Development and Reproduction of Eucyclops Serrulatus (Copepoda: Cyclopoida) in the Laboratory Culture

Korean J. Limnol. 33 (1): 1~8 (2000)

Development and Reproduction of Eucyclops serrulatus (Copepoda: Cyclopoida) in the Laboratory Culture

Yoon, Kyonga and Won Kim*

(Department of Molecular Biology, Seoul National University, Seoul 151-741, Korea)

실험실에서 배양한 Eucyclops serrulatus (검물벼룩목 요각류)의 생식과 성장에 관한 연구 윤경아∙김 원* (서울대학교 자연과학대학 분자생물학과)

실험실에서배양을하기위해담수에서광범위하게서식하는 4종류의검물벼룩목요각류를실험 하였다. 이중Eucyclops serrulatus만이성공적으로배양되었다 . 다음으로 9가지먹이조건중어 떤 것에서유생발달이정상적으로이루어지는지를살펴보았다 . 이렇게선정된실험실배양환경 하에서 E. serrulatus의 생식과성장을관찰하였다 . E. serrulatus는 상대적으로짧은세대간격을 가지고많은수의자손을낳는것으로밝혀졌다 . 배양방법이간단하여짧은시간동안많은순수 배양된개체들을얻을수있다는점으로볼때 Eucyclops serrulatus는 담수 생태계의오염을측정 하는데 bioindicator 생물로서응용가능성이크다고할수 있겠다 .

Key words : Eucyclops serrulatus, Laboratory culture, Test animal, Development, Reproduction

enormous number of genera and species from all INTRODUCTION kinds of freshwater habitats including standing water bodies such as lakes, ponds, ditches, tem- Copepods have many ecological importances. porary pools and wells, and running water such Calanoid and cyclopoid copepods form the first as streams and rivers. The other free-living vital link in the food chain that leads from the cyclopoids are primarily marine in distribution; minute algal cells of the phytoplankton up to the the family Cyclopoidae being predominantly ben- large fish and mammals. These phytoplankton- thic and the Oithonidae planktonic (Kim and feeding copepods are by far the most important Chang, 1989; Huys and Boxshall, 1991). primary consumers in marine planktonic com- Because of the economic value of copepods, munities and, as such, form the base of virtually their important ecological status, and broad app- all pelagic food chains. Also, copepods are plenty lications in ecotoxicology (Hoffman et al., 1995), in freshwater planktonic communities. Members an aquaculture of copepods is generally conduct- of the families Cyclopoidae in the Cyclopoida, ed and have developed greatly in Europe and the Canthocamptidae in the Harpacticoida, and Dia- US. Although the laboratory culture of these ptomidae in the Calanoida are particularly suc- species is the basic step to the study of their ecol- cessful in all kinds of freshwater habitats (Huys ogy, there is no information especially on the cul- and Boxshall, 1991). ture of freshwater copepods. The problems of en- Cyclopoids are especially by far the most abun- vironmental pollution make us develop the cul- dant and successful group of copepods in the ture method of endemic species for evaluating freshwater. The family Cyclopoidae contains an our environment. The biology of test species sho-

* Corresponding author: Tel: 02) 887-0752, Fax: 02) 872-9113, E-mail: [email protected]

