebuffer, dehydrated in a graded series of ethanol, andfinally critical-point dried. After fixation, singlecapsules were sectioned with a razor blade, so thatthe sperm mass was cut in the middle (ideallythrough the flagella of the germ cells; Fig. 1a). Theventral halves of the spermatophores were coatedwith carbon, sputtered with gold, and then scannedwith a Cambridge-250 scanning electron microscope(Cambridge Instruments GmbH, Vienna, Austria) atacceleration voltages of 10–30 kV. To quantifysperm, a micrograph of the apical section of the

Invertebrate Biologyvol. 130, no. 4, December 2011

Sperm number in Teleogryllus commodus 363

sperm mass was covered with a 50950-mesh grid,and the numbers of germ cells within single gridunits were carefully counted (Fig. 1b). Based uponthe number of sperm per grid unit and the numberof grid units required to completely covering thesection of the sperm mass, the total number of cellscontained in the spermatophore was computed andcorrelated with the inner diameter of the capsule atthe section site.

For the following investigations, the number ofsperm stored within a single, unfixed spermatophore,Nsp, was determined according to the equation

Nsp¼dsm2=ðdsp2cÞ

where, dsm denotes the maximal diameter of thesperm mass measured with a stereomicroscope, anddsp the diameter of the sperm flagellum (c. 0.5 lm).To consider possible gaps between the spermatozoa,which may occur despite the dense packaging of thesperm mass, differential shrinking effects between“fresh” and dried spermatophores, and possiblefolding of the long flagella, a correction factor c wasdefined. Computation of c was carried out by firstusing the above formula without c, using the esti-mate of dsm made using light microscopy, and calcu-lating Nsp. After the spermatophore was fixed andhalved, Nsp was computed using scanning electronmicroscopy as described above. For each of the tenspermatophores, the factor c was calculated as thequotient between Nsp from the light microscopicmeasurement and Nsp derived from scanning elec-tron microscopy. Estimates of c ranged from 5.0 to6.3, with a mean of 5.7; the mean value was appliedin the equation above for estimating sperm numberin all other unfixed spermatophores examined.

Statistical analysis

Statistical analyses were conducted using Micro-soft EXCEL® (V. 2007) and SPSS® (V. 18).Theeffect of intermating interval length on the numberof sperm contained in the spermatophores wasexamined with one-way ANOVA. Post hoc pairwisecomparisons were conducted using a Student–Newman–Keuls multiple range test. The ad libitummating experiments were examined by repeated-measures ANOVA, with post hoc comparison ofmain effects by confidence interval adjustmentaccording to the Bonferroni correction.

Results

Effect of intermating interval on sperm number

Variation in sperm number per spermatophore wascorrelated with male weight: heavier males commonlyproduced a higher number of germ cells than lightermales (Fig. 2). The ampullae of virgin males with anadult age of 8 d contained 1.52 9 105 ± 3.2 9 104

spermatozoa (n=80; Fig. 3). Sperm number inspermatophores produced during the second matingwas not influenced significantly by the length of theintermating interval (df=159, F=2.03, p=0.093).

Effect of ad libidum mating on sperm number

When males were offered continual, ad libitumaccess to virgin females, the time between subsequent

a

b c

Fig. 1. Estimating sperm number in spermatophores ofTeleogryllus commodus. a. Cross section through theampulla (diameter da) of a spermatophore with its innermembrane (ima) and the sperm mass (sm). b. Schematicof a cross section through a spermatophore illustratingthe stereological technique that was applied for the esti-mation of sperm number. c. Schematic longitudinal sec-tion through the ampulla showing the relationshipbetween ampulla diameter (da), ampulla height (ha), diam-eter of sperm mass (dsm) and sperm number. da, ampulladiameter; dsm, diameter of sperm mass; ha, ampullaheight; ima, inner membrane of ampulla; sm, sperm mass.


