Appl. Entomol. Zool. 42 (2): 241–246 (2007) http://odokon.org/

Size, hatching rate, and hatching period of sexually and asexually produced eggs in the facultatively parthenogenetic speratus (Isoptera: )

Kenji MATSUURA* and Norimasa KOBAYASHI Laboratory of Ecology, Graduate School of Environmental Science, Okayama University; Okayama 700–8530, Japan (Received 20 June 2006; Accepted 7 December 2006)

Abstract Facultative parthenogenesis, or condition-dependent alternation of sexual and , is widespread in . Parthenogenesis enables unmated females to reproduce and thus has a great adaptive significance, especially under low pairing efficiency. In the termite Reticulitermes speratus Kolbe, females that fail to pair with males found colonies cooperatively with partner females and reproduce parthenogenetically. We compared the quality of partheno- genetic and sexual eggs in terms of size, hatching rate, and hatching period. We developed a method to culture iso- lated eggs until hatching under sterile conditions and in appropriate humidity. We successfully isolated, sterilized, and maintained the eggs on agar plates containing 200 ppm tetracycline. Females of female-female (FF) pairs began to lay eggs at the same time as those of female-male (FM) pairs. Nevertheless, the parthenogenetic eggs were significantly larger than sexual eggs. While the two types of eggs had similar hatching rates, parthenogenetic eggs had longer hatching periods (36.360.16 [SE] days) than sexual eggs (34.950.12 SE). We conclude that primary queens invest more resources into each parthenogenetic egg than each sexual egg, and that parthenogenetic eggs are as viable as sexual eggs.

Key words: Colony foundation; social ; automixis; inbreeding depression; developmental disadvantage

found colonies cooperatively with partner females, INTRODUCTION and reproduce by parthenogenesis (Matsuura and Facultative parthenogenesis enables females Nishida, 2001; Matsuura et al., 2002). The cooper- to produce offspring by themselves, preventing ation between two females promotes their survival reproductive failure when they do not find a mate rates, much like monogamous foundation, whereas before dying (Cuellar, 1977). Therefore, facultative single females rarely survive due to extremely high parthenogenesis is likely to be advantageous in cer- pathogenic infection rates (Matsuura and Nishida, tain situations, even though it may be ultimately in- 2001; Matsuura et al., 2002). A grooming partner ferior to sexual reproduction in terms of long-term is essential to survival during colony foundation fitness. Recently, the importance of studying facul- because unlike ants, can only clean their tative parthenogenesis has been recognized as a antennae during self-grooming (Matsuura et al., means to investigate the evolution and maintenance 2002). In asexual colonies of R. speratus, diploid of sex because it is possible to directly compare female progeny produced via thelytokous partheno- individuals with the capacity to reproduce both genesis develop in the same way as sexually pro- sexually and asexually (e.g., Corley and Moore, duced offspring. According to Matsuura et al. 1999; Corley et al., 2001; Matsuura and Nishida, (2004), two facultatively parthenogenetic species, 2001; Ball, 2002). R. speratus and R. virginicus, develop mature Facultative parthenogenesis has important adap- female-only parthenogenetic colonies in the wild. tive significance in Reticulitermes termites. In R. In the parthenogenesis of Reticulitermes ter- speratus Kolbe, females that fail to pair with males mites, ploidy restoration occurs via terminal fu-

*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2007.241

241 242 K. MATSUURA and N. KOBAYASHI sion, in which two haploid pronuclei that divide were divided into two groups according to sex and during meiosis II, fuse (Matsuura et al., 2004). maintained in Petri dishes containing moist filter Parthenogenesis involves both genetic and develop- paper until they shed their wings. Then, individual mental constraints, and therefore, the survival rates dealates were randomly chosen from each colony of parthenogens (individuals produced by partheno- and assigned to either FM or FF pairs. FM pairs in- genesis) are lower than those of sexually produced cluded a female and male from the same colony offspring (Lamb and Willey, 1979; Corley et al., (FAMA and FBMB), or a female and male from dif- 1999). Such constraints could arise from ploidy ferent colonies (FAMB and FBMA). Similarly, FF differences or mutation accumulation (Kondrashov, pairs included two females from the same colony

