Fisheries Science 61(4), 618-622 (1995)

Some Reproductive Characteristics of the Broods of the Harpacticoid Tigriopus japonicus Cultured in Different Salinities

Atsushi Hagiwara,*1 Cheng-Sheng Lee,*2 and Debra J. Shiraishi*2 *1Faculty of Fisheries , Nagasaki University, Bunkyo, Nagasaki 852, Japan *2Oceanic Institute , Makapu'u Point, Waimanalo, HI 96795, USA (Received November 18, 1994)

After the harpacticoid copepod Tigriopus japonicus was cultured at 25 •Ž on Tetraselmis tetrathele for several generations at 4, 8, 16 and 32 ppt, reproductive characteristics such as brood size, sex ratio, survival and mature age were studied. Results showed that 32 ppt was the best salinity for the popula tion growth since significantly more nauplii were produced, reached the mature stage earlier, and had higher survival rates and longer lifesan. Sex ratio of a brood was not affected by different salinities. Sex ual maturation occurs at younger stage with higher survival in offspring from broods produced by youn ger mothers. The male is dominant in the population. Instrinsic rate of increase (r) was 0.150, 0.218, 0.273 and 0.276 at 4, 8, 16 and 32 ppt, respectively. Key words: copepod, Tigriopus japonicus, salinity, reproduction, population growth, sex ratio, aging

The harpacticoid copepod Tigriopus japonicus Mori is We have conducted a bibliographic survey on rearing ex an intertidal organism that can be cultured easily and sur periments of this , and only a few studies have been vive in extreme environmental conditions.1,2) Importance conducted on the population growth of T. japonicus,4,10) of harpacticoid in the marine meiobenthic food most of which were conducted in mass-culture tanks rang web was reviewed by Hicks and Coull.3) ing in volume from 0.5 m3 to 200 ml.3 10,17)Such studies of Nutritional analysis 4)showed that T. japonicus is high in batch cultures are limited, however, in their ability to polyunsaturated fatty acids, 20:5n3 and 22:6n3, that are es obtain precise demographic data.18) The present study ex sential to marine fish larvae.5) Fish larvae fed on T. japoni amined the reproductive characteristics of individual cop cus generally show higher viability than those fed on epods to determine whether changes in brood reproduc rotifers or brine shrimps.) This species has been widely tion occur in water of constant temperature and different mass cultured by aquaculturists in Japan to provide an in salinities. The parameters investigated were (1) number of termediate-size class of live food for larval fish.7) Culture nauplii per spawn, (2) sex ratios, (3) percent survival at the experiments have provided fundamental information that onset of maturation and (4) intrinsic rate of increase and could be utilized for improving mass culture of this spe generation time. Data on individual T. japonicus are use cies. Lately, the development of artificial diets has enabled ful for evaluating production potential in culture systems. aquaculturists to provide intermediate size food with cheaper cost.8) Recently, mass cultures of harpacticoid cop Materials and Methods epods have markedly diminished.) Fundamental informa tion on T. japonicus accumulated till 1980, 10)but little has This study consisted of two experiments. The first experi been done during the last decade. Basic information on in ment (February-April, 1987) studied the brood production termediate size zooplankton, however, may be important of individual adult females at different salinities. The sec again in the near future.11-15)Some aquaculture facilities in ond experiment (July-September, 1987) investigated addi Japan (e.g., Nagasaki Pref. and Kanagawa Pref., personal tional parameters including sex ratio, generation time, communication) still utilize these accidentally grown cop reproductive period and survival. epods for feeding fish larvae. Our copepod stock originated from an outdoor pond of Low biomass of harpacticoid copepods are commonly the Fisheries Laboratory, Univ. of Tokyo near Lake found in rotifer mass culture tanks or in larval rearing Hamana. This was identified as Tigriopus japonicus.19-23) tanks as contaminants. Contamination of copepods brings Prior to the experiments, copepods were batch-reared in interspeciflc interaction with rotifers or fish larvae for sup salinities of 4, 8, 16, and 32 ppt for three to six genera plied food.16) Even small numbers of copepods produce sig tions. Temperature was maintained at 25•}1•Ž. Tetrasel nificant amounts of fecal pellets that inhibit aquaculturists mis tetrathele was provided as food, cultured in 21 flasks from harvesting rotifers for feeding fish larvae and make containing Guillard F media24) at salinities of 4, 8, 16, and the proper management of cultures more difficult. General 32 ppt under fluorescent lighting. methods to exterminate copepods has not been developed. For each experiment, 10 pairs of clasping copepods were Thus, from the aquacultural viewpoint, harpacticoid cop randomly selected from the batch cultures in the four salin epods have now both aspects as a live feed and as a pest ities. The precopulatory behavior of pairing was described . by Burton.25) To ensure the female's initial spawn, the Reproduction of Harpacticoid Copepod 619 pairs were isolated when the females were sexually imma Table 1. Lifespan and active spawning period of female T. japoni ture (in copepodite stages ‡U-‡X). Determination of the de cus at four salinities velopmental sttage followed the description of Igarashi.26) Each pair was transferred to a petri dish filled with 10-12 ml of water of the designated salinity. T. tetrathele was maintained at approximately 5•~105 cells/ml in each cul ture container. Water was partially replaced every three to five days or as needed to maintain the algal density. Experi ments were conducted in an incubator at 25.0•} 1.0•Ž with Active spawning period=Spawning period with the offspring production. The fluorescent lighting on a 12L: 12D light-dark regimen. The numbers in parenthesis indicate the number of replicates. Values are meant SD. male was removed when the female exuded her first eg

