The Japanese Spineless Cuttlefish, Sepiella japonica, 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. The wild population of Sepiella japonica has declined since the 1980s due to over-fishing and pollution. To enhance production, artificial breeding methods are being developed in China and successful aquaculture techniques have been established in recent years. However, studies have revealed that the populations and individual genetic diversity in this species has declined under artificial conditions.An important factor that affects the genetic diversity of a population is the effective population size which in turn is greatly influenced by the mating system of a species. The mating system influences effective population size through changing the number of individuals contributing to subsequent generations. 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. In such a mating system, effective population size increases, and, as a result, maximizes the genetic diversity of the offspring within a single reproductive season. Some studies have confirmed that a polyandrous mating system is frequent in marine Cephalopods including the Common Octopus, Octopus vulgaris, Deep-sea Octopus, Graneledone boreopacifica, the Southern Reef Squid, Sepioteuthis australis, the Giant Cuttlefish, Sepia apama, the Longfin Inshore Squid, Loligo pealeii, and the Spear Sqiuid, Loligo bleekeri. The female of all these species carries stored sperm from more than one male, and the effective population size is therefore significantly higher. Previous studies have shown that female Sepiella japonica store sperm in the seminal receptacle found in the buccal membrane. All else being equal, long-term sperm storage enhances the opportunity for multiple matings of this species. Moreover, multiple matings of female Sepiella japonica has actually been observed. Polyandry, coupled with sperm storage, is therefore potentially an important reproductive strategy for maximizing the genetic diversity of offspring in Sepiella japonica.
In a paper published in the journal ZooKeys on 14 October 2019, Liqin Liu of the National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization and the National Engineering Research Center for Facilitated Marine Aquaculture at Zhejiang Ocean University, Yao Zhang and Xiaoyu Hu, also of the National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhenming Lü, also of the National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization and the National Engineering Research Center for Facilitated Marine Aquaculture, Bingjian Liu, again of the National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Li Hua Jiang, again of the National Engineering Research Center for Facilitated Marine Aquaculture, and Li Gong, once again of the National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, examine the mating system of Sepiella japonica experimentally, in order to understand the role of polyandry in maintaining effective population size.
In recent years, multiple paternity in several marine species has been documented using different genetic markers including allozymes, DNA fingerprinting, Random Amplification of Polymorphic DNA, and microsatellites. Microsatellites are the preferred marker because they are widely distributed in the genomes of most organisms and are highly polymorphic. Paternity studies based on microsatellites have become increasingly common, and the number of studies using microsatellites has increased. Several microsatellite markers have been isolated and characterized for Sepiella japonica and used to evaluate the genetic structure of its populations. Liu et al. used the previously described microsatellite markers to investigate whether multiple paternity occurs in Sepiella japonica.
Sexually mature adult Sepiella japonica were obtained from the Fujian Shacheng Harbor Cultivation Base in 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.
Total genomic DNA was isolated from each offspring and from the muscular tissue of the respective parents. Three microsatellite loci, chosen from four loci (CL168, CL327, CL3354, CL904) developed specifically for Sepiella japonica were used to study genotypes for parents and their offspring.
Parents and their offspring were genotyped by determining alleles at three of the four microsatellite loci. Liu et al. 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. They determined paternal alleles through subtracting the maternal alleles from offspring in a brood. 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.
Mating behavior in Sepiella 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. 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. Liu et al. 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. They 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 Sepiella 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.
Sepiella japonica mating in the head-to-head position. Liu et al. (2019).
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. Lui et al. 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. Lui et al. analyzed paternity including sampled males and non-sampled males that had copulated with females prior to capture.
Almost all females were heterozygous at these loci (CL168, CL327, CL3354, CL904), except for CL327 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.
Lui et al. observed female Sepiella japonica mating with different males during the reproductive period, a behavior also recorded in other species of Cephalopods. The benefits of multiple mating not only may raise the potential for genetic diversity but also increases the possibility of offspring survival. It has previously been shown that female Southern Dumpling Squid, Euprymna tasmanica, 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. Male Cephalopods exhibit 'flushing behavior' in which they remove fresh spermatangia from previous males. In the Golden Cuttlefish, Sepia esculenta, the males remove sperm by using the hectocotylus (a modified arm used by some male Cephalopods to transfer sperm to the female) instead of flushing water. The males in Lui et al.'s 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 and the Deep-sea Octopus, Graneledone boreopacifica. 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 previous studies where multiple paternity was also found in the Giant Cuttlefish, Sepia apama. Multiple paternity in Sepiella 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, after a process of sperm competition or mediation by female choice.
Despite the prevalence of multiple paternity in cephalopod species, these studies show widely differing incidences of multiple paternity. In Lui et al.'s study, multiple paternity was demonstrated in all sampled clutches (100%). In the Giant Cuttlefish, Sepia apama, one-third of the females mated with multiple males and 67% of females’ eggs had multiple sires. 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. Moreover, as suggested for the Spear Squid, Loligo bleekeri, males who were the last to mate fertilized 85–100% eggs in four broods tested. However, in the multiple paternity study of the Longfin Inshore Squid Loligo pealeii, the mate order is not the most important factor in determining paternity; however, no clear hypothesis has yet emerged to explain which factor is essential in the multiple paternity of Sepiella japonica. Further work should be carried out to understand paternity patterns and to investigate different factors affecting multiple paternity in this species.
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