Long-term rearing of Cynops pyrrhogaster and Cynops ensicauda popei confirmed that the longevities of the two species were longer than 36 years; C. pyrrhogaster longer than 37 years and C. e. popei longer than 36 years, respectively. The male C. e. popei that was 34 years old or older showed reproductive behavior with a conspecific young female and successfully bred. The paternal relationships between the old male and the six offspring were also genetically proven, indicating that the male C. e. popei retained its fertility at the age of 34 or older. Since physiological longevity and reproductive ability are important parameters for life history studies, our findings are meaningful for the conservation of the species.
Introduction
An organism's longevity is closely related to many other parameters of its life history, such as lifetime reproductive success, age of sexual maturation, and survivorship. Thus, longevity is one of the most important parameters for considering the life history characteristics of different species (de Magalhāes and Costa, 2009; Niemiller et al., 2016). The longevity of amphibians in the wild is often estimated by skeletochronology (Wells, 2010). However, it is difficult to estimate the exact age of long-lived species because lines of arrested growth (LAGs) can be compressed, resulting in underestimated ages (Staub, 2016). Therefore, the records of longevity based on reliable rearing and those on recapturing of individually marked ones should be considered.
The aging process is known as a factor that determines the longevity of organisms. Aging leads to a decline in physiological functions and is associated with increased mortality and decreased fertility (Jones and Vaupel, 2017). Although aging has hitherto been considered a common feature in organisms, increasing empirical data indicate that some organisms have substantial resistance to aging process (Schaible et al., 2015). In salamanders, three species of Salamandridae (Lyciasalamandra fazilae, Salamandra salamandra, and S. perspicillata) are known to show only negligible aging (Cayuela et al., 2019).
The longevity of animals is strongly correlated with their body sizes, but some salamanders have longer longevities than those expected from their body sizes (Yun, 2021) (e.g., Proteus anguinus are: 100 years [Voituron et al., 2011; Tacutu et al., 2018], S. salamandra: 50 years [Böhme, 1979], Andrias japonicus: 55 years, Ambystoma maculatum: 25 years, Am. tigrinum: 25 years, Amphiuma means: 27 years, Cryptobranchus alleganiensis: 25 years, Siren lacertina: 25 years [Carey and Judge, 2000; de Magalhāes and Costa, 2009; Wells, 2010]).
The genus Cynops is a group of modern newts, occurring in Japan (Frost, 2023). The sword-tailed newt, Cynops ensicauda is a newt endemic to the Amami Islands and Okinawa Islands of the Ryukyu Archipelago (Tominaga et al., 2010). There are two subspecies of this species, C. e. ensicauda and C. e. popei (Inger, 1947). The red-bellied newt C. pyrrhogaster occupies mainland Japan and is a sister species of C. ensicauda.
The longevity of C. pyrrhogaster have been reported to be 25 years (Sato, 1943). In captivity, an individual of C. pyrrhogaster has been recorded as surviving to about 30 years of age (Eguchi et al., 2011). In other cases, two individuals also reported to have survived for at least 33 and 42 years, one individual survived more than 34 years, and four individuals have been kept in captivity for more than 47 years, two of which are likely to have reached more than 50 years old (Köhler et al., 2023).
The age structure of wild populations of C. pyrrhogaster was studied by Marunouchi et al. (2000) based on skeletochronology, and the age of mature individuals was estimated to be 3–23 years. The maximum age of C. pyrrhogaster was also estimated to be 31 years for males and 23 for females based on the 27-year-long field study by mark and recapture method (Kusano, 2020). However, there have been no reports on the age and longevity of C. ensicauda. In this paper, we report on the longevity of the two species, C. pyrrhogaster and C. ensicauda, and the maximum reproductive ages of C. ensicauda, based on long-term rearing records.
Materials and Methods
Procurement and rearing conditions of the newts
The female of C. pyrrhogaster (hereafter referred to as CpF) was purchased by the third author at an aquarium fish shop in Saitama Prefecture in the summer of 1987 (Fig. 1). It already had a mature body size at the time of purchase. The individual of C. ensicauda is a male (hereafter referred to as CeM) purchased by the third author from the same aquarium fish shop in the summer of 1988 (Fig. 1). At the time of purchase, the body size had already reached a mature size. Four other C. ensicauda were purchased at the same time and reared, but CeM was easily distinguishable from these four as the male with most white mottles. CeM and CpF were kept in the same aquarium.
