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Testicular development and modes of apoptosis during spermatogenesis in various castes of the termite Reticulitermes labralis (Isoptera:Rhinotermitidae)
Arthropod Structure & Development 44 (2015) 630e638
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Testicular development and modes of apoptosis during spermatogenesis in various castes of the termite Reticulitermes labralis (Isoptera:Rhinotermitidae) Xiao Hong Su*, Jiao Ling Chen, Xiao Jing Zhang, Wei Xue, He Liu, Lian Xi Xing College of Life Sciences, Northwest University, Xi'an, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2015 Accepted 25 August 2015 Available online 4 September 2015
The separation of reproductive and non-reproductive roles based on caste differentiation is the most prominent characteristic of termites. However, little is known about the mechanism of male reproductive division that underlies caste differentiation. In the present study, testicular development and stagespecific apoptotic patterns were investigated and compared during spermatogenesis in reproductives, workers and soldiers of the termite Reticulitermes labralis. The results showed that male workers were divided into two types, the workers with spermatozoa (WS) and the workers without spermatozoa (WN). Spermatogenesis in WN and soldiers arrested at the spermatocyte stage. Moreover, there were significant differences in testicular size and spermatogenesis among the various castes. The mode of apoptosis in late instar WS was similar to the reproductives, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis. First, the majority of apoptotic cells were spermatogonia, and the spermatogonia of both late instar WS and reproductives exhibited lower apoptotic rates compared with late instar WN and soldiers. Second, the spermatocytes and spermatids showed very little apoptosis in the late instar WS and reproductives, and no TUNEL signal was detected in any of the examined spermatozoa. Our findings suggest that the male workers undergo a basal developmental schema comprising two undifferentiated larval instars, followed by a bifurcated development into either (i) the sexual lineage, in which the workers are able to provide normal spermatozoa to queens, or (ii) the neuter lineage, in which the male workers lose reproductive options. The level of testicular development may explain the significant discrepancies in reproductive capacity among the reproductives, workers and soldiers and reveal the reproductive division in male workers. These differences are controlled by apoptosis during early spermatogenesis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Reproductive division Termite Spermatogenesis Apoptosis TUNEL
1. Introduction All existing termites are eusocial insects. Generally, eusociality is defined by the (i) overlap of generations, (ii) cooperative brood care, and (iii) reproductive division of labour. The latter is the key feature of eusocial insects (Michener, 1969). The reproductive division among the different castes of a termite colony is primarily triggered by environmental stimuli, allowing reproductives, workers, and soldiers to develop within a colony with essentially the same genetic background (Korb et al., 2009). In addition, genetic diversity may be related to the differentiation of intraspecific
* Corresponding author. Tel.: þ86 029 88302411. E-mail address: [email protected] (X.H. Su). http://dx.doi.org/10.1016/j.asd.2015.08.009 1467-8039/© 2015 Elsevier Ltd. All rights reserved.
groups (Frati et al., 1992). Generally, reproductives lay eggs, and sterile workers and soldiers care for younger siblings and provide colony defence, respectively (Korb, 2015). Lower termites are characterized by a unique flexibility in development. In the genus Reticulitermes within Rhinotermitidae, the workers can develop in three ways: (i) via moults into higher instar workers, (ii) via two successive moults into soldiers, or (iii) via one moult into apterous neotenic reproductives that develop in the absence of or at a great distance from the primary reproductive to provide for continued or additional growth of the colony (Korb and Hartfelder, 2008; Korb et al., 2009). In addition, it was reported that the male workers of Reticulitermes speratus can develop into inconspicuous reproductive males without undergoing a moult and without exhibiting any significant change in morphology (Fujita and Watanabe, 2010). The reproductive plasticity of the workers gives colonies tremendous
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flexibility in response to environmental change and the deterioration of the nest and food resources. Dominant individuals typically have well-developed gonads (Geva et al., 2005). In many social species of Hymenoptera, such as ants, wasps and honeybees, reproductive differentiation is controlled by gonadal activity (Koudji and Doumandji, 2008; Okada et al., 2010). In the termite R. speratus, the testis size of the workers increases from approximately 0.2 mme0.4 mm during development into neotenic reproductives (Fujita and Watanabe, 2010). Therefore, gonadal development in the termite would appear to be the most significant biological process that leads to the formation of caste-specific differences in tasks and status. Spermatogenesis is a complex process of proliferation and differentiation, transforming spermatogonia into mature spermatozoa. In insects, this process involves spermatogonial proliferation by repeated mitotic divisions, resulting in a group of primary spermatocytes. These spermatocytes undergo two successive meiotic divisions and give rise to spermatids, which, after deep morphological changes (spermiogenesis), form sperm bundles (Dallai, 2014). Functional spermatogenesis requires an intrinsic regulation, which is managed by a conserved genetic program that underlies the development of germ cells, ensuring sperm viability. The lack of an efficient control mechanism may lead to spermatogenetic alterations, which, in turn, can result in the production of damaged spermatozoa (Dias et al., 2013, 2015). Aberrant spermatogenesis in the two species Stenognathellus denisi and Sminthurides aquaticus (Collembola) is described, in which the secondary spermatocytes with a small size and reduced cytoplasm are not able to perform the second meiotic division and degenerate (Dallai et al., 2004). The number of sperm produced can be affected by environmental conditions, such as food deprivation, a condition capable of determining a significant reduction in the number of sperm produced by the moths Plodia interpunctella and by the dipterans Sarcophaga stercoraria and Drosophila melanogaster (Gage and Cook, 1994; Hellriegel and Blanckenhorn, 2002; Amitin and Pitnick, 2007). In addition, the number of sperm also depends on the size of both sperm and testis (Pitnick et al., 2009). However, there have been no studies to examine the characteristics of spermatogenesis in workers and soldiers of termites. During spermatogenesis, the balance between proliferation and cell death signals determines the maturation fate of the germ cells. Apoptosis, also known as programmed cell death, often denotes an active and highly choreographed process of cell suicide and may be triggered by several factors, including intrinsic signals and external stimuli (Baum et al., 2005). Invertebrates and vertebrates display many similarities in the use of apoptosis to eliminate dangerous or damaged cells and regulate cell numbers by removing excess cells during spermatogenesis. Failure of apoptosis to selectively deplete abnormal germ cells earmarked for programmed cell death would lead to abnormal spermatozoa in the ejaculate and male infertility (Sakkas, 2003). In the insect Lepidoptera, the degenerative changes in the germ cells of the testis during pupal diapause are controlled by apoptosis, and apyrene sperm development occurs by the apoptosis of their nuclei (Polanska et al., 2005; Shimoda et al., 2007). One of the best studied aspects of apoptosis in social insects occurs during caste determination in bees (Baum et al., 2005). In the honeybee Apis mellifera, an adult queen ovary contains as many as 200 ovarioles, whereas the ovary of a worker has less than 10. This large discrepancy in ovariole number is a result of apoptosis (Capella and Hartfelder, 1998). In the termite Reticulitermes aculabialis, our previous studies also indicate that apoptosis during early oogenesis is an efficient and crucial regulatory mechanism affecting the number of oocytes and the reproductive capacity of the reproductives, soldiers and workers (Su et al., 2014). Therefore, the available evidence suggests that apoptosis is not only necessary for