─ 1 ─ 2 윤경아∙김원 uld be known first if they are to be used as test Table 1. The food regime. species. So laboratory culture is needed to devel- Single species op the test species. Chlorophyceae flagellates Purposes of this study was threefold: (a) To Chlamydomonas reinhardii screen appropriate species for laboratory culture Chlorophyceae non-flagellates among four species in family Cyclopoidae; (b) Scenedesmus falcatus After screening, to determine a method for pure Selenastrum capricornum culture of the selected animal; (c) To investigate Chlorella sp. its development and reproductivity in the labora- Heterotropic flagellates tory culture. Euglena gracilis Protozoan ciliates MATERIALS AND METHODS Paramecium aurelia 1. Culturing and routine techniques Combination of two species Copepods were sampled from the pond and the Chlamydomonas reinhardii + Paramecium aurelia dam in Seoul National University, Seoul, Korea Euglena gracilis + Paramecium aurelia (37.27�N, 126.57�E). For collecting egg-sacs, Chlamydomonas reinhardii + Euglena gracilis copepods were anaesthetized with carbonated water and eggs-sacs were removed with a need- le. Egg-sacs of 10 individuals were transferred kers (~50 eggs/beaker). The beakers were filled to 50 ml beaker with M4 media, the media for with M4 media and contained one diet per bea- Daphnia continuous culture. After hatching, lar- ker at excess concentration of daily requirements vae were transferred to 50 ml beakers contain- with 5×105 cells ml-1 (Vijerberg, 1989). Three ing M4 media. The animals were maintained at replicates were carried out for each diet. The 20�C, 16L : 8D, and 10,000 Lux. They were fed in food regime is shown in Table 1. After two days, excess of daily requirements (Vijerberg, 1989). the contents of the beakers were filtered on a 50 They were observed every six hours before cope- µm sieve and rinsed in a Petri dish. Remaining podid stage and every 12 hours from copepodid eggs were removed and the hatched nauplii were stage. Among cultured copepods, only one gravid pipetted into beakers containing fresh food sus- female which moved actively was chosen for con- pension. Every other day, the animals were check- tinuous pure culture. ed under a microscope and pipetted into beakers 2. Experiments with new food suspension. Dead animals were removed. The experiments ran until all animals Experiment I : Screening proper species to a were dead or grown to adults. laboratory culture Experiment III : The development and Four species in family Cyclopidae; Macrocy- reproduction of E. serrulatus in the laboratory clops fuscus, Macrocyclops albidus, Tropocyclops culture prasinus, and Eucyclops serrulatus were tested Developmental times were determined by mo- for culture in the laboratory condition. After the nitoring the extrusion of egg sacs and the time selection of the proper species, an active one of taken for hatching. Newly hatched nauplii were this species was picked and cultured continu- isolated into groups of five to ten animals in ously until the number of progeny was enough beakers and observed every six hours. Food was for the next experiment. added daily and media were changed daily. The Experiment II : Suitability of various diets for period from hatching to death was determined as the development of E. serrulatus’ nauplii longevity. The number of days required to reach The experimental method of Amarasinghe each stage was calculated. (1997) was taken to determine suitability of diets Newly moulted adult males and females were for the development of nauplii. From continuous kept together in 50 ml beakers containing M4 pure culture of E. serrulatus, adult females car- media with algal food and were observed daily. rying egg sacs were kept and eggs were taken To determine the clutch size, several gravid fem- from them and distributed in 100 ml glass bea- ales were removed and anesthetized with car- Development and Reproduction of Eucyclops serrulatus 3 bonated water. Eggs were removed with a need- Table 2. Suitability of various diets for the development le, and counted. of E. serrulatus’ nauplii; + indicates successful Newly moulted females were isolated as they development of newly hatched nauplii to adults, became gravid and kept singly in nylon mesh - indicates no development. sieves in 50 ml beakers. The nylon mesh sieve Development allowed separation of the female from newly Chlorophyceae flagellates + hatched nauplii (Vijerberg, 1989). When the eggs Chlamydomonas reinhardii, hatched, the females were transferred to other Chlorophyceae non-flagellates beakers containing fresh media. Every six hours Scenedesmus falcatus - copepods were checked to ascertain the time of Selenastrum capricornum - their egg release. The culture media were re- Chlorella sp. - freshed every day. The copepod nauplii hatched Heterotropic flagellates from the released eggs were fixed by adding a Euglena gracilis drop of 5% formalin before they were counted Protozoan ciliates under a microscope. Egg appearance and hatch- Paramecium aurelia - ing were recorded. Embryonic development time Chlamydomonas reinhardii + was determined as the period between egg ap- Paramecium aurelia - pearance and hatching. Fecundity was the mean Euglena gracilis + Paramecium aurelia - number of nauplii produced in each clutch. Chlamydomonas reinhardii + Fifteen to thirty gravid females were isolated Euglena gracilis - and kept singly in nylon mesh sieves in 50 ml beakers. Every female that liberated a clutch of nauplii was transferred to another 50 ml beaker. born nauplii (Table 2). Complete development to Interclutch period was determined as the mean the adult stage and egg production occurred with time interval from hatching of one brood to the this algae. The newborn nauplii in the other die- extrusion into sacs of the next. Brood number ts died after a few days. was the total number of brood which one female produced during its life time. The reproductive Experiment III : The development and the duration was time from carrying the first clutch reproduction of E. serrulatus in the laboratory to the last. The experiment continued till all culture female copepods died. Animals from the same Duration of each developmental stage and clutch were reared to maturity. All newly moult- longevity ed adult females were observed daily and the appearance of first clutches of eggs noted and All developmental data are presented in Table recorded. The maturation time was time between 3. Mean duration of nauplius stage was the shor- hatching and carrying the first clutch. test among the three developmental stages. Cop- For evaluation of the data, statistical inferenc- epodid stage is divided in six substages. But es of mean were drawn using SAS Version 4.10. because individuals have reproductivity in cope- program (SAS Institute Inc.). podid 6 stage, in this study, copepodid 6 stage was included in the adults stage. In this cope- RESULTS podid stage, the mean time of copepodid 1 stage and 2 stage were longer than the others (Fig. 1). Experiment I : Screening proper species to a The lifetime of E. serrulatus was longer than two laboratory culture months. The longest was about three months, After screening proper species for laboratory but the shortest was one month. The life-time of culture among four species in family Cyclopidae, E. serrulatus consisted mainly of the copepod only one species, E. serrulatus could be cultured stage (Fig. 2). in the laboratory condition. Brood number and reproductive duration Experiment II : Suitability of various diets for the development of E. serrulatus’ nauplii The mean brood number was 3.8 (Table 4). Only the flagellate algae, Chlamydomonas re- Mean reproductive duration is also presented in inhardii was sufficient as a food source for new- Table 4. It could be calculated from the period 4 윤경아∙김 원