In October 2020, a captive bred female of C. ensicauda, which was born in November 2018 (hereafter referred to as CeF), was introduced and kept with CeM and CpF from November 2020.
Snout vent length (SVL) and body weight of CeM and CeF were measured on February 18, 2023. The SVL of CpF was measured from the photograph taken on March 9, 2022, using ImageJ software (version 1.53a). SVL and body weight of these individuals were not measured at the time of purchase.
Breeding conditions in captivity
Between 1987 and 2015, the newts were kept in Saitama Prefecture in a 60×30×36 cm fish tank with a 2 cm layer of gravel on the bottom and a 5 cm depth of water. One loach (Misgurnus anguillicaudatus) was cohabitated during 1987–2015. They were kept in an indoor environment at room temperature. The temperature exceeded 30°C on several days in summer. In winter, rearing temperatures often dropped to below 10°C. The water supplied from a well (pumping depth 180 m) was changed approximately once a week. Live larvae of nonbiting midges (Chironomidae) were fed mainly as food.
Fig. 1.
A: A photograph of CpF and CeM taken on November 5, 1991. Black arrow indicates CeM. White arrow indicates CpF. B: CeM on September 7, 2021. C: CpF on March 16, 2023.
In 2015, the rearing environment has been changed because of moving the house to Hokkaido. They were kept indoors in the same fish tank as mentioned above, but aged tap water for more than one day was used as rearing water. The rearing temperature was warmer than 20°C, even in winter, and in summer was about 23°C–28°C. Commercial feed for turtles (Kamepros: Kyorin) and supplementary thawed frozen larvae of nonbiting midges have been fed since 2015. CeM and CeF were reared in the same tank since November 2020 and CeF bred thereafter.
Confirmation of parent–child relationship between offspring and CeM
Since CeF was reared with other C. ensicauda before introduction, it could have retained sperm received from other individuals in the seminal vesicles before introduction to the aquarium tank. In order to clarify the paternity, the parent–child relationship between juveniles obtained and CeM was investigated using microsatellite markers.
The tail tips of CeM, CeF, and six juveniles were cut under MS222 anesthesia and placed in 99% ethanol for cryopreservation. Tissue from each individual was added to a mixed solution of 660 µL of 1×STE Buffer, 60 µL of 10% SDS solution, and 10 µL of proteinase K (23 ng/µL) and incubated at 55°C overnight. Then, total DNA was extracted according to the protocol of the Promega Wizard Genomic DNA Purification Kit. Finally, it was dissolved in 50 µL of 1×TE Buffer and stored in a refrigerator.
For microsatellite analysis, 15 nuclear microsatellite loci (CP_NGS21, CP_EST30, CP_ EST10, CP_NGS36, CP_NGS19, CP_NGS26, CP_EST05, CP_NGS11, CP_NGS35, CP_ NGS24, CP_EST03, CP_EST21, CP_NGS43, CP_EST02, and CP_NGS46) developed by Tominaga et al. (2014) were used. Microsatellite analysis experiments followed the method of Tominaga et al. (2021). The amplified microsatellite samples were run through a sequencer (ABI3130) for fragment analysis, and fragment lengths were determined for each locus in every individual using Geneious 9.0.2 software for genetic analysis.
Paternal relationships between CeM and six offspring were confirmed by the fragment length of each locus. In all amplified loci, if both the allele of CeM and the allele of CeF were shared by a juvenile for all loci, CeM was assumed to be the father of the juvenile.
Identification of subspecies of C. ensicauda
To determine the subspecies of CeM and CeF, mitochondrial DNA (mtDNA) sequencing was performed. Part of the cytochrome b (cytb) gene of mtDNA was amplified by PCR. PCR was performed using primers SLD_cytb_L1 (5′TGACCTGAAAATCAAYGTTGTA 3′) and SLD_cytb_H 2 (5′TAGCRAATARRAARTAY CAYTCTGG 3′) (Tominaga et al., 2010) according to the standard protocol of GoTaq Green Master Mix (Promega). Cycle-sequencing reactions were conducted using a BigDye Terminator v. 3.1 (Thermo Fisher Scientific), and DNA sequencing was performed using a DNA sequencer (ABI3130). The sequence of each individual was searched for homology in the DNA sequence database of the DNA Data Bank of Japan (DDBJ) to identify the subspecies.