364 Sturm

matings was affected by the number of previous mat-ings (Fig. 4: df=4,76, F=11.04, p<0.001). The inter-mating interval increased with number of previousmatings for the first few rematings. The intervalbetween the first and second mating was115 ± 12 min (n=20; Fig. 4), which presumably cor-responds to the time required for the production of anew spermatophore.

Remating also affected the number of spermdelivered in each spermatophore (Fig. 5: df=4,76,F=15.49, p<0.001). Post hoc tests showed significant

declines in spermatophore sperm content for mostmatings after the first (Fig. 5). In later matings (e.g.,matings 4 and 5), there was substantial variation insperm number, with some spermatophores contain-ing more than 105 sperm, but others being nearlyempty.

Discussion

Spermatophores of Teleogryllus commodus con-tained between 0.8 9 105 and 2 9 105 sperm cells,which is similar to that number measured in sper-matophores of other orthopterans (e.g., 1.6 9 105–2 9 105 in Gryllodes supplicans (WALKER 1859): Sak-aluk 1984; 2 9 105 in Kawanaphila nartee RENTZ

1993: Simmons & Gwynne 1991; 8 9 105–20 9 105

in Requena verticalis (WALKER 1869): Gwynne 1986;Simmons et al. 1993; and 63–105 9 105 in Poecili-mon veluchianus: Reinhold 1994; Reinhold & vonHelversen 1997). Interspecific variability in spermnumber has been suggested to correlate with varia-tion in the typical time period between matingsrather than with the body size of the investigatedspecies (Reinhold & Heller 1993). Regarding free-living individuals of T. commodus, the intervalbetween two copulations is on the order of severalhours (Loher & Rence 1978). A decrease of thisinterval for males given unlimited access to virginfemales results, however, in fewer sperm being

Fig. 2. Relationship between the number of spermatozoaper produced spermatophore and the weight of males ofTeleogryllus commodus. By linear regression analysis,sperm number and weight (mg) are related as follows:sperm number=243.9 9 weight�30,086 (n=20, p<0.001).

Fig. 3. Graph illustrating the relationship between spermnumber delivered by males of Teleogryllus commodus(mean ± standard deviation) and intermating interval. Atime interval of 0 h corresponds to the first mating.Sperm number is not significantly related to the timeinterval between copulations (p>0.05). For each timeinterval, n=20.

Fig. 4. Effect of ad libidum mating on the length of theintermating period (mean ± standard deviation) in malesof Teleogryllus commodus. For each mating event, n=20males. “1-2” denotes the time span between first and sec-ond mating, “2-3” the time span between second andthird mating, etc. Brackets indicate significant differencesbetween mean values (*p<0.05, **p<0.001).



transferred into the spermatophore in subsequentmatings.

In males of T. commodus, the number of spermenclosed in a spermatophore is positively correlatedwith body size (measured as weight: Fig. 2). Thisresult seems to be plausible insofar as body size maypositively correlate with the dimensions of internalorgans, rather than reflecting only variation in thefatty tissue content. On the other hand, male crick-ets seem to have the ability to modify sperm numberin response to both intraspecific competition andfemale size (Gage & Barnard 1996). Although bothfactors were controlled as much as possible duringthe experiments reported on here (mating betweenone male and one female, selection of equally sizedfemales), the sperm number-size correlation foundhere requires further investigation to be either sup-ported or rejected. However, according to the find-ings presented here, males of T. commodus exhibit adifferent pattern than males of several bushcricketspecies (e.g., P. veluchianus, R. verticalis), wheresperm number delivered by the male during matingis independent of male’s body size (Simmons et al.1993; Reinhold & von Helversen 1997).