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the normal development of germ cells in many organisms but also plays a vital role in the caste differentiation in social insects. Although Reticulitermes is an economically important genus and is probably the most studied of all termites, the precise development pattern in workers and soldiers remains uncertain. The separation of reproductive and non-reproductive roles based on caste differentiation is the most prominent characteristic of termites, and the clarification of the mechanism underlying this separation is an important topic in termites. The difference in the physiological detail of testis development among workers, soldiers and reproductives is unclear, although the general morphology of the male reproductive system and sperm of termites have received attention (Sieber and Leuthold, 1982; Riparbelli et al., 2009; Hartke and Baer, 2011). To obtain a better understanding of the development of the testes underlying reproductive division in various castes and to reveal whether each male worker has reproductive flexibility, spermatogenesis and apoptotic patterns were investigated and compared in reproductives, workers and soldiers of the termite Reticulitermes labralis. 2. Materials and methods 2.1. Termites R. labralis colony fragments were collected from the monasteries of Xi'an, China, in May 2011 when the alate adults were swarming. The colonies were comprised of workers, soldiers, and alate reproductives. Newly hatched individuals (larvae) developed into workers (third instar workers) or nymphs (third instar nymphs) through two successive moults, distinguished by the absence or presence of wing pads, respectively. The third instar workers (W3), fourth instar workers (W4) and fifth instar workers (W5) (early instar workers) were identified by antennal segments, the width of the heads and the length of the bodies. Sixth instar workers (W6) (late instar workers, WL) were identified by the presence of 16 or more antennal segments. The male reproductive and late instar worker individuals were discriminated according to the seventh sternite, and the male early instar workers and soldier individuals were discriminated by longitudinal sections. After collection from the field, the abdomens were immediately removed and fixed in Bouin's solution for HE (haematoxylin and eosin) staining. They were also fixed in 4% paraformaldehyde in PBS overnight at 4 C for terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) analysis. 2.2. Measurement of testis development in reproductives, workers and soldiers The testes of the reproductives and W6 were dissected out in PBS under a stereomicroscope to examine the testis morphology of R. labralis. The testicular development of the workers, soldiers and reproductives was evaluated with HE staining. The fixed samples were dehydrated in an ascending ethanol series and embedded in paraffin. Longitudinal sections 6 mm thick were collected on polylysine-coated slides. Deparaffinised and rehydrated sections were stained in HE. Because the testes of R. aculabialis had a mostly round shape, the diameter of each testis at its widest point was measured using a digital microscope in accordance with a previous study on R. speratus (Shimada and Maekawa, 2010). 2.3. Spermatogenesis in various castes Thin sections through the testes showed different stages of spermatogenesis in the testicular follicles of the reproductives,
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Fig. 1. The testes in various castes of the termite Reticulitermes labralis. (A) Testis and vas deferens of a fifth instar worker. (B) Testis of a sixth instar worker. (C) Testis of a reproductive. (D) Testis in longitudinal section of abdomen of a reproductive. (E) Testis in longitudinal section of abdomen of a third instar WN. (F) Testis in longitudinal section of abdomen of a fourth instar WN. (G) Testis in longitudinal section of abdomen of a fifth instar WN. (H) Testis in longitudinal section of abdomen of a sixth instar WN. (I) Extremely degenerate testis in longitudinal section of abdomen of a soldier. Te, testis; VD, vas deferens.
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3. Results 3.1. Testicular development in the reproductives, workers and soldiers
Fig. 2. The mean size of testes in reproductives, workers and soldiers of the termite Reticulitermes labralis (n ¼ 10). The columns represent the means; bars represent SD. The letters (aed) above the columns indicate significant differences (p < 0.05) among the various castes. R, reproductives; S, soldiers; WS, workers with spermatozoa; WN, workers without spermatozoa.
workers and soldiers. The spermatogenic stages were categorized based on the morphogenetic criteria of germ cells (Sieber and Leuthold, 1982; Chawanji et al., 2007; Kubo-Irie et al., 2011; Paoli et al., 2013; Carcupino et al., 1998). The number of spermatozoa per longitudinal section of the testes was counted, and the sum of the results was used as the number of spermatozoa per testis. The diameter of the germ cells at each stage was measured based on the captured digital images. 2.4. TUNEL assay Detection of the DNA fragments was performed in paraffinembedded sections by TUNEL analysis using the ApopTag peroxidase in situ apoptosis detection kit (Roche Applied Science, Germany). The paraffin-embedded sections were dewaxed and incubated with 3% H2O2 for 10 min at room temperature and washed in distilled water. Following this step, the sections were incubated with PBS containing 20 mg/ml proteinase K for 10 min at 37 C and washed three times in PBS for 5 min each. The TUNEL reaction mixture (30 ml of 3 ml enzyme solution and 27 ml label solution) (Roche Applied Science, Germany) was added to the sections for 60 min at 37 C, and the slides were rinsed in PBS. Then, 30 ml converter-POD (peroxidase) was added to the sample for 30 min at 37 C and washed in PBS. The sections were incubated in 3, 30 -diaminobenzidine tetrahydrochloride (DAB, Boster Co. Ltd., Wuhan, China) solution for 5e10 min at room temperature. Finally, the slides were stained with haematoxylin counterstain. For the negative control, 30 ml label solution, instead of the TUNEL reaction mixture, was used. The sections were analysed by light microscopy, and the apoptotic cells were identified by brown nuclei. The apoptotic rate during spermatogenesis was defined as the ratio of the number of TUNEL-positive germ cells within the total germ cells per longitudinal section of the testes. 2.5. Statistical analysis A statistical test was performed by the usual methods for multiple comparisons (Tukey's test) using SPSS version 16.0 (SPSS Inc. Chicago, Illinois). All values are expressed as the mean ± SD. P values less than 0.05 were considered statistically significant.
The male individuals of R. labralis have a pair of round testes (Fig. 1A, B and C). Each testis is composed of a varying number of testicular follicles, in which spermatogenesis is synchronous. In the male reproductives, sixth instar workers with spermatozoa (W6s) and soldiers, the mean size of the testes was 204.3 ± 26.3 mm, 162.2 ± 28.1 mm and 36.0 ± 2.8 mm, respectively. The mean size of the testis of the reproductives and late instar workers was approximately six-fold and four-fold longer compared with the soldiers, respectively. Compared with the early instar workers, the testes of the soldiers were also extremely degenerate. The difference in testis development among the reproductives, late instar workers and soldiers was significant (Figs. 1DeI and 2). The workers were divided into two types: the workers (WS) in which testes contained spermatozoa (Fig. 3CeF) and the workers (WN) in which the testes did not produce spermatozoa (Fig. 1EeH). In early instar workers (W3,W4 and W5), there was no significant difference in the mean diameter of the testes between WS and WN individuals. The size of the testes slightly increased during W3 developing into W5. However, the testis size increased substantially from W5 to W6. The mean diameter of the testes in W6S and W6N was approximately three-fold and two-fold longer compared with early instar workers, respectively, indicating that the testis in late instar workers developed significantly (Fig. 2). 3.2. Spermatogenesis in reproductives, workers and soldiers Thin sections through the testes showed different stages of spermatogenesis in testicular follicles. Spermatogenesis in R. labralis was similar to that described for other insect orders. Spermatogenesis is divided into four stages: mitotic proliferation of spermatogonia, meiotic division of spermatocytes, differentiation of spermatids and final transformation of spermatozoa (Dallai, 2014). In the reproductives and WS, the spermatogenetic process occurred synchronously in the testicular follicles where germ cells arranged in an ascending order of development. By comparison, spermatogenesis in soldiers and WN only reached the spermatocyte stage. Therefore, spermatozoa were absent in the soldiers and some of the workers (Fig. 3). In the soldiers, no spermatids and spermatozoa were observed (Fig. 3G), and the size of the spermatogonia and spermatocytes (3.8 ± 0.5 mm and 2.7 ± 0.3 mm, respectively) was significantly smaller compared with W6s and reproductives (Fig. 4). The soldiers had a few germ cells, approximately 90e120 per testes. In the reproductives, the spermatogonia 6.2 ± 0.9 mm in diameter appeared irregular in shape and had the largest diffuse chromatin (4.2 ± 0.4 mm). The spermatocytes 5.1 ± 0.6 mm in diameter were characterized by clearly visible condensed chromatin and roundish nuclei (3.2 ± 0.2 mm). Compared to the spermatocytes, the size of spermatids and their nuclei was significantly smaller (2.4 ± 0.2 mm and 1.7 ± 0.2 mm, respectively). The spermatids became smaller progressively, the nuclear size was reduced, the excess cytoplasm was eliminated, and they were finally transformed into the globular aflagellar spermatozoa 1.5 ± 0.1 mm in size (Fig. 4). The reproductives had 5724 ± 1683 spermatozoa per testis (Fig. 5). During the third instar to the fifth instar stage, the number of spermatozoa of Ws gradually increased by approximately 30e200 in a small number per testes. However, the number of spermatozoa in the W6S was approximately ten-fold higher compared with the W5S (Fig. 5). Although there was no significant difference in the size
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Fig. 3. Spermatogenesis in various castes of the termite Reticulitermes labralis. (A) Spermatozoa in the testes of reproductives. (B) The process of spermatogenesis, transforming spermatogonia into mature spermatozoa are shown in the testicular follicles of reproductives. (C) Spermatozoa in third instar Ws. (D) Spermatozoa in fourth instar Ws. (E) Spermatozoa in fifth instar Ws. (F) Spermatozoa in sixth instar Ws. (G) Spermatogenesis in soldiers is arrested at the spermatocyte stage. Sg, spermatogonia; Sc, spermatocytes; St, spermatids; Sp, spermatozoa.