Results
Long-term rearing of C. pyrrhogaster and C. ensicauda
In July 2022, CpF escaped from the cage and died accidentally. CeM is still alive as of October 2022. All C. ensicauda except CeM, reared since 1988, died by 2015. CpF was an adult size when purchased in 1987 and thus was considered to be at least two years old (Marunouchi et al., 2000). It survived 35 years until the escape, making it at least 37 years old. CeM had also reached the mature size of this species when purchased in 1988 and was assumed to be at least two years old at that time (Tanaka, 2005). CeM is surviving until the end of 2022 and has been in captivity for 34 years, which, combined with the previous two years, makes it at least 36 years old.
Fig. 1A is a photograph of the newts taken in 1991 (four years after rearing). A comparative photograph of CeM was taken in September 2021 (Fig. 1B). Some mottles (yellowish-white spots) have faded over time, while new ones have appeared in other areas. However, the lateral red line on the right side of the body, the areas without spots on the dorsum, and the patterns of spotting defects on the right abdomen and spotting defects on the right caudal indicate that this is the same individual (Figs. 1A and B). Except for the CeF, which was introduced in 2020, all of C. ensicauda involved in this study were purchased in 1988. Since we have not kept any other C. pyrrhogatser except for CpF, there is no possibility that it was replaced by other individuals.
The SVL of CeM and CeF were 65 and 62 mm, respectively, on February 18, 2023. The body weight of CeM and CeF were 10.0 and 8.0 g, respectively. The SVL of CpF on March 9, 2022 was estimated to be 67 mm from a photograph.
Reproduction of long-reared C. ensicauda
When CeF was introduced to CeM in November 2020 (=the beginning of the breeding season of this newt in wild), CeM showed courtship behavior toward CeF. CeF responded to this behavior, taking in spermatophores deposited by CeM. Some spermatophores that failed to be received were also observed during rearing. From December 2020 to January 2021, CeF laid eggs on the Brazilian waterweed Egeria densa, eventually yielding 70 eggs. In December 2020, when oviposition began, CeM and CeF were separated from each other.
Of the 70 eggs, 65 hatched (92.9% survival to hatching). Of the 65 larvae, one had cervical spine curvature, and another had thoracic spine curvature malformations (malformation rate 3.1%). Five individuals died during the larval stage, including that with thoracic spine curvature. Sixty individuals metamorphosed from mid-May to June 2021 (85.7% survival to metamorphosis). The individuals with cervical spine curvature also metamorphosed. Fifty-five individuals were alive until the end of September 2021 (survival rate 78.5%), and then they were transferred to acquaintances at the beginning of October 2021. Six individuals, including the one with cervical curvature, have remained in possession of the original owner and were alive as of the end of 2021.
Subspecies identification by mtDNA analysis
Sequence data for the 824 bp cytb gene of mtDNA for CeM and CeF were obtained. A homology search revealed that the CeM sequence showed 100% homology with the registered sequence of C. e. popei (DDBJ registration number: AB754750). The sequence of CeF also showed 100% homology with the registered sequence of C. e. popei (DDBJ registration number: AB754747). Thus, both CeM and CeF were confirmed to be C. e. popei.
Parent–child determination by microsatellite analysis
The paternity of CeM were confirmed by microsatellite analysis for the six offspring born from CeF. Fourteen loci as previously listed, except for CP_NGS35, were successfully genotyped for CeM and the six offspring. Thirteen loci, except for CP_NGS35 and CP_NGS43, were successfully genotyped for CeF. Of these, four loci were not informative in determining paternity because of the lack of allelic diversity. The analysis confirmed that all six offspring had one allele of CeM origin and one allele of CeF origin at all loci, indicating that the paternity of all six offspring of CeF was contributed by CeM (Table 1).
Discussion
Most estimates of amphibian longevity have been made using skeletochronology (Wells, 2010). However, in skeletochronology, lines of arrested growth (LAGs) seem to be compressed when the growth rate becomes slow in the adult stage. Therefore, it has been pointed out that it is hard to discriminate LAGs in old individuals, and that such old individuals in which LAGs could not be reliably counted may be excluded from the data (Staub, 2016). Thus, the estimates of longevity based on skeletochronology may be underestimated.