gsac. Eggsac development and spawning were monitored daily. After each brood hatched, the female was trans hatched. The actual lifespan of the female copepod ranged ferred by glass pipette to a new petri dish. from 56-101 days. Female copepods had longer lifespans In the first experiment, the newly-hatched nauplii were at 32 ppt than at 4 ppt (Table 1). The lifespan of the cop counted but not reared. The frequency of nauplii produc epods did not differ when cultured at 4, 8, or 16 ppt. tion of the female was recorded throughout her lifespan. The average brood size and total number of nauplii pro In the second experiment, the nauplii of the first, fifth duced per female varied significantly with salinity and ninth brood were counted and transferred to 50 ml (p<0.01, Table 2). The average brood size increased 2.27 beakers for rearing. If the brood size was greater than 40 times, from 23 nauplii at 4 ppt to 52.2 nauplii at 32 ppt. nauplii, only the first 40 nauplii were transferred and The average total number of nauplii produced increased reared. The water volume in each beaker was 1 ml/ from 270 nauplii at 4 ppt to 831 nauplii at 32 ppt. At 32 nauplius. T. tetrathele was supplied at a density of 5•~105 ppt, females had significantly larger broods and produced cells/ml. Stage of nauplius and copepodite were moni significantly more nauplii more frequently than at the low tored daily. The brood was considered to have completed er salinities of 4 and 8 ppt (Table 2). T. japonicus had larg the naupliar stage when 50% or more were copepodites. er broods and produced more nauplii after one mating at The brood was considered sexually mature at first observa 32 ppt than at 16 ppt, but the differences were not sig tion of an ovisac bearing female. At this point, the entire nificant. Individual females at 32 ppt produced the highest brood was fixed with a few drops of 10% formalin, sexed cumulative number of nauplii (Table 2). and counted. The sex ratio was expressed as the percentage The duration of the naupliar stage was significantly lon of females in a brood. ger (p<0.01) at the lower salinities of 4 and 8 ppt than at Life table parameters of net reproductive rate (R0), in the higher salinities of 16 and 32 ppt (Table 3). At lower trinsic rate of natural increase (r) and mean generation salinities, the naupliar stage lasted 4.4-4.5 days, at higher time (To) were computed after Birch.17) Namely, R0= salinities, 3.7-4.0 days. The duration from the first E Ixmx., where lx is the number of females alive during a naupliar stage to the onset of sexual maturation (the first given age interval and mx is the number of living females observation of an egg sac bearing female) was significantly born per female in each age interval. For nauplius and shorter in higher salinities (Table 3). At 16 and 32 ppt, this copepodite ‡T-‡V stages, their sex could not be determined duration was about 27% shorter than at 4 and 8 ppt. This morphologically. Thus, these lx and mx were determined duration for hatched nauplii from successive broods was from the daily count of total individuals and the data of inconsistent, but significantly longer among the offspring sex ratio obtained after the sexual maturation, assuming of the ninth brood than the first brood (Table 4, p<0.05). that the death rates before sexual maturation are equal between females and males. The r is computed from Survival Ee-rxlxmx=1 and T0=loseRo/r. Survival of copepods that reached the mature stage was A one-way analysis of variance (ANOVA) was used to significantly different at various salinities (p<0.01; Table test the significant effects of different salinities on various reproductive characteristics.27) The generation time and Table 2. Reproduction of female T. japonicus at four salinities survivorship of sequential broods from one female were also subjected to an ANOVA test. Multiple comparison (post-hoc test) was used to compare the individual salinity differences.281

Results

Reproduction The reproductive period after one mating ranged from 27-34 days and was not significantly different among salini ties (Table 1). On average, each female spawned 11-15 times during its reproductive period. After the reproduc tive period, several females continued to exude egg sacs The ANOVA tests the hypothesis that salinity has an effect on broodsize and num ber of offspring in the lifetime of a female. that did not mature; spawning frequency was intermittent. When the egg sac did mature, only one to four nauplii 620 Hagiwara et at.