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Fig. 4. The mean size of the germ cells at each stage in the various castes of the termite Reticulitermes labralis (n ¼ 30). The columns represent the means; bars represent the SD. Letters (a and b) above the columns of the same colour indicate significant differences (p < 0.05) among the various castes. R, reproductives; S, soldiers; W6S, sixth instar workers with spermatozoa; W6N, sixth instar workers whithout spermatozoa.
of the germ cells between the reproductives and W6S, the number of spermatozoa in reproductives was approximately three-fold higher than that in W6S. There was a significant difference in the size of the spermatogonia and spermatocytes between the W6S and W6N (Fig. 4). 3.3. Apoptosis in the testes of reproductives, workers and soldiers Apoptosis in testes was confirmed using a TUNEL assay, which identified nuclei with clear signs of DNA fragmentation, a hallmark of apoptosis. In the reproductives, the majority of positive nuclei were located in spermatogonia, indicating that apoptosis occurred mainly at early stages of spermatogenesis. Most of the apoptotic spermatogonia in the testes were not randomly distributed throughout the testicular follicles, but cohorts of 2e4 neighbouring apoptotic spermatogonia were often noted to be degenerating synchronously. The spermatocytes and spermatids showed very little apoptosis, and no TUNEL signal was detected in any of the examined spermatozoa (Fig. 6A, B and C).
Fig. 5. The mean number of spermatozoa per testis in reproductives and each instar workers of the termite Reticulitermes labralis (n ¼ 10). The columns represent the means; bars represent the SD. The letters (aee) above the columns indicate significant differences (p < 0.05). W3S, third instar workers with spermatozoa; W4S, fourth instar workers with spermatozoa; W5S, fifth instar workers with spermatozoa; W6S, sixth instar workers with spermatozoa, R, reproductives.
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The mode of apoptosis in W6S was similar to that of the reproductives. The apoptosis of germ cells mainly took place in the spermatogonia, and only one or two spermatocytes and spermatids per longitudinal section of the testes were TUNEL positive (Fig. 6D). There was no significant difference in the apoptotic rate of the spermatogonia between the workers (11.6 ± 2.8) and the reproductives (9.5 ± 2.3) (Fig. 7). Compared with the W6S, in the W6N, the testes exhibited stronger TUNEL-positive staining and a higher apoptotic rate of spermatogonia (21.8 ± 3.7) (Figs. 6E and 7). In the soldiers, the testes exhibited the strongest TUNELpositive staining (Fig. 6F) and the highest apoptotic rate of spermatogonia (Fig. 7). Apoptotic cells occupied the entire region of the testicular follicles. The apoptotic rate of spermatogonia was approximately four-fold higher compared with the W6S and reproductives, and two-fold higher compared with the W6N, indicating that a greater number of germ cells in the soldiers had undergone degeneration. 4. Discussion For R. labralis male workers, we established a basal developmental schema comprising two undifferentiated larval instars, followed by a bifurcated development into either (i) the sexual lineage, in which the workers maintained normal spermatogenesis during the third instar to late instar stage, and at the late instar stage became either apterous neotenic reproductives via a single moult or inconspicuous reproductive males, or (ii) the neuter lineage, in which the workers did not produce spermatozoa and their spermatogenesis stopped at the spermatocyte stage. This study of testis development in individual workers of R. labralis indicates that there is a significant difference in spermatogenesis, but not in testis size, among early instar worker individuals and that the degenerated testis can be characterized by the absence of spermatids and spermatozoa. As in WN and soldiers of R. labralis, it has been previously reported that in the testis of diapausing insects, the meiotic divisions that lead to the formation of spermatids do not occur or are somehow altered (Polanska et al., 2005). Our results show that in R. labralis, a sharp increase in the testis size of workers occurs at the sixth instar stage. In Reticulitermes, the last three instar nymphs can moult into brachypterous neotenic reproductives, whereas late instar workers can moult into apterous neotenic reproductives (Buchli, 1958). Therefore, the male W6S of R. labralis are defined as a functional category of individuals performing work, while retaining the reproductive potential to copulate with female reproductives. This latter property distinguishes them from W6N, which result from an early and irreversible developmental bifurcation. A late instar WS is able to copulate with and provide spermatozoa to queens, suggesting that there is a fundamental difference in the reproductive capability between male and female workers in R. labralis. Although the presence of oocytes is a key mechanism for female workers having the potential for differentiation into neotenic reproductives through a moult, the absence of vitellogenic oocytes in workers results from oogenesis stopping at the previtellogenesis stage, which is similar to those in the last instar nymphs (Su et al., 2014). Therefore, the female late instar workers are not able to produce mature eggs unless they moult into conspicuous reproductive females (apterous neotenic reproductives), which have significant differences in morphology compared with the female workers. Interestingly, a strongly female-biased sex ratio among the emerging neotenic reproductives has been observed in R. speratus. However, when the numbers of inconspicuous mature males were added to the number of the conspicuous male neotenic reproductives, the sex ratio was almost 1:1 (Fujita and Watanabe, 2010). Therefore, insurance against the loss of male
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Fig. 6. Apoptosis during spermatogenesis of the termite Reticulitermes labralis. (A) Apoptotic spermatogonia and spermatids in the testes of reproductives. Two neighbouring apoptotic cells apparently are degenerating synchronously. TUNEL positive germ cells were identified by brown nuclei. (B) A TUNEL positive spermatocyte was observed in the testes of reproductives. (C) An apoptotic spermatogonium in the testis of a reproductive. (D) Apoptotic spermatogonia in sixth instar Ws. (E) In sixth instar W6N, the testes exhibit strong TUNEL-positive staining. (F) In soldiers, the testes exhibit strong TUNEL-positive staining. (G) Negative control. Sg, spermatogonia; Sc, spermatocytes; St, spermatids.
