2urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7Eurn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727AZooKeysZK1313-29891313-2970Pensoft Publishers10.3897/zookeys.880.3356933569Research ArticleCephalopodaSepiidaConservation BiologyPopulation geneticsCenozoicAsiaMultiple paternity assessed in the cuttlefish Sepiellajaponica (Mollusca, Cephalopoda) using microsatellite markersLiuLiqin1ZhangYao1HuXiaoyu1LüZhenming1LiuBingjian1JiangLi Hua1GongLigongli1027@163.com1National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, College of Marine Sciences and Technology, Zhejiang Ocean University, Zhoushan 316022, ChinaZhejiang Ocean UniversityZhoushanChinaNational Engineering Research Center for Facilitated Marine Aquaculture, Zhejiang Ocean University, Zhoushan 316022, ChinaZhejiang Ocean UniversityZhoushanChina
Corresponding author: Li Gong (gongli1027@163.com)
Academic editor: Jiri Frank
2019141020198803342C0877440-9CDE-5EB6-BE94-F8B6C35AE342B748E660-232D-4292-BFCF-BBFBA8B8614835156100102201930082019Liqin Liu, Yao Zhang, Xiaoyu Hu, Zhenming Lü, Bingjian Liu, Li Hua Jiang, Li GongThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/B748E660-232D-4292-BFCF-BBFBA8B86148
Multiple paternity was demonstrated for seven clutches of eggs and 40 offspring sampled from these clutches in the cuttlefish Sepiellajaponica from Fujian Shacheng Harbor Cultivation Base (Fujian Province, China), using four microsatellite DNA markers. It was observed that female cuttlefish copulated with different males. In this study, genotyping data suggest that at least three paternal allele genotypes were present in all seven clutches indicating that at least two males were responsible for each brood. Combined with behavioral observations, this study provides evidence for sperm competition and multiple paternity in S.japonica.
Liu L, Zhang Y, Hu X, Lü Z, Liu B, Jiang LH, Gong L (2019) Multiple paternity assessed in the cuttlefish Sepiella japonica (Mollusca, Cephalopoda) using microsatellite markers. ZooKeys 880: 33–42. https://doi.org/10.3897/zookeys.880.33569
Introduction
The cuttlefish Sepiellajaponica Sasaki, 1929 (Mollusca, Cephalopoda) is a commercially important marine species in China. Production from wild stocks reached 60,000 tons in Zhejiang Province and accounted for more than 9.3% of provincial fishing catches in 1957 (Liu 2002; Wu et al. 2010). The resource of S.japonica has declined since the 1980s due to over-fishing and pollution (Jiang et al. 2014). To enhance production, artificial breeding methods are being developed in China and successful aquaculture techniques have been established in recent years (Yin et al. 2013). However, studies have revealed that the populations and individual genetic diversity in this species has declined under artificial conditions (Song and Wang 2009; Xu et al. 2011). The factors affecting the maintenance of genetic diversity have been a primary concern of conservation biologists.
An important factor that affects the genetic diversity of a population is the effective population size (Ne) which in turn is greatly influenced by the mating system of a species (Hoekert et al. 2002). The mating system influences Ne through changing the number of individuals contributing to subsequent generations (Brown et al. 2005). In a polyandrous mating system, females mate with several males within a single reproductive cycle in which the clustered offspring are descended from multiple males (Pearse and Anderson 2009). In such a mating system, Ne increases, and, as a result, maximizes the genetic diversity of the offspring within a single reproductive season (Sugg and Chesser 1994; Balloux and Lehmann 2003). Some studies have confirmed that a polyandrous mating system is frequent in marine cephalopods including Octopusvulgaris Sasaki, 1929 (Quinteiro et al. 2011), Graneledoneboreopacifica Nesis, 1982 (Voight and Feldheim 2009), Sepioteuthisaustralis Quoy & Gaimard, 1832 (van Camp et al. 2004), Sepiaapama Gray, 1849 (Naud et al. 2005), Loligopealeii LeSueur, 1821 (Buresch et al. 2001), and Loligobleekeri Keferstein, 1866 (Iwata et al. 2005). It is worth noting that the female of these species carries stored sperm from more than one male, and Ne will therefore be significantly higher (Pearse and Anderson 2009). Previous studies have shown that female S.japonica store sperm in the seminal receptacle found in the buccal membrane (Hanlon et al. 1999; Naud et al. 2005). All else being equal, long-term sperm storage enhances the opportunity for multiple matings of this species (Olsson et al. 1994; Ross 2001). Moreover, multiple matings of female S.japonica has actually been observed (Wada et al. 2006). Polyandry, coupled with sperm storage, is an important reproductive strategy for maximizing the genetic diversity of offspring in S.japonica.