The longevity of C. pyrrhogaster in captive conditions has been reported to be 25 to 50 years old (Sato, 1943; Eguchi et al., 2011; Köhler et al., 2023). The longevity of the wild individuals has been reported to be 23 years as estimated by skeletochronology (Marunouchi et al., 2000) and 31 and 23 years old for males and females, respectively, based on the mark and recapture study (Kusano, 2020). In this study, we confirmed that a female C. pyrrhogaster survived for at least 37 years and that a male C. e. popei did for more than 36 years. Thus, the very long lifespan of the species in this genus is not exceptional, as Köhler et al. (2023) pointed out. These long longevity records of the genus Cynops are particularly long compared with the longevities of most other salamanders, although P. anguinus is known to survive longer than this, at 100 years (Voituron et al., 2011; Tacutu et al., 2018), Japanese giant salamander, A. japonicus, has survived more than 55 years (Goin et al., 1978) and S. salamandra has been known to live for more than 50 years (Böhme, 1979).
Table 1.
Genotyping data from 14 microsatellite loci for the eight individuals (CeM, CeF, and six offspring) used in this study. URE: Faculty of Education, University of the Ryukyus. –9: Missing data. The alleles derived from CeM were in bold letters.
It has also been reported that female newts close to 50 years old stopped laying eggs many years ago, while similarly aged males have exhibited courtship behavior toward females (Köhler et al., 2023). We confirmed that a male of C. e. popei older than 34 years retained its fertility. Thus, at least the males of this genus may remain reproductive in old age.
The longevities of animals correlate with their body size, but some salamanders have longer longevities relative to body sizes (Sousounis et al., 2014). A factor contributing to their relatively long longevities might be their resistance to aging process (Yun, 2021). A study examining the aging pattern of three species of Salamandridae (L. fazilae, S. salamandra, and S. perspicillata) found that their survival rates declined slowly with age, and their mortality rate remained constant regardless of age (Cayuela et al., 2019). Therefore, an age-related physiological decline commonly observed among mammals occurs only slightly in these species (Cayuela et al., 2019). It is possible that the resistance to senescence is also retained in many other salamanders. The two species of the genus Cynops observed in this study might also have strong resistance to senescence.
The aging of cells in organisms is induced by various types of cellular stresses, including DNA damage, telomere shortening, oxidative attack, and activation of oncogenes (Gorgoulis et al., 2019). The mechanism of resistance to senescence in salamanders is still not well understood, but the high regenerative capacity of this animal group is thought to be a factor (Seifert and Voss, 2013). Salamanders have a very high regenerative capacity compared to other animal groups (Yun et al., 2015). C. pyrrhogaster is also known to have an extremely high regenerative capacity, even in old age (Eguchi et al., 2011; Sousounis et al., 2015). A strong macrophage-dependent immune surveillance mechanism is significant for eliminating senescent cells as a factor in maintaining the regenerative capacity of salamanders (Yun et al., 2015). It has also been suggested that the newts and axolotls are highly resistant to cancer (Ingram, 1971; Tsonis, 1983), which may also be a factor in their resistance to aging (Yun, 2021). Future research is expected to confirm whether these factors are related to the resistance to aging and long longevity of salamanders.
Our result indicates that C. pyrrhogaster and C. e. popei are long-lived species capable of living for over 34 years. It also showed that the male of C. e. popei remains its fertility at that age at least. Long-lived species are generally less vulnerable than short-lived species to temporary deterioration in reproductive success caused by climate change and other factors (Morris et al., 2008). Even if there are several years with low reproductive success, long-lived species can reproduce in other years as long as adults survive. Therefore, C. pyrrhogaster and C. e. popei might be able to maintain their populations even in an environment where they are not able to reproduce in some years by poor environmental conditions (e.g., insufficient water at the breeding site, temporary disturbance of the breeding environment), as long as adults can survive. On the other hand, it has been noted that long-lived species tend to have optimistic estimates of population extinction, as there is a considerable time lag between the decline in new recruitment and the extinction of the population (Jachowski and Hopkins, 2018). Both two Cynops species are collected in significant numbers for the domestic and international pet trade (IUCN, 2021a, b). There are also disappearances of populations in both species due to habitat destruction. It is necessary to focus on multiple age classes and assess population status when designing conservation strategies for C. pyrrhogaster and C. e. popei.
Acknowledgments
The genetic analysis of this study was approved by the University of the Ryukyus Animal Experimentation Committee (approval number 2021055).
© 2023 by The Herpetological Society of Japan