reproductives, possibly in the form of the presence of inconspicuous males as a male reproductive “backup,” would appear to be important for the functionality of the termite society. It would be reasonable to suppose that the termite colony could maintain a 1:1
sex ratio among reproductives by producing a supplemental inconspicuous type of male neotenic. In addition, the male reproductive individuals of termites cannot store a large number of spermatozoa due to the absence of seminal vesicles, whereas a
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Fig. 7. Apoptotic rates of spermatogonia in the reproductives, W6S, W6N and soldiers of the termite Reticulitermes labralis (n ¼ 10). The columns represent the means; the bars represent the SD. Letters (a, b, and c) above the columns indicate significant differences (p < 0.05) among the various castes. W6S, sixth instar workers with spermatozoa; W6N, sixth instar workers without spermatozoa.
physogastric queen needs approximately 40,000 sperm per day, which likely cannot be produced by a male (Sieber and Leuthold, 1982). Thus, one female reproductive mates with several male reproductives in a short time (Wu et al., 2013). Participation of the male workers in sexual reproduction has been confirmed by genetic analysis in R. labralis (Wu et al., 2013). Therefore, we believe that in Reticulitermes, the inconspicuous male reproductives are the late instar workers with spermatozoa, and the spermatozoa of late instar workers are able to participate in fertilisation. A higher frequency of apoptosis in the spermatogonia of R. labralis indicates that a critical checkpoint occurs in the germarium of the testicular follicles in response to an imbalance in cell numbers. In the zone of the germarium, the spermatogonia undergo several mitotic divisions to form primary spermatocytes. Therefore, the results of the present study support previous studies in which the apoptosis of germ cells coincides with mitotic peaks (Heninger et al., 2004). Degeneration during normal spermatogenesis is also observed in the testes of reproductives in R. labralis, where some spermatogonia display characteristics of apoptosis. In R. labralis, the majority of apoptotic spermatogonia were not randomly distributed throughout the follicles, but cohorts of neighbouring apoptotic spermatogonia degenerated synchronously. This observation was consistent with groups of cells interconnected by cytoplasmic bridges that were synchronously undergoing apoptosis (Gilboa and Lehmann, 2004; Heninger et al., 2004). Intercellular bridges derived from polyfusomes are common in spermatogenesis of animals, but it is only in the ovarioles of female insects where such bridges are also formed and where their function is best studied, much better than in males. Furthermore, the consequences of the disorganization of polyfusomes have been suggested as a major cause for autophagic cell death in the larval ovaries of honey bee workers (Capella and Hartfelder, 2002). Significantly, slight increases in the level of apoptosis in spermatogonia can have dramatic effects on overall germ cell numbers (Heninger et al., 2004). Therefore, the apoptotic rate in various castes of R. labralis suggests that apoptosis during early spermatogenesis is an efficient and crucial regulatory mechanism affecting both the number of germ cells and the reproductive capacity of labour in termites. Apoptosis also occurs during the spermatocyte and spermatid stages of spermatogenesis in the reproductives and W6S of
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R. labralis. Although only one or two spermatocytes and spermatids appear to be apoptotic in the testes of reproductives and WS, the checkpoints of spermatocytes and spermatids provide a final opportunity for the testes to eliminate defective or abnormal germ cells before developing into spermatozoa. In rodents, as well as cats and rabbits, high levels of germ cell apoptosis are also observed in spermatocytes, which proceed through meiosis, suggesting that the meiotic checkpoint may be present to help correct for the number of germ cells and to account for problems such as rearrangement during meiosis or unrepaired breaks in DNA by inducing apoptosis of these damaged cells (Baum et al., 2005). However, in the reproductives and W6S of R. labralis, only one or two apoptotic spermatocytes and spermatids were randomly distributed in the testes, indicating that the two apoptosis checkpoints are used as “quality control” to eliminate defective spermatocytes and spermatids with incomplete genetic components or chromosomal abnormalities. No TUNEL-positive spermatozoa are observed in the reproductives and W6S of R. labralis, indicating that during normal spermatogenesis, apoptosis can eliminate all defective germ cells before the spermatids transform into spermatozoa. The presence of spermatozoa that have damaged nuclear DNA or loosely packaged chromatin has been confirmed in male infertility. In humans, a strong association has been found between abnormal semen parameters and the presence of apoptosis in ejaculated spermatozoa (Sakkas, 2003). The presence of nuclear DNA strand breaks in spermatozoa is due to anomalies in apoptosis during spermatogenesis, and high levels of apoptosis in spermatozoa result in male infertility (Sakkas, 2003). Sakkas et al. (1999) also reported that abnormal spermatozoa could exist and express various apoptotic markers because they have escaped programmed cell death, a procedure termed ‘abortive apoptosis.’ These findings allow us to extend the identification of abnormal or normal spermatogenesis in R. labralis and to suggest that in R. labralis, the W6S produce the same spermatozoa as reproductives. The histological data demonstrate that male late instar WN and soldiers have lost the ability to develop into neotenic reproductives or inconspicuous reproductive males in R. labralis. Compared with the reproductives and late instar WS, the size of the testes and germ cells in the late instar WN and soldiers are significantly smaller, and their testes exhibited strong apoptotic staining. Similarly, Dallai et al. (2004) reported abnormal spermatogenesis in Collembola, in which the aberrant secondary spermatocytes are characterized by small size, reduced cytoplasm and nuclei with very condensed chromatin, and these secondary spermatocytes are not able to perform the second meiotic division and degenerate. The degeneration of germ cells in the testis and ovaries are controlled by apoptosis (Capella and Hartfelder, 1998; Baum et al., 2005; Polanska et al., 2005; Shimoda et al., 2007). Therefore, the spermatogonia and spermatocytes of the WN and soldiers in R. labralis are abnormal and degenerate. The involvement of apoptosis is extremely important for WN and soldiers to eliminate germ cells and remain sterile. Some of the male workers lose reproductive options earlier in development, which may enhance the ability to complete tasks and require less energy. Acknowledgments We thank Li Gang for help with collecting termites. Financial support was provided by the National Natural Scince Foundation of China (31370428 and 31170363) and the Scientific Research Program Funded by Shaanxi Provincial Education Department (2013JK0715).