Table 3. Development time, sex ratio and survival of T. japonicus offsprings reared at four salinities

*1 Nauplii stage= Days from hatching until 50% of the brood reached the copepodite stage. *2 Sexual maturation=Days from hatching until first female in a brood developed an ovisac.* 3 Sex ratio=Ratio of females/total individuals.* 4 Survival rate=Surviving individuals/nauphi hatched, at age of sexual maturation.

The number in parenthesis indicates the number of replicates.Values are mean•}SD.

Table 4. Effect of the age of a parental female on the development Table 5. The effect of salinity on life table parameters of T. japoni rate and survival of T. japonicus offspring cus

(Table 2). This difference might be attributed to the genetic difference or interactive effect of warmer temperature,

The number in parenthesis indicates the number of replicates. Values are higher salinity or the different algae fed. Takano30) indicat mean •} SD. ed that the optimal temperature for T. japonicus growth ranged from 23-25•Ž. The duration of the naupliar stage and the time to the 3). Survival at 8, 16, and 32 ppt was similar and significant onset of sexual maturation were significantly shortened at ly greater than at 4 ppt. Except at 4 ppt, survival rates at the higher salinities (Table 3). It was reported that the different salinities were 74% or more. Survival was also growth rate peaked at 27 ppt and decreased at lower or different among broods. The offspring of the first brood higher salinities.311 This difference among salinities was not had a significantly higher percentage of survival at matura tested statistically. The growth rate tended to be slower in tion than those of the ninth brood (p<0.01) (Table 4). diluted seawater, however. In the current study, copepods were not cultured in seawater more saline than 32 ppt, so Sex Ratio their development rate at higher salinities is unknown. The average percentage of females in the broods ranged In this study, the reproductive period represented only from 38.9% to 43.6% (Table 3) with a coefficient of varia 33-48% of the lifespan; this may be the result of a single tion of 36-56%. The very high probability indicates that mating, since the male copepod was removed after mating. salinity does not affect the sex ratio of T. japonicus If the reproductive period could be extended through most (p=0.70). of the lifespan, the total number of nauplii produced would increased, especially at 32 ppt. At 32 ppt, the cop Intrinsic Rate of Natural Increase and Generation Time epod has a longer lifespan and larger brood size than un The intrinsic rate of natural increase was high at higher der other salinity conditions. The difference in total salinities (32 and 16 ppt) and low at lower salinities (8 and nauplii produced per female under different salinities 4 ppt) (Table 5). The mean generation time was the short would be much greater than the results in Table 2. The lar est at 16 ppt, followed by 32, 8, and 4 ppt (Table 5). gest r value was 0.276 at 32 ppt. Hicks and Coull3) summa rized reproductive potential of harpacticoid copepod spe Discussion cies. Among them, large r values were reported for Tachidius discipes321 (r=0.239) and Tisbe holothuriae33) A highly significant effect of salinity upon the brood size (r=0.268). of T. japonicus was observed by Lee and Hu.291 The largest Seasonal changes in sex ratios in natural populations average brood size among females fed Chlorella sp. was 36 have been observed '31)but their cause is unknown. The sex nauplii at 30.1 ppt and 15-20•Ž. In the present study, a ratio of the broods was not influenced by salinity (Table 3). higher average brood size, 52 nauplii, was obtained among Igarashi31) also concluded that the sex ratio was not altered females fed T. tetrathele and reared at 32 ppt and 25•Ž by different salinities of the culture medium. In harpacti Reproduction of Harpacticoid Copepod 621 coid copepods, females generally outnumber males in field the future for controlling copepods contaminating live samples.) The higher culture density in the current food cultures. research (1 ind./ml) may have caused male dominance.3) The male dominance in the T. japonicus population was Acknowledgments This research was supported by United States Agen also reported in previous studies. Igarashi1) indicated that cy for International Development (DAN-4161-A-00-4055-00). Authors the primary sex ratio, which is always male dominant, is wish to thank Takashi Onbe, David Por and H.-S. Kim for identification of the experimental copepod, and Terry W. Snell for reviewing the genetically determined. Female dominant populations, manuscript. however, will increase the overall amount of nauplii pro duced. A reliable method of manipulating sex is needed to References control the copepod population. Takeda34) reported that sexratio was manipulated by the addition of several agents 1) S. Igarashi: The primary sex ratio of a marine copepod, Tigriopus such as KCl and KClO into culture media. Adding KCl, japonicus. Sci. Rep. Tohoku Univ. Ser. ‡W (Biol.), 29, 73-81 (1963). which raised the salinity to 42 ppt, affected the sex ratio. 2) C. Kitajima: Experimental trials on mass culture of copepods. Bull. Egami35)did not find the same results with KCI, but other Plankton Soc. Japan., 20, 54-60 (1973) (in Japanese). factors might account for this lack of effect. The survival 3) G. R. F. Hicks and B. C. 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