In recent years, multiple paternity in several marine species has been documented using different genetic markers including allozymes, DNA fingerprinting, RAPDs, and microsatellites. Microsatellites are the preferred marker because they are widely distributed in the genomes of most organisms and are highly polymorphic (Jarne and Lagoda 1996). Paternity studies based on microsatellites have become increasingly common, and the number of studies using microsatellites has increased (Hoekert et al. 2002; Laloi et al. 2004; Takagi et al. 2008). Several microsatellite markers have been isolated and characterized for S.japonica and used to evaluate the genetic structure of its populations (Wu et al. 2010; Lü et al. 2017). In this study, we used the previously described microsatellite markers to investigate whether multiple paternity occurs in S.japonica. We observed multiple mating and paternity in this species and discussed the possible factors contributing to this reproductive strategy.
Materials and methodsSample collection
Sexually mature adult S.japonica were obtained from the Fujian Shacheng Harbor Cultivation Base (Fujian Province, China). A sample of 200 wild adults was captured using traps and kept mixed into a cage (9 m3). Seawater parameters were continuously maintained at 25–27 °C and 23‰ salinity. From this sample, seven mating pairs were randomly chosen as breeders to produce the next generation. All behavioral interactions were recorded using closed-circuit television with infrared to observe individual animals. Each mating pair was gently captured and placed in a spawning tank until oviposition. Egg strings derived from each clutch were transferred to a hatchery tank. After hatching, 280 offspring were randomly collected for population genotyping, maintained in a tank until they reached a pre-determined age. The muscles from the mantle cavity of parents and offspring were taken and placed in 95% ethanol and stored at –20 °C until DNA extraction. Seven clutches (called A–G) were analyzed.
DNA extraction and amplification
Total genomic DNA was isolated from each offspring and from the muscular tissue of the respective parents using the standard method of phenol-chloroform (Towner 1991). The concentration of DNA was estimated by a spectrophotometer (Nanodrop ND-2000, Thermo Electron Corporation, USA) and then the quality was assessed in 0.8% agarose gel. Three microsatellite loci, chosen from four loci (CL168, CL327, CL3354, CL904) developed specifically for S.japonica by Lü et al. (2017) were used to study genotypes for parents and their offspring.
The amplifications were carried out in a 2720 thermal cycler (ABI, USA) and in a 10 uL reaction volume: 2–10 ng DNA (0.5 µL), 0.5 µL of each forward and reverse primers, 5 µL 2×Es Taq MasterMix and 3.5 µL of double distilled water. The Polymerase Chain Reaction (PCR) conditions were initial denaturation for 5 min at 94 °C, followed by 30 cycles of denaturation for 40 s at 94 °C, annealing for 40 s at a primer-specific annealing temperature, extension for 40 s at 72 °C. PCR products were detected using capillary electrophoresis (BIOptic’s Qsep100 dna-CE, Taiwan) and allele size was estimated using Q-Analyzer software.
Data analyses
Parents and their offspring were genotyped by determining alleles at three of the four microsatellite loci. We considered evidence from at least two loci to be necessary for estimation of multiple paternity, because evidence from one locus may have been caused by mutations or genotyping error (Davy et al. 2011). We determined paternal alleles through subtracting the maternal alleles from offspring in a brood following the technique of FitzSimmons (1998). The minimum number of sires for a clutch was assigned by counting the number of paternal alleles at each locus. Any instance of more than two possible paternal alleles at any loci indicated multiple paternity in a clutch (Buresch et al. 2001). In addition to manual reconstruction, we attempted to estimate paternal number, as genotypes, to corroborate our results using GERUD 2.0 (Jones 2005). Progeny genotypes were tested for conformity with Mendelian inheritance patterns using the X2 test (P < 0.05). Exclusion probabilities were assessed using the program CERVUS v. 2.0 (Marshall et al. 1998).