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Korb, J., Hartfelder, K., 2008. Life history and development-a framework for understanding developmental plasticity in lower termites. Biol. Rev. 83, 295e313. Korb, J., Hoffmann, K., Hartfelder, K., 2009. Endocrine signatures underlying plasticity in postembryon development of a lower termite, Cryptotermes secundus (Kalotermitidae). Evol. Dev. 11, 269e277. Koudji, M., Doumandji, S.E., 2008. Ultrastructural characterization of the ovarian stages of the worker bee, Apis mellifera mellifera Linne (Hymenoptera, Apidae). C. R. Biol. 33, 185e197. Kubo-Irie, M., Yamaguchi, T., Tanaka, Y., Yamazaki, I., Irie, M., Mohri, H., Shimoda, M., 2011. Identification of the starting point for spermatogenesis resumption in the post-diapause deveiopment of the sweet potato hornworm. J. Insect Physiol. 57, 784e790. Michener, C.D., 1969. Comparative social behavior of bees. Annu. Rev. Entomol. 14, 299e342. Okada, Y., Miyazaki, S., Miyakawa, H., Ishikawa, A., Tsuji, K., Miura, T., 2010. Ovarian development and insulin-signaling pathways during reproductive differentiation in the queenless ponerine ant Diacamma sp. J. Insect Physiol. 56, 288e295. Paoli, F., Gottardo, M., Dallai, R., Roversi, P.F., 2013. Morphology of the male reproductive system and sperm ultrastructure of the egg parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera, Platygastridae). Arthropod Struct. Dev. 42, 297e308. Pitnick, S., Hosken, D.J., Birkhead, T., 2009. Sperm morphological diversity. In: Birkhead, T., Hosken, D.J., Pitnick, S. (Eds.), Sperm Biology: an Evolutionary Perspective. Academic Press, Oxford, pp. 69e149. Polanska, M.A., Ciuk, M.A., Cymborowski, B., Bebas, P., 2005. Germ cell death in the testis and its relation to spermatigenesis in the wax moth,Galleria mellonella (Lepidoptera:Pyralidae), effects of facultative diapause. J. Exp. Zool. A Comp. Exp. Biol. 303, 1013e1029. Riparbelli, M.G., Callaini, G., Mercati, D., Hertel, H., Dallai, R., 2009. Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera). Cell Motil. Cytoskelet. 66, 248e259. Shimada, K., Maekawa, K., 2010. Changes in endogenous cellulase gene expression levels and reproductive characteristics of primary and secondary reproductives with colony development of the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). J. Insect Physiol. 56, 1118e1124. Shimoda, M., Kubo-Irie, M., Ohta, K., Irie, M., Mohri, H., 2007. Spermatogenesis in the testes of diapause and non-diapause pupae of the sweet potato hornworm, Agrius convolvuli (L.) (Lepidoptera: Sphingidae). Zool. Sci. 24, 1036e1044. Sieber, R., Leuthold, R.R., 1982. Repeated copulation and testes enlargement in Mactrotermes michaelseni. Physiol. Entomol. 7, 457e465. Sakkas, D., 2003. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod. Biomed. 4, 428e432. Sakkas, D., Mariethoz, E., St John, J.C., 1999. Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the fas-mediated pathway. Exp. Cell Res. 251, 350e355. Su, X.H., Wei, Y.H., Liu, M.H., 2014. Ovarian development and modes of apoptosis during oogenesis in various castes of the termite, Reticulitermes aculabialis. Physiol. Entomol. 39, 44e52. Wu, J., Su, X., Kong, X., Liu, M., Xing, L., 2013. Multiple male and female reproductive strategies and the presence of a polyandric mating system in the termite Reticulitermes labralis (Isoptera: Rhinotermitidae). Sociobiology 60, 459e465.
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Testicular development and modes of apoptosis during spermatogenesis in various castes of the termite Reticulitermes labralis (Isoptera:Rhinotermitidae) Xiao Hong Su*, Jiao Ling Chen, Xiao Jing Zhang, Wei Xue, He Liu, Lian Xi Xing College of Life Sciences, Northwest University, Xi'an, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2015 Accepted 25 August 2015 Available online 4 September 2015
The separation of reproductive and non-reproductive roles based on caste differentiation is the most prominent characteristic of termites. However, little is known about the mechanism of male reproductive division that underlies caste differentiation. In the present study, testicular development and stagespecific apoptotic patterns were investigated and compared during spermatogenesis in reproductives, workers and soldiers of the termite Reticulitermes labralis. The results showed that male workers were divided into two types, the workers with spermatozoa (WS) and the workers without spermatozoa (WN). Spermatogenesis in WN and soldiers arrested at the spermatocyte stage. Moreover, there were significant differences in testicular size and spermatogenesis among the various castes. The mode of apoptosis in late instar WS was similar to the reproductives, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis. First, the majority of apoptotic cells were spermatogonia, and the spermatogonia of both late instar WS and reproductives exhibited lower apoptotic rates compared with late instar WN and soldiers. Second, the spermatocytes and spermatids showed very little apoptosis in the late instar WS and reproductives, and no TUNEL signal was detected in any of the examined spermatozoa. Our findings suggest that the male workers undergo a basal developmental schema comprising two undifferentiated larval instars, followed by a bifurcated development into either (i) the sexual lineage, in which the workers are able to provide normal spermatozoa to queens, or (ii) the neuter lineage, in which the male workers lose reproductive options. The level of testicular development may explain the significant discrepancies in reproductive capacity among the reproductives, workers and soldiers and reveal the reproductive division in male workers. These differences are controlled by apoptosis during early spermatogenesis. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Reproductive division Termite Spermatogenesis Apoptosis TUNEL
1. Introduction All existing termites are eusocial insects. Generally, eusociality is defined by the (i) overlap of generations, (ii) cooperative brood care, and (iii) reproductive division of labour. The latter is the key feature of eusocial insects (Michener, 1969). The reproductive division among the different castes of a termite colony is primarily triggered by environmental stimuli, allowing reproductives, workers, and soldiers to develop within a colony with essentially the same genetic background (Korb et al., 2009). In addition, genetic diversity may be related to the differentiation of intraspecific
* Corresponding author. Tel.: þ86 029 88302411. E-mail address: [email protected] (X.H. Su). http://dx.doi.org/10.1016/j.asd.2015.08.009 1467-8039/© 2015 Elsevier Ltd. All rights reserved.
groups (Frati et al., 1992). Generally, reproductives lay eggs, and sterile workers and soldiers care for younger siblings and provide colony defence, respectively (Korb, 2015). Lower termites are characterized by a unique flexibility in development. In the genus Reticulitermes within Rhinotermitidae, the workers can develop in three ways: (i) via moults into higher instar workers, (ii) via two successive moults into soldiers, or (iii) via one moult into apterous neotenic reproductives that develop in the absence of or at a great distance from the primary reproductive to provide for continued or additional growth of the colony (Korb and Hartfelder, 2008; Korb et al., 2009). In addition, it was reported that the male workers of Reticulitermes speratus can develop into inconspicuous reproductive males without undergoing a moult and without exhibiting any significant change in morphology (Fujita and Watanabe, 2010). The reproductive plasticity of the workers gives colonies tremendous
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flexibility in response to environmental change and the deterioration of the nest and food resources. Dominant individuals typically have well-developed gonads (Geva et al., 2005). In many social species of Hymenoptera, such as ants, wasps and honeybees, reproductive differentiation is controlled by gonadal activity (Koudji and Doumandji, 2008; Okada et al., 2010). In the termite R. speratus, the testis size of the workers increases from approximately 0.2 mme0.4 mm during development into neotenic reproductives (Fujita and Watanabe, 2010). Therefore, gonadal development in the termite would appear to be the most significant biological process that leads to the formation of caste-specific differences in tasks and status. Spermatogenesis is a complex process of proliferation and differentiation, transforming spermatogonia into mature spermatozoa. In insects, this process involves spermatogonial proliferation by repeated mitotic divisions, resulting in a group of primary spermatocytes. These spermatocytes undergo two successive meiotic divisions and give rise to spermatids, which, after deep morphological changes (spermiogenesis), form sperm bundles (Dallai, 2014). Functional spermatogenesis requires an intrinsic regulation, which is managed by a conserved genetic program that underlies the development of germ cells, ensuring sperm viability. The lack of an efficient control mechanism may lead to spermatogenetic alterations, which, in turn, can result in the production of damaged spermatozoa (Dias et al., 2013, 2015). Aberrant spermatogenesis in the two species Stenognathellus denisi and