A primary result presented in this study is thatthe number of spermatozoa per spermatophore isnot influenced by the time interval between copula-tions (Fig. 3). Any intermating interval � 2 henables the males to produce the initial number ofgerm cells. Generation of a new sperm capsule isstarted immediately after copulation (Spann 1934;

Hall et al. 2000; Sturm 2003), and sperm supply ofthe papilla (the apical compartment of the ampulla)is finished within 20–30 min. Hence, under theconditions of this experiment, the content of a sper-matophore was determined well before the male wasallowed to copulate again. As there is no relation-ship between intermating interval and the number ofsperm transferred, it seems unlikely that the numberof sperm available controls the timing of remating.This is similar to what has been found in othercricket species, especially those producing bipartitespermatophores (Reinhold & von Helversen 1997).

An interesting question concerns the influence ofunlimited mating, i.e., the subjection of males topermanent mating stress, on both sperm numbersproduced for each copulation, and the lengths ofintermating intervals. As found here for T. commo-dus, the number of spermatozoa delivered declinesafter the initial mating (Fig. 5), as does the intercop-ulatory period (Fig. 4). Although comparable stud-ies are rare, it is well-known that males of somecricket species may mate up to 12 times a day(Spann 1934), if intraspecific competition and avail-ability of females are within optimal ranges. Formales of T. commodus, an average number of fourmatings per day has been observed under naturalconditions (Loher & Rence 1978), suggesting aphase of retreat and rest after each copulation. Ifthis important phase is not available to the animals,sperm and spermatophore production may be nega-tively affected.

The results of this study suggest that the manu-facture of ejaculates is costly, and that males requirea minimum recovery period of about 2 h to producespermatophores that contain the full sperm comple-ment. The results also suggest that males are willingto suffer a decrease in the number of sperm they areable to transfer to females at mating if a matingopportunity presents itself before the male can makea complete recovery. Hence, in high-density popula-tions, male crickets may often face a tradeoffbetween maximizing their number of mating oppor-tunities and maximizing their fertilization successfrom individual matings.

References

Bateman AJ 1948. Intra-sexual selection in Drosophila.Heredity 2: 349–368.

Dewsbury DA 1982. Ejaculate cost and male choice. Am.Nat. 119: 601–610.

Gage AR & Barnard CJ 1996. Male crickets increasesperm number in relation to competition and femalesize. Behav. Ecol. Sociobiol. 38: 349–353.

Fig. 5. Effect of ad libidum mating on the number ofspermatozoa delivered by males of Teleogryllus commo-dus. “1” refers to the first mating, “2” to the second, etc.Brackets indicate significant differences between meanvalues (*p<0.05, **p<0.001).


366 Sturm

George JA & Howard MG 1968. Insemination withoutspermatophore in the Oriental fruit moth, Grapholithamolesta (Lepidoptera: Tortricidae). Can. Entomol. 100:190–192.

Gwynne DT 1986. Courtship feeding in katydids (Orthop-tera: Tettigoniidae): investment in offspring or inobtaining fertilizations. Am. Nat. 128: 342–352.

Gwynne DT 1990. Testing parental investment and thecontrol of sexual selection in katydids: the operationalsex ratio. Am. Nat. 136: 474–484.

Hall MD, Beck R, & Greenwood M 2000. Detailed devel-opmental morphology of the spermatophore of theMediterranean field cricket, Gryllus bimaculatus (DE

GEER) (Orthoptera: Gryllidae). Arthropod Struc. Dev.29: 23–32.

Heller KG & Reinhold K 1994. Mating effort function ofthe spermatophore in the bushcricket Poecilimon velu-chianus (Orthoptera, Phaneropteridae): support fromthe comparison of the mating behaviour of two subspe-cies. Biol. J. Linnean Soc. 53: 153–163.

Karnovsky MJ 1965. A formaldehyde-glutaraldehyde fix-ative of high osmolality for use in electron microscopy.J. Cell Biol. 27: 137A–138A.

Loher W & Rence B 1978. The mating behaviour of Tele-ogryllus commodus (WALKER) and its central andperipheral control. Z. Tierpsychol. 46: 225–259.