ResultsBehavioral observations
Mating behavior in S.japonica involves courtship of a female by a male, and females may copulate with multiple males. Mating pairs mated in the head-to-head position during which males transfer spermatophores to the buccal membrane of the females or to an internal seminal receptacle (Fig. 1). The spermatophores that are deposited around the buccal area extrude the sperm mass to form spermatangium. Then the spermatangia attach to the buccal membrane where slowly released sperm are used for fertilization. We found that the male flushed water strongly when he was close to the female buccal area prior to mating with the female. This behavior is thought to dislodge sperm from previous males. We also found obvious courtship rituals and agonistic behaviors after sexual maturity. Males are generally capable of mating early in life (3–6 months maturity) and will continue to mate until senescence. However, the females do not generally lay eggs after copulating until fully mature. The duration of spawning in S.japonica varied from 21 to 30 days. Females lay multiple eggs (from tens to hundreds of thousands) by extruding them from the ovary and then they die shortly after spawning.
Microsatellite loci used for paternity assessment in Sepiellajaponica.
Locus
Repeat motif
Primer Sequences(5’-3’)
Ta(°C)
GenBank Accession
CL168
(AAC)6
F:ACAATCAACGGCTGTAAAGTCA
55
KU306816
R:GACTATGGTTTGGATTTGGCAT
CL3354
(CTG)5…(TGC)5
F:CCTCGGCTTCTGATGAAAAT
55
KU306828
R:AGCCTTACTTCTGCAACATG
CL904
(AT)8
F:TCTAGGCCTGTGGTTAATGT
55
KU306823
R:TGATCGTTACTTGATGGCAG
CL327
(TA)6
F: ACAGCATCTTCTGGTAAGCCAT
58
KX839255
R: TAGTCCTGTCACCACAGTTATGC
Paternity analysis
Three of the four microsatellite markers were chosen to test paternity in seven offspring clutches. These loci exhibited three or more alleles and were polymorphic in each individual. We chose the locus which followed Mendelian inheritance to analyze paternity. Two hundred and eighty-seven individuals were genotyped at three loci, seven adult females and 280 offspring. The analysis was highly reproducible. We analyzed paternity including sampled males and non-sampled males that had copulated with females prior to capture. The exclusionary power of paternity assignments varied between 0.951 and 0.981. Maternal and offspring genotypes for each clutch are given in Table 2.
Genotypes of maternal cuttlefish, offspring and estimated paternal cuttlefish of Sepiellajaponica.
Maternal Genotype
Offspring Genotype
Estimated Paternal Genotype
Clutch Code
Locus
Genotype
I
II
III
IV
V
1
2
3
4
A
CL168
155/170
155/160(21)
155/165(10)
175/170(9)
160/165
175/160
CL3354
240/260
240/250(3)
260/270(18)
240/230(19)
250/270
230/270
CL327
140/170
130/170(2)
140/160(15)
140/170(17)
160/170(6)
130/170
160/140
B
CL168
175/185
175/180(12)
180/185(13)
185/200(5)
160/185(10)
180/180
180/200
160/200
CL3354
230/250
230/240(21)
235/250(9)
230/235(9)
230/250(1)
230/230(1)
240/230
235/235
240/235
CL327
160/160
145/160(16)
155/160(13)
150/160(11)
145/155
145/150
155/150
C
CL168
150/160
140/150(14)
150/155(10)
140/160(12)
136/150(4)
140/140
140/136
150/136
CL3354
200/230
195/230(16)
195/200(6)
200/225(11)
210/230(3)
225/230(4)
200/195
230/195
195/225
CL327
140/154
136/140(13)
140/150(6)
136/154(13)
140/140(3)
150/154(5)
136/136
150/140
136/150
D
CL168
160/180
175/180(13)
160/175(15)
165/180(5)
160/165(6)
160/160(1)
175/165
175/160
175/165
CL3354
220/240
220/235(14)
230/240(7)
255/240(2)
220/225(8)
220/245(9)
235/255
235/245
230/225
CL327
150/180
145/150(14)
160/180(6)
145/180(15)
150/160(1)
180/180(4)
145/160
145/160
160/180
E
CL3354
260/270
250/260(29)
260/263(10)
260/265(1)
250/263
250/265
CL904
210/230
205/230(2)
200/210(4)
200/230(26)
205/210(4)
210/220(4)
200/220
205/200
CL327
140/160
135/140(24)
140/145(8)
135/160(4)
140/150(4)
135/135
145/150
F
CL168
150/160
140/150(15)
140/160(8)
145/150(9)
150/180(7)
160/180(1)
140/180
145/145
140/145
CL3354
240/250
240/250(26)
240/240(1)
240/250(1)
250/260(10)
240/260(2)
250/260
240/250
240/260
CL904
220/230
220/230(9)
215/230(15)
210/230(9)
230/230(7)
215/210
230/215
230/210
G
CL168
160/150
160/170(5)
140/160(20)
160/160(2)
150/160(4)
130/160(9)
170/160
170/150
150/130
130/140
CL3354
220/250
240/250(16)
225/250(1)
220/240(12)
220/225(10)
220/250(1)
240/220
240/225
225/220
250/240
CL904
260/280
250/280(7)
250/260(14)
260/270(15)
240/260(1)
260/280(2)
250/270
240/250
240/270
280/270
Notes: The numbers in the brackets represent number of offspring.