Sminthurides aquaticus (Collembola) is described, in which the secondary spermatocytes with a small size and reduced cytoplasm are not able to perform the second meiotic division and degenerate (Dallai et al., 2004). The number of sperm produced can be affected by environmental conditions, such as food deprivation, a condition capable of determining a significant reduction in the number of sperm produced by the moths Plodia interpunctella and by the dipterans Sarcophaga stercoraria and Drosophila melanogaster (Gage and Cook, 1994; Hellriegel and Blanckenhorn, 2002; Amitin and Pitnick, 2007). In addition, the number of sperm also depends on the size of both sperm and testis (Pitnick et al., 2009). However, there have been no studies to examine the characteristics of spermatogenesis in workers and soldiers of termites. During spermatogenesis, the balance between proliferation and cell death signals determines the maturation fate of the germ cells. Apoptosis, also known as programmed cell death, often denotes an active and highly choreographed process of cell suicide and may be triggered by several factors, including intrinsic signals and external stimuli (Baum et al., 2005). Invertebrates and vertebrates display many similarities in the use of apoptosis to eliminate dangerous or damaged cells and regulate cell numbers by removing excess cells during spermatogenesis. Failure of apoptosis to selectively deplete abnormal germ cells earmarked for programmed cell death would lead to abnormal spermatozoa in the ejaculate and male infertility (Sakkas, 2003). In the insect Lepidoptera, the degenerative changes in the germ cells of the testis during pupal diapause are controlled by apoptosis, and apyrene sperm development occurs by the apoptosis of their nuclei (Polanska et al., 2005; Shimoda et al., 2007). One of the best studied aspects of apoptosis in social insects occurs during caste determination in bees (Baum et al., 2005). In the honeybee Apis mellifera, an adult queen ovary contains as many as 200 ovarioles, whereas the ovary of a worker has less than 10. This large discrepancy in ovariole number is a result of apoptosis (Capella and Hartfelder, 1998). In the termite Reticulitermes aculabialis, our previous studies also indicate that apoptosis during early oogenesis is an efficient and crucial regulatory mechanism affecting the number of oocytes and the reproductive capacity of the reproductives, soldiers and workers (Su et al., 2014). Therefore, the available evidence suggests that apoptosis is not only necessary for
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the normal development of germ cells in many organisms but also plays a vital role in the caste differentiation in social insects. Although Reticulitermes is an economically important genus and is probably the most studied of all termites, the precise development pattern in workers and soldiers remains uncertain. The separation of reproductive and non-reproductive roles based on caste differentiation is the most prominent characteristic of termites, and the clarification of the mechanism underlying this separation is an important topic in termites. The difference in the physiological detail of testis development among workers, soldiers and reproductives is unclear, although the general morphology of the male reproductive system and sperm of termites have received attention (Sieber and Leuthold, 1982; Riparbelli et al., 2009; Hartke and Baer, 2011). To obtain a better understanding of the development of the testes underlying reproductive division in various castes and to reveal whether each male worker has reproductive flexibility, spermatogenesis and apoptotic patterns were investigated and compared in reproductives, workers and soldiers of the termite Reticulitermes labralis. 2. Materials and methods 2.1. Termites R. labralis colony fragments were collected from the monasteries of Xi'an, China, in May 2011 when the alate adults were swarming. The colonies were comprised of workers, soldiers, and alate reproductives. Newly hatched individuals (larvae) developed into workers (third instar workers) or nymphs (third instar nymphs) through two successive moults, distinguished by the absence or presence of wing pads, respectively. The third instar workers (W3), fourth instar workers (W4) and fifth instar workers (W5) (early instar workers) were identified by antennal segments, the width of the heads and the length of the bodies. Sixth instar workers (W6) (late instar workers, WL) were identified by the presence of 16 or more antennal segments. The male reproductive and late instar worker individuals were discriminated according to the seventh sternite, and the male early instar workers and soldier individuals were discriminated by longitudinal sections. After collection from the field, the abdomens were immediately removed and fixed in Bouin's solution for HE (haematoxylin and eosin) staining. They were also fixed in 4% paraformaldehyde in PBS overnight at 4 C for terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) analysis. 2.2. Measurement of testis development in reproductives, workers and soldiers The testes of the reproductives and W6 were dissected out in PBS under a stereomicroscope to examine the testis morphology of R. labralis. The testicular development of the workers, soldiers and reproductives was evaluated with HE staining. The fixed samples were dehydrated in an ascending ethanol series and embedded in paraffin. Longitudinal sections 6 mm thick were collected on polylysine-coated slides. Deparaffinised and rehydrated sections were stained in HE. Because the testes of R. aculabialis had a mostly round shape, the diameter of each testis at its widest point was measured using a digital microscope in accordance with a previous study on R. speratus (Shimada and Maekawa, 2010). 2.3. Spermatogenesis in various castes Thin sections through the testes showed different stages of spermatogenesis in the testicular follicles of the reproductives,
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Fig. 1. The testes in various castes of the termite Reticulitermes labralis. (A) Testis and vas deferens of a fifth instar worker. (B) Testis of a sixth instar worker. (C) Testis of a reproductive. (D) Testis in longitudinal section of abdomen of a reproductive. (E) Testis in longitudinal section of abdomen of a third instar WN. (F) Testis in longitudinal section of abdomen of a fourth instar WN. (G) Testis in longitudinal section of abdomen of a fifth instar WN. (H) Testis in longitudinal section of abdomen of a sixth instar WN. (I) Extremely degenerate testis in longitudinal section of abdomen of a soldier. Te, testis; VD, vas deferens.
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3. Results 3.1. Testicular development in the reproductives, workers and soldiers
Fig. 2. The mean size of testes in reproductives, workers and soldiers of the termite Reticulitermes labralis (n ¼ 10). The columns represent the means; bars represent SD. The letters (aed) above the columns indicate significant differences (p < 0.05) among the various castes. R, reproductives; S, soldiers; WS, workers with spermatozoa; WN, workers without spermatozoa.
workers and soldiers. The spermatogenic stages were categorized based on the morphogenetic criteria of germ cells (Sieber and Leuthold, 1982; Chawanji et al., 2007; Kubo-Irie et al., 2011; Paoli et al., 2013; Carcupino et al., 1998). The number of spermatozoa per longitudinal section of the testes was counted, and the sum of the results was used as the number of spermatozoa per testis. The diameter of the germ cells at each stage was measured based on the captured digital images. 2.4. TUNEL assay Detection of the DNA fragments was performed in paraffinembedded sections by TUNEL analysis using the ApopTag peroxidase in situ apoptosis detection kit (Roche Applied Science, Germany). The paraffin-embedded sections were dewaxed and incubated with 3% H2O2 for 10 min at room temperature and washed in distilled water. Following this step, the sections were incubated with PBS containing 20 mg/ml proteinase K for 10 min at 37 C and washed three times in PBS for 5 min each. The TUNEL reaction mixture (30 ml of 3 ml enzyme solution and 27 ml label solution) (Roche Applied Science, Germany) was added to the sections for 60 min at 37 C, and the slides were rinsed in PBS. Then, 30 ml converter-POD (peroxidase) was added to the sample for 30 min at 37 C and washed in PBS. The sections were incubated in 3, 30 -diaminobenzidine tetrahydrochloride (DAB, Boster Co. Ltd., Wuhan, China) solution for 5e10 min at room temperature. Finally, the slides were stained with haematoxylin counterstain. For the negative control, 30 ml label solution, instead of the TUNEL reaction mixture, was used. The sections were analysed by light microscopy, and the apoptotic cells were identified by brown nuclei. The apoptotic rate during spermatogenesis was defined as the ratio of the number of TUNEL-positive germ cells within the total germ cells per longitudinal section of the testes. 2.5. Statistical analysis A statistical test was performed by the usual methods for multiple comparisons (Tukey's test) using SPSS version 16.0 (SPSS Inc. Chicago, Illinois). All values are expressed as the mean ± SD. P values less than 0.05 were considered statistically significant.