1988) or a reduction in heterozygosity associated (FAFA and FBFB), or one female from each of the with automictic parthenogenesis (Maynard Smith, two colonies (FAFB). Each combination was repli- 1986; Kondrashov, 1988). Specifically, terminal fu- cated five times. Pairs were placed in a 5276-mm sion causes a rapid reduction in heterozygosity glass cell (see Matsuura, 2002; Matsuura et al., (Templeton, 1982). Therefore, parthenogens would 2004). A mixed sawdust food block was placed in suffer severe inbreeding depression by expressing the upper half of the cell, and a 10-mm-diameter any recessive deleterious genes. However, supple- hole with a 1.5-mm opening was cut out. The cell mentary (i.e., secondary or replacement) reproduc- was covered with three 2676-mm glass micro- tives usually reproduce by inbreeding (Thorne scope slides, and maintained at 25°C in the dark. et al., 1999). The frequent inbreeding cycle of R. We monitored the glass cells every day through- speratus may remove most recessive lethal genes out the production of the first brood and collected from the population; the inbreeding cycle itself the eggs. Eggs were placed in a 1.5-ml microtube may act as a preadaptive selection for the evolution with 1 ml distilled water and voltexed for 30 s. of parthenogenesis (Cuellar, 1977). Then they were washed three times, placed on a The number of offspring per female is signifi- 1.5% agar plate (90 mm) containing 200 ppm tetra- cantly less in female-female (FF) than in monoga- cycline under sterile conditions, and maintained at mous female-male (FM) colonies (Matsuura and 25°C in the dark. The length (a) and diameter (b) Nishida, 2001), which may be attributable to the of each egg were measured soon after isolation reduced fecundity of FF pairs and the lower hatch- under a stereomicroscope (Olympus, Tokyo, Japan) ing rate of parthenogenetic eggs compared to sex- with a digital imaging system (FLVFS-LS; Flovel, ual eggs. Per capita investment may also be differ- Tokyo, Japan). Approximate egg volumes were cal- ent between sexual and parthenogenetic eggs. It is culated using the formula V4pab2/3. Isolated difficult to quantify actual egg survival rates in eu- eggs were observed daily until hatching. social insects, especially in termites; the eggs need Data on the size of eggs and hatching period to be isolated from the colony soon after oviposit- were analyzed separately by nested ANOVA fol- ing because eggs are laid daily and dead ones are lowed by Scheffe’s test. The survival rates of eggs immediately removed. Therefore, we first devel- among pairs were compared using Fisher’s exact oped a method to culture eggs until hatching, under probability test with Holm’s sequential Bonferroni sterile conditions and appropriate humidity. In this correction (Rice, 1989). Eggs that died unexpect- paper, we focus only on egg production and quality edly before hatching (e.g., bacterial contamination) because post-hatching survivorship has previously were excluded from the analysis. been addressed (Matsuura et al., 2004). We com- pared the quality of parthenogenetic eggs and sex- RESULTS ual eggs in terms of size, survival rates, and hatch- ing period. When both primary reproductives survived, FM colonies produced 28.0 (1.49 SE) first-brood eggs, and FF colonies produced 34.2 (1.88 SE) MATERIALS AND METHODS first-brood eggs. The period between pairing and The nest wood of two termite colonies (A and B) first oviposition was not significantly different be- in Onoda, Yamaguchi, Japan, was sampled in April tween FF pairs (4.441.81 [SD] days) and FM 2005. After alates emerged from the wood, they pairs (5.803.14; t-test, df22, t1.18, p0.25). Termite Parthenogenesis 243

Fig. 1. Embryo development of sexually produced eggs by female-male pairs (FM) and parthenogenetically produced eggs by female-female pairs (FF) in Reticulitermes speratus. Eggs were observed under a stereomicroscope with a transmitted light bases (Olympus SZX7), photographed and measured by using a digital imaging system (FLVFS-LS; Flovel, Tokyo, Japan). No visible difference occurred between parthenogenetic eggs and sexual eggs. Stage I: eggs soon after oviposition, Stage II: germ band forma- tion, Stage III: elongation of the embryo, Stage IV: dorsal closure, Stage V: fully developed embryo just before hatching. GB: germ band, HL: head lobe, HC: head capsule, Se: serosa, Am: amnion. The day after oviposition was shown for each stage.

Fig. 3. Egg hatching rate of sexually produced eggs and Fig. 2. The size of sexually produced eggs and partheno- parthenogenetic eggs of Reticulitermes speratus. The number genetic eggs of Reticulitermes speratus, soon after oviposition. of eggs examined is shown in parentheses. No significant dif- Bars indicate standard errors. The number of eggs examined is ferences among pair combinations were observed at p0.05, shown in parentheses. Different letters (a vs. b) represent sig- using Fisher’s exact probability test with Holm’s sequential nificant differences at p0.05, using Sheffe’s test. A and B in- Bonferroni correction. Eggs that died accidentally before dicate the natal colonies of founders. hatching (e.g., bacterial contamination) were excluded from the comparative analysis of hatching rates. A and B indicate The embryos of both parthenogenetic eggs and the natal colonies of founders. sexually produced eggs developed normally, and no visible differences were observed in development ference in hatching rate (survival rate until hatch- between the two types of egg (Fig. 1). However, ing) was detected between sexually produced eggs parthenogenetic eggs were significantly larger than (N302) and parthenogenetically produced eggs sexual eggs (nested ANOVA, FF/FM: F1 31.23, (N 195) (Fisher’s exact probability test, p 0.206; MS 0.058, p 0.0001; pair combination: F5 Fig. 3). The hatching period of parthenogenetic 6.68, MS0.012, p0.0001; Fig. 2). Details of the eggs was significantly longer than sexually pro- data set are presented in Appendix 1 (FM colonies) duced eggs (nested ANOVA, FF/FM: F1 25.43, and Appendix 2 (FF colonies). No significant dif- MS 97.58, p 0.0001; pair combination: F5 244 K. MATSUURA and N. KOBAYASHI