Almost all females were heterozygous at these loci (CL168, CL327, CL3354, CL904), except for CL327 (160/160) in the clutch B female. For clutches A and E, three different alleles which the father contributed were observed at the three chosen loci, suggesting that these two clutches had been sired by at least two males. The offspring of four females (B, C, D, and F) had three or four paternal alleles in each locus, and three paternal genotypes were observed in all loci. The number of paternal genotypes at these three loci indicated that females B, C, D, and F had mated with three different males. Within clutch G, five different alleles were detected at loci CL168 and CL3354, two of which were from maternal alleles. Clutch G showed four alleles for the locus CL904 in addition to the two alleles detected in the female. Four different paternal genotypes were estimated in clutch G, suggesting the female G was fertilized by at least four different males.
Discussion
We observed female S.japonica mating with different males during the reproductive period, a behavior also recorded in other species of cephalopods (Hall and Hanlon 2002; Naud et al. 2005). The benefits of multiple mating not only may raise the potential for genetic diversity but also increases the possibility of offspring survival (Mann et al. 1966; Jennions and Petrie 2000). Female Euprymnatasmanica Pfeffer, 1884 that mated with different males had larger eggs than those that mated with one male, indicating that females may obtain nourishment from the seminal fluid of several males (Squires et al. 2012). Male cephalopods exhibit “flushing behavior” in which they remove fresh spermatangia from previous males (Hanlon et al. 1999). In Sepiaesculenta Hoyle, 1885, the males remove sperm by using the hectocotylus instead of flushing water (Wada et al. 2005). The males in this study also exhibited such behavior, flushing the buccal area of the female with water, when mating with a previously mated female.
Microsatellite markers are particularly useful in paternity studies because of their polymorphism, codominance, and repeatability. Cephalopod biologists have determined multiple paternity in many species, including squid (van Camp et al. 2004; Shaw and Sauer 2004; Iwata et al. 2005), and Graneledoneboreopacifica Nesis, 1982 (Voight and Feldheim 2009). In this study, at least three paternal allele genotypes were found in all seven clutches indicating that at least two males were responsible for each brood. This result was in accordance with that of Naud et al. (2004), where multiple paternity was also found in Sepiaapama. Multiple paternity in S.japonica offspring indicates that sperm from different males must be mixed within the female’s reproductive tract. These sperm deposited around the buccal mass were used differentially to fertilize eggs (Shaw and Sauer 2004; Walker et al. 2006), after a process of sperm competition (Hanlon et al. 1999; Hall and Hanlon 2002) or mediation by female choice (Eberhard 1996).
Despite the prevalence of multiple paternity in cephalopod species, these studies show widely differing incidences of multiple paternity. In our study, multiple paternity was demonstrated in all sampled clutches (100%). In Sepiaapama, one-third of the females mated with multiple males and 67% of females’ eggs had multiple sires (Naud et al. 2004). Several factors have been confirmed to be related to the variance in incidence of multiple paternity observed in cephalopod species, e.g., sperm allocation, mating systems, sperm competition, and female choice (Wada et al. 2005; Wada et al. 2010). Moreover, as suggested for the squid Loligobleekeri by Iwata et al (2005), males who were the last to mate fertilized 85–100% eggs in four broods tested. However, in the multiple paternity study of Loligopealeii, the mate order is not the most important factor in determining paternity (Naud et al. 2004; Buresch et al. 2009); however, no clear hypothesis has yet emerged to explain which factor is essential in the multiple paternity of S.japonica. Further work should be carried out to understand paternity patterns and to investigate different factors affecting multiple paternity in this species.
Acknowledgements
This work was financially supported by the Chinese National Natural Science Foundation (41406138), Natural Science Foundation of Zhejiang Province (LY130190001).
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