The male individuals of R. labralis have a pair of round testes (Fig. 1A, B and C). Each testis is composed of a varying number of testicular follicles, in which spermatogenesis is synchronous. In the male reproductives, sixth instar workers with spermatozoa (W6s) and soldiers, the mean size of the testes was 204.3 ± 26.3 mm, 162.2 ± 28.1 mm and 36.0 ± 2.8 mm, respectively. The mean size of the testis of the reproductives and late instar workers was approximately six-fold and four-fold longer compared with the soldiers, respectively. Compared with the early instar workers, the testes of the soldiers were also extremely degenerate. The difference in testis development among the reproductives, late instar workers and soldiers was significant (Figs. 1DeI and 2). The workers were divided into two types: the workers (WS) in which testes contained spermatozoa (Fig. 3CeF) and the workers (WN) in which the testes did not produce spermatozoa (Fig. 1EeH). In early instar workers (W3,W4 and W5), there was no significant difference in the mean diameter of the testes between WS and WN individuals. The size of the testes slightly increased during W3 developing into W5. However, the testis size increased substantially from W5 to W6. The mean diameter of the testes in W6S and W6N was approximately three-fold and two-fold longer compared with early instar workers, respectively, indicating that the testis in late instar workers developed significantly (Fig. 2). 3.2. Spermatogenesis in reproductives, workers and soldiers Thin sections through the testes showed different stages of spermatogenesis in testicular follicles. Spermatogenesis in R. labralis was similar to that described for other insect orders. Spermatogenesis is divided into four stages: mitotic proliferation of spermatogonia, meiotic division of spermatocytes, differentiation of spermatids and final transformation of spermatozoa (Dallai, 2014). In the reproductives and WS, the spermatogenetic process occurred synchronously in the testicular follicles where germ cells arranged in an ascending order of development. By comparison, spermatogenesis in soldiers and WN only reached the spermatocyte stage. Therefore, spermatozoa were absent in the soldiers and some of the workers (Fig. 3). In the soldiers, no spermatids and spermatozoa were observed (Fig. 3G), and the size of the spermatogonia and spermatocytes (3.8 ± 0.5 mm and 2.7 ± 0.3 mm, respectively) was significantly smaller compared with W6s and reproductives (Fig. 4). The soldiers had a few germ cells, approximately 90e120 per testes. In the reproductives, the spermatogonia 6.2 ± 0.9 mm in diameter appeared irregular in shape and had the largest diffuse chromatin (4.2 ± 0.4 mm). The spermatocytes 5.1 ± 0.6 mm in diameter were characterized by clearly visible condensed chromatin and roundish nuclei (3.2 ± 0.2 mm). Compared to the spermatocytes, the size of spermatids and their nuclei was significantly smaller (2.4 ± 0.2 mm and 1.7 ± 0.2 mm, respectively). The spermatids became smaller progressively, the nuclear size was reduced, the excess cytoplasm was eliminated, and they were finally transformed into the globular aflagellar spermatozoa 1.5 ± 0.1 mm in size (Fig. 4). The reproductives had 5724 ± 1683 spermatozoa per testis (Fig. 5). During the third instar to the fifth instar stage, the number of spermatozoa of Ws gradually increased by approximately 30e200 in a small number per testes. However, the number of spermatozoa in the W6S was approximately ten-fold higher compared with the W5S (Fig. 5). Although there was no significant difference in the size
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Fig. 3. Spermatogenesis in various castes of the termite Reticulitermes labralis. (A) Spermatozoa in the testes of reproductives. (B) The process of spermatogenesis, transforming spermatogonia into mature spermatozoa are shown in the testicular follicles of reproductives. (C) Spermatozoa in third instar Ws. (D) Spermatozoa in fourth instar Ws. (E) Spermatozoa in fifth instar Ws. (F) Spermatozoa in sixth instar Ws. (G) Spermatogenesis in soldiers is arrested at the spermatocyte stage. Sg, spermatogonia; Sc, spermatocytes; St, spermatids; Sp, spermatozoa.
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Fig. 4. The mean size of the germ cells at each stage in the various castes of the termite Reticulitermes labralis (n ¼ 30). The columns represent the means; bars represent the SD. Letters (a and b) above the columns of the same colour indicate significant differences (p < 0.05) among the various castes. R, reproductives; S, soldiers; W6S, sixth instar workers with spermatozoa; W6N, sixth instar workers whithout spermatozoa.
of the germ cells between the reproductives and W6S, the number of spermatozoa in reproductives was approximately three-fold higher than that in W6S. There was a significant difference in the size of the spermatogonia and spermatocytes between the W6S and W6N (Fig. 4). 3.3. Apoptosis in the testes of reproductives, workers and soldiers Apoptosis in testes was confirmed using a TUNEL assay, which identified nuclei with clear signs of DNA fragmentation, a hallmark of apoptosis. In the reproductives, the majority of positive nuclei were located in spermatogonia, indicating that apoptosis occurred mainly at early stages of spermatogenesis. Most of the apoptotic spermatogonia in the testes were not randomly distributed throughout the testicular follicles, but cohorts of 2e4 neighbouring apoptotic spermatogonia were often noted to be degenerating synchronously. The spermatocytes and spermatids showed very little apoptosis, and no TUNEL signal was detected in any of the examined spermatozoa (Fig. 6A, B and C).
Fig. 5. The mean number of spermatozoa per testis in reproductives and each instar workers of the termite Reticulitermes labralis (n ¼ 10). The columns represent the means; bars represent the SD. The letters (aee) above the columns indicate significant differences (p < 0.05). W3S, third instar workers with spermatozoa; W4S, fourth instar workers with spermatozoa; W5S, fifth instar workers with spermatozoa; W6S, sixth instar workers with spermatozoa, R, reproductives.
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The mode of apoptosis in W6S was similar to that of the reproductives. The apoptosis of germ cells mainly took place in the spermatogonia, and only one or two spermatocytes and spermatids per longitudinal section of the testes were TUNEL positive (Fig. 6D). There was no significant difference in the apoptotic rate of the spermatogonia between the workers (11.6 ± 2.8) and the reproductives (9.5 ± 2.3) (Fig. 7). Compared with the W6S, in the W6N, the testes exhibited stronger TUNEL-positive staining and a higher apoptotic rate of spermatogonia (21.8 ± 3.7) (Figs. 6E and 7). In the soldiers, the testes exhibited the strongest TUNELpositive staining (Fig. 6F) and the highest apoptotic rate of spermatogonia (Fig. 7). Apoptotic cells occupied the entire region of the testicular follicles. The apoptotic rate of spermatogonia was approximately four-fold higher compared with the W6S and reproductives, and two-fold higher compared with the W6N, indicating that a greater number of germ cells in the soldiers had undergone degeneration. 4. Discussion For R. labralis male workers, we established a basal developmental schema comprising two undifferentiated larval instars, followed by a bifurcated development into either (i) the sexual lineage, in which the workers maintained normal spermatogenesis during the third instar to late instar stage, and at the late instar stage became either apterous neotenic reproductives via a single moult or inconspicuous reproductive males, or (ii) the neuter lineage, in which the workers did not produce spermatozoa and their spermatogenesis stopped at the spermatocyte stage. This study of testis development in individual workers of R. labralis indicates that there is a significant difference in spermatogenesis, but not in testis size, among early instar worker individuals and that the degenerated testis can be characterized by the absence of spermatids and spermatozoa. As in WN and soldiers of R. labralis, it has been previously reported that in the testis of diapausing insects, the meiotic divisions that lead to the formation of spermatids do not occur or are somehow altered (Polanska et al., 2005). Our results show that in R. labralis, a sharp increase in the testis size of workers occurs at the sixth instar stage. In Reticulitermes, the last three instar nymphs can moult into brachypterous neotenic reproductives, whereas late instar workers can moult into apterous neotenic reproductives (Buchli, 1958). Therefore, the male W6S of R. labralis are defined as a functional category of individuals performing work, while retaining the reproductive potential to copulate with female reproductives. This latter property distinguishes them from W6N, which result from an early and irreversible developmental bifurcation. A late instar WS is able to copulate with and provide spermatozoa to queens, suggesting that there is a fundamental difference in the reproductive capability between male and female workers in R. labralis. Although the presence of oocytes is a key mechanism for female workers having the potential for differentiation into neotenic reproductives through a moult, the absence of vitellogenic oocytes in workers results from oogenesis stopping at the previtellogenesis stage, which is similar to those in the last instar nymphs (Su et al., 2014). Therefore, the female late instar workers are not able to produce mature eggs unless they moult into conspicuous reproductive females (apterous neotenic reproductives), which have significant differences in morphology compared with the female workers. Interestingly, a strongly female-biased sex ratio among the emerging neotenic reproductives has been observed in R. speratus. However, when the numbers of inconspicuous mature males were added to the number of the conspicuous male neotenic reproductives, the sex ratio was almost 1:1 (Fujita and Watanabe, 2010). Therefore, insurance against the loss of male
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Fig. 6. Apoptosis during spermatogenesis of the termite Reticulitermes labralis. (A) Apoptotic spermatogonia and spermatids in the testes of reproductives. Two neighbouring apoptotic cells apparently are degenerating synchronously. TUNEL positive germ cells were identified by brown nuclei. (B) A TUNEL positive spermatocyte was observed in the testes of reproductives. (C) An apoptotic spermatogonium in the testis of a reproductive. (D) Apoptotic spermatogonia in sixth instar Ws. (E) In sixth instar W6N, the testes exhibit strong TUNEL-positive staining. (F) In soldiers, the testes exhibit strong TUNEL-positive staining. (G) Negative control. Sg, spermatogonia; Sc, spermatocytes; St, spermatids.