Mann T 1984. Spermatophore. Development, Structure,Biochemical Attributes and Role in the Transfer ofSpermatozoa. Springer, Berlin, Heidelberg, New York,Tokyo. 224 pp.

Markow TA, Quaid M, & Kerr S 1978. Male matingexperience and competitive courtship success in Dro-sophila melanogaster. Nature 276: 821–822.

Outram I 1971. Aspects of mating in the sprucebudworm, Choristoneura fumiferana (Lepidoptera: Tor-tricidae). Can. Entomol. 103: 1121–1128.

Reinhold K 1994. Inheritance of body and testis size inthe bushcricket Poecilimon veluchianus RAMME (Orthop-tera: Tettigoniidae) examined by means of subspecieshybrids. Biol. J. Linn. Soc. 52: 305–316.

Reinhold K & Heller KG 1993. The ultimate function ofnuptial feeding in the bushcricket Poecilimon veluchi-anus (Orthoptera: Tettigoniidae: Phaneropterinae).Behav. Ecol. Sociobiol. 32: 55–60.

Reinhold K & von Helversen D 1997. Sperm number,spermatophore weight and remating in the bushcricketPoecilimon veluchianus. Ethology 103: 12–18.

Rutkowski RL 1979. The butterfly as an honest salesman.Anim. Behav. 27: 1269–1270.

Sakaluk SK 1984. Male crickets feed females to ensurecomplete sperm transfer. Science 223: 609–610.

Sakaluk SK 1985. Spermatophore size and its role in thereproductive behaviour of the cricket, Gryllodes suppli-cans (Orthoptera: Gryllidae). Can. J. Zool. 63: 1652–1656.

Simmons LW 1990. Nuptial feeding in tettigoniids: malecosts and the rates of fecundity increase. Behav. Evol.Sociobiol. 27: 43–47.

Simmons LW & Bailey WJ 1990. Resource influenced sexroles of zaphrochiline tettigoniids (Orthoptera: Tettigo-niidae). Evolution 44: 1853–1868.

Simmons LW & Gwynne DT 1991. The refractory periodof female katydids (Orthoptera: Tettigoniidae): sexualconflict over the remating internal. Behav. Ecol. 2:276–282.

Simmons LW, Craig M, Llorens T, Schinzig M, & Hos-ken D 1993. Bushcricket spermatophore vary in accordwith sperm competition and parental investmenttheory. Proc. R. Soc. Lond. B 251: 183–186.

Sims SR 1979. Aspects of mating frequency and repro-ductive maturity in Papilio zelicaon. Am. Midl. Nat.102: 36–50.

Sivinsiki J 1984. Effect of sexual experience on malemating success in a lek forming tephritid Anastrephasuspensa (Loew). Fla. Entomol. 67: 126–130.

Spann L 1934. Studies on the reproductive system ofGryllus assimilis FABRICIUS. Trans. Kansas Acad. Sci.66: 299–339.

Sturm R 2002. Development of the accessory glands inthe genital tract of female Teleogryllus commodusWALKER (Insecta, Orthoptera). Arthropod Struc. Dev.31: 231–241.

Sturm R 2003. The spermatophore of the black fieldcricket Teleogryllus commodus (Insecta: Orthoptera:Gryllidae): size, structure and formation. Ent. Abh. 61:227–232.

Sturm R & Pohlhammer K 2000. Morphology and devel-opment of the female accessory sex glands in thecricket Teleogryllus commodus (Saltatoria: Ensifera:Gryllidae). Inv. Reprod. Dev. 38: 13–21.

Trivers RL 1972. Parental investment and sexual selection.In: Sexual Selection and the Descent of Man. CampbellB, ed., pp. 136–179. Aldine/Atherton Inc., Chicago.



the effect of remating on sperm number in the spermatophores of teleogryllus commodus (gryllidae)

Documents