sexually produced eggs, suggesting that females might be able to change the per capita investment in each egg, depending on conditions. The trade- off between size and number of eggs may partially explain the reduced number of eggs per female in FF colonies. Facultative parthenogenesis is widespread among Polyneoptera, especially in cockroaches (Roth and Willis, 1956; Corley and Moore, 1999), grasshoppers (Pardo et al., 1995; Zhu and Ando, 1998) and walking sticks (Law and Crespi, 2002). Yet few species are particularly fecund when repro- ducing parthenogenetically. In most of facultatively parthenogenetic species, the viability of partheno- genetic offspring is much less than that of sexually Fig. 4. Egg hatching period of sexually produced eggs produced offspring. In the facultatively partheno- and parthenogenetic eggs of Reticulitermes speratus. Bars in- genetic cockroach Periplaneta americana, hatching dicate standard errors. The number of eggs examined is shown rate of unfertilized eggs is 40.5%, which is about in parentheses. Different letters (a vs. b) represent significant half that of fertilized eggs (Roth and Willis, 1956). differences at p 0.05, using Sheffe’s test. A and B indicate the Among rice grasshoppers, hatching rates of unfer- natal colonies of founders. tilized eggs are 17.8% in Oxya japonica, 10.4% in O. chinensis formosana and 5.4% in O. yezoensis 0.59, MS2.24, p0.71; Fig. 4). (Zhu and Ando, 1998). Parthenogenetic offspring The mean hatching period of colony FMBB-2 often take longer to develop (Roth and Willis, was considerably longer than the other FM colonies 1956; Lamb and Willey, 1979; Corley and Moore, (two-tailed t-test, p0.01), and similar to FF 1999). A longer time for embryo development has colonies, most likely due to male infertility (Ap- been reported in the locust Locusta migratoria pendix 1). Because the eggs in FMBB-2 might (Pardo et al., 1995). It has been also reported that have been produced by parthenogenesis, the data the preoviposition period of unmated females is from this group were excluded from statistic analy- longer than that of mated females. Fertilized fe- ses. males of the cockroach P. americana produced their first oothecae 13 days after emergence, while unfertilized females produced their first oothecae DISCUSSION 25 days after emergence (Roth and Willis, 1956). In a previous study using glass cells without iso- Similarly, the preoviposition period of unmated fe- lating eggs, FM colonies produced 11.3 (1.34 males is nearly three times longer than that of SE) first-brood eggs and FF colonies produced mated females in the facultatively parthenogenetic 18.0 (2.00 SE) first-brood eggs (Matsuura et al., cockroach Nauphoeta cinerea (Corley and Moore, 2004), far fewer than the results of the present 1999). study (t-test, FM: df13, t8.24, p0.0001; FF: In comparison with other facultatively partheno- df7, t2.91, p0.05). The 147% increased fe- genetic polyneopteran insects, parthenogenesis of cundity in FM colonies and 90% increase in FF R. speratus is remarkable for the high viability and colonies are likely not the result of differences in the short preoviposition period. Importantly, no the natal colonies used in the experiments. Females significant difference in hatching rate was observed should be able to recognize the present number of between the two types of egg in R. speratus, eggs in a colony to decide resource allocation. whereas the hatching period of parthenogenetic Therefore, the removal of eggs in the present study eggs was significantly longer than sexually pro- may have stimulated egg production. All females duced eggs. This high viability of parthenogenetic began to lay eggs simultaneously. Nevertheless, eggs clearly shows that parthenogenesis is not an parthenogenetic eggs were significantly larger than accidental event but a normal strategy in this ter- Termite Parthenogenesis 245 mite. Among termites, parthenogenesis has been cifugus Rossi. Z. Angew. Entomol. 33: 69–77. demonstrated in Zootermopsis angusticollis (Light, Howard, R. W., E. J. Mallette, M. I. Haverty and R. V. Smythe 1944), Z. nevadensis (Light, 1944), Kalotermes (1981) Laboratory evaluation of within-species, be- tween-species, and parthenogenetic reproduction in Retic- flavicollis (Grassé, 1949), Reticulitermes virginicus ulitermes flavipes and Reticulitermes virginicus. Psyche (Howard et al., 1981), R. speratus (Matsuura and 88: 75–87. Nishida, 2001; Matsuura et al., 2002, 2004; Kondrashov, A. S. (1988) Deleterious mutations and the evo- Hayashi et al., 2003), and Velocitermes sp. (Stansly lution of sexual reproduction. Nature 336: 435–440. and Korman, 1993), and is suspected in R. lucifu- Lamb, R. Y. and R. B. Willey (1979) Are parthenogenetic and related bisexual insects equal in fertility? Evolution gus (Herfs, 1951) and Bifiditermes beesoni 33: 774–775. (Chhotani, 1962). Further research may reveal Law, J. H. and B. J. Crespi (2002) The evolution of geo- parthenogenesis in many more termite species. graphic parthenogenesis in Timema walking-sticks. Comparative studies on parthenogenetic ability Mol. Ecol. 11: 1471–1489. among various species will help elucidate the evo- Light, S. F. (1944) Parthenogenesis in termites of the genus lution of parthenogenesis in termites. Zootermopsis. Univ. Calif. Pub. Zool. 43: 405–412. Matsuura, K. (2002) Colony-level stabilization of soldier ACKNOWLEDGEMENTS head width for head-plug defense in the termite Retic- ulitermes speratus (Isoptera: Rhinotermitidae). Behav. We thank T. Yashiro, S. Tatsumi, A. Sato for the assistance Ecol. Sociobiol. 51: 172–179. in the course of this study, K. Okada, F. Nakasuji, T. Miyatake, Matsuura, K., M. Fujimoto and K. Goka (2004) Sexual and T. Nishida, K. Fujisaki and K. Tsuji for useful discussion. This asexual colony foundation and the mechanism of faculta- work was funded by the Japan Society for the Promotion of tive parthenogenesis in the termite Reticulitermes spera- Science (JSPS) and the Program for Promotion of Basic Re- tus (Isoptera: Rhinotermitidae). 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Appendix 1. Hatching rates and hatching period of inseminated eggs produced by FM colonies