reproductives, possibly in the form of the presence of inconspicuous males as a male reproductive “backup,” would appear to be important for the functionality of the termite society. It would be reasonable to suppose that the termite colony could maintain a 1:1
sex ratio among reproductives by producing a supplemental inconspicuous type of male neotenic. In addition, the male reproductive individuals of termites cannot store a large number of spermatozoa due to the absence of seminal vesicles, whereas a
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Fig. 7. Apoptotic rates of spermatogonia in the reproductives, W6S, W6N and soldiers of the termite Reticulitermes labralis (n ¼ 10). The columns represent the means; the bars represent the SD. Letters (a, b, and c) above the columns indicate significant differences (p < 0.05) among the various castes. W6S, sixth instar workers with spermatozoa; W6N, sixth instar workers without spermatozoa.
physogastric queen needs approximately 40,000 sperm per day, which likely cannot be produced by a male (Sieber and Leuthold, 1982). Thus, one female reproductive mates with several male reproductives in a short time (Wu et al., 2013). Participation of the male workers in sexual reproduction has been confirmed by genetic analysis in R. labralis (Wu et al., 2013). Therefore, we believe that in Reticulitermes, the inconspicuous male reproductives are the late instar workers with spermatozoa, and the spermatozoa of late instar workers are able to participate in fertilisation. A higher frequency of apoptosis in the spermatogonia of R. labralis indicates that a critical checkpoint occurs in the germarium of the testicular follicles in response to an imbalance in cell numbers. In the zone of the germarium, the spermatogonia undergo several mitotic divisions to form primary spermatocytes. Therefore, the results of the present study support previous studies in which the apoptosis of germ cells coincides with mitotic peaks (Heninger et al., 2004). Degeneration during normal spermatogenesis is also observed in the testes of reproductives in R. labralis, where some spermatogonia display characteristics of apoptosis. In R. labralis, the majority of apoptotic spermatogonia were not randomly distributed throughout the follicles, but cohorts of neighbouring apoptotic spermatogonia degenerated synchronously. This observation was consistent with groups of cells interconnected by cytoplasmic bridges that were synchronously undergoing apoptosis (Gilboa and Lehmann, 2004; Heninger et al., 2004). Intercellular bridges derived from polyfusomes are common in spermatogenesis of animals, but it is only in the ovarioles of female insects where such bridges are also formed and where their function is best studied, much better than in males. Furthermore, the consequences of the disorganization of polyfusomes have been suggested as a major cause for autophagic cell death in the larval ovaries of honey bee workers (Capella and Hartfelder, 2002). Significantly, slight increases in the level of apoptosis in spermatogonia can have dramatic effects on overall germ cell numbers (Heninger et al., 2004). Therefore, the apoptotic rate in various castes of R. labralis suggests that apoptosis during early spermatogenesis is an efficient and crucial regulatory mechanism affecting both the number of germ cells and the reproductive capacity of labour in termites. Apoptosis also occurs during the spermatocyte and spermatid stages of spermatogenesis in the reproductives and W6S of
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R. labralis. Although only one or two spermatocytes and spermatids appear to be apoptotic in the testes of reproductives and WS, the checkpoints of spermatocytes and spermatids provide a final opportunity for the testes to eliminate defective or abnormal germ cells before developing into spermatozoa. In rodents, as well as cats and rabbits, high levels of germ cell apoptosis are also observed in spermatocytes, which proceed through meiosis, suggesting that the meiotic checkpoint may be present to help correct for the number of germ cells and to account for problems such as rearrangement during meiosis or unrepaired breaks in DNA by inducing apoptosis of these damaged cells (Baum et al., 2005). However, in the reproductives and W6S of R. labralis, only one or two apoptotic spermatocytes and spermatids were randomly distributed in the testes, indicating that the two apoptosis checkpoints are used as “quality control” to eliminate defective spermatocytes and spermatids with incomplete genetic components or chromosomal abnormalities. No TUNEL-positive spermatozoa are observed in the reproductives and W6S of R. labralis, indicating that during normal spermatogenesis, apoptosis can eliminate all defective germ cells before the spermatids transform into spermatozoa. The presence of spermatozoa that have damaged nuclear DNA or loosely packaged chromatin has been confirmed in male infertility. In humans, a strong association has been found between abnormal semen parameters and the presence of apoptosis in ejaculated spermatozoa (Sakkas, 2003). The presence of nuclear DNA strand breaks in spermatozoa is due to anomalies in apoptosis during spermatogenesis, and high levels of apoptosis in spermatozoa result in male infertility (Sakkas, 2003). Sakkas et al. (1999) also reported that abnormal spermatozoa could exist and express various apoptotic markers because they have escaped programmed cell death, a procedure termed ‘abortive apoptosis.’ These findings allow us to extend the identification of abnormal or normal spermatogenesis in R. labralis and to suggest that in R. labralis, the W6S produce the same spermatozoa as reproductives. The histological data demonstrate that male late instar WN and soldiers have lost the ability to develop into neotenic reproductives or inconspicuous reproductive males in R. labralis. Compared with the reproductives and late instar WS, the size of the testes and germ cells in the late instar WN and soldiers are significantly smaller, and their testes exhibited strong apoptotic staining. Similarly, Dallai et al. (2004) reported abnormal spermatogenesis in Collembola, in which the aberrant secondary spermatocytes are characterized by small size, reduced cytoplasm and nuclei with very condensed chromatin, and these secondary spermatocytes are not able to perform the second meiotic division and degenerate. The degeneration of germ cells in the testis and ovaries are controlled by apoptosis (Capella and Hartfelder, 1998; Baum et al., 2005; Polanska et al., 2005; Shimoda et al., 2007). Therefore, the spermatogonia and spermatocytes of the WN and soldiers in R. labralis are abnormal and degenerate. The involvement of apoptosis is extremely important for WN and soldiers to eliminate germ cells and remain sterile. Some of the male workers lose reproductive options earlier in development, which may enhance the ability to complete tasks and require less energy. Acknowledgments We thank Li Gang for help with collecting termites. Financial support was provided by the National Natural Scince Foundation of China (31370428 and 31170363) and the Scientific Research Program Funded by Shaanxi Provincial Education Department (2013JK0715).
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