Pair No. of eggs No. of eggs No. of eggs Hatching Hatching Colony examined hatched died rate period (d) MF

FM AA-1 A A 10 6 4 0.60 34.33 0.33SE FM AA-2 A A 16 14 2 0.88 33.710.77 FM AA-3 A A 17 15 2 0.88 34.600.43 FM AA-4 A A 20 17 3 0.85 34.940.31 FM AA-5 A A 27 26 1 0.96 35.650.51 Total A A 90 78 12 0.87 34.850.25

FM AB-1 A B 12 11 1 0.92 34.360.31 FM AB-2 A B 8 8 0 1.00 34.000.42 FM AB-3 A B 10 6 4 0.60 34.170.17 FM AB-4 A B 28 26 2 0.93 34.920.30 FM AB-5 A B 25 23 2 0.92 35.430.38 Total A B 83 74 9 0.89 34.840.18

FM BA-1 B A 24 23 1 0.96 35.220.51 FM BA-2 B A 28 12 16 0.43 34.750.55 FM BA-3 B A 24 21 3 0.88 35.860.48 FM BA-4 B A 22 16 6 0.73 34.250.39 Total B A 98 72 26 0.74 35.110.26

FM BB-1 B B 31 24 7 0.77 35.130.37 FM BB-2* B B 30* 25* 5* 0.83* 38.120.60*

Grand total 302 248 54 0.82 34.950.12

* Data of FM BB-2 was excluded from the statistic analysis because of the unusually long hatching period most likely due to the infertility of the male. FM: female-male pair.

Appendix 2. Hatching rates and hatching period of parthenogenetic eggs produced by FF colonies

Pair No. of eggs No. of eggs No. of eggs Hatching Hatching Colony examined hatched died rate period (d) F1 F2

FF AA-1 A A 23 19 4 0.83 36.37 0.36SE FF AA-2 A A 38 31 7 0.82 36.550.31 FF AA-3 A A 39 31 8 0.80 36.480.31 FF AA-4 A A 17 10 7 0.59 35.500.62 FF AA-5 A A 17 13 4 0.77 35.150.34 Total A A 134 104 30 0.78 36.220.17

FF AB-1 A B 10 9 1 0.90 36.330.29 FF AB-2 A B 32 25 7 0.78 37.080.59 FF AB-3 A B 6 6 0 1.00 35.500.34 Total A B 48 40 8 0.83 36.680.38

FF BB-1 B B 13 7 6 0.35 36.710.52

Grand total 195 151 44 0.75 36.360.16

FF: female-female pair.