Fig. 1. The time rate of copepodid stage in E. serrulatus in the laboratory culture.

Fig. 2. The time rate of each developmental stage in E. serrulatus in the laboratory culture. from the extrusion of the first egg-sacs to the last egg-sacs. Clutch size Because mean brood number is 3.8, clutch sizes were measured only from the first to the fourth egg sacs. Clutch sizes were related to their order (Table 5). On the average, they tend to grow up to the third clutch and decline after the fourth clutch (Fig. 3). Fecundity and embryonic development time The duration of embryonic development is gi- Fig. 3. Mean clutch size in the order of clutch. ven in Table 3. Fecundity was the mean number of nauplii produced in each clutch. These are shown in Table Hatching rate of each clutch was the value 6. There is also a same tendency as that of clutch from the mean number of nauplii produced in sizes (Fig. 4). each clutch divided by the mean of each clutch Development and Reproduction of Eucyclops serrulatus 5

Table 4. Mean time interval between hatching of one brood to the extrusion into sacs of the next, mean brood number, and mean reproductive duration; Std Dev = Standard deviation, N = the number of observations, CLM = Confidence Limit of Mean. Lower Upper Std N Mean 95.0% 95.0% Dev CLM CLM Mean time interval (day) between hatching of one brood 20 1.400 0.681 1.082 1.719 to the extrusion into sacs of the next Mean brood number 20 3.800 0.616 3.512 4.088 Fig. 4. Mean No. of nauplii produced in the order of clutch. Mean reproductive 10.64 duration (day)

Table 3. The development of E. serrulatus in the labora- Table 5. Clutch sizes of E. serrulatus in the laboratory tory culture; Std Dev = Standard deviation, N = culture; Std Dev = Standard deviation, N = the the number of observations, CLM = Confidence number of observations, CLM = Confidence Li- Limit of Mean. mit of Mean. Lower Upper Mean Std Lower Upper N 95.0% 95.0% Std (day) Dev N Mean 95.0% 95.0% CLM CLM Dev CLM CLM Mean duration of Mean 1st clutch 5 20.000 3.536 15.610 24.390 embryonic 10 1.900 0.738 1.372 2.428 size development Mean 2nd clutch Mean duration of 5 37.400 9.813 25.215 49.584 21 7.238 0.768 6.889 7.588 size nauplius stage Mean 3rd clutch 5 57.800 14.342 39.992 75.608 Mean duration of size copepodid stage 6 9.500 0.837 8.622 10.378 (C1~C5) Mean 4th clutch 5 47.600 4.276 29.874 65.326 size Mean time of C1 6 2.200 0.707 1.458 2.942 Total clutch size 162.8 Mean time of C2 6 3.900 0.253 3.635 4.165 Mean time of C3 6 2.100 0.469 1.608 2.592 Table 6. Mean fecundities of E. serrulatus in the labora- Mean time of C4 6 1.400 0.509 0.865 1.935 tory culture; Std Dev = Standard deviation, N = Mean time of C5 6 1.900 0.724 1.140 2.660 the number of observations, CLM = Confidence Limit of Mean. Mean duration of 44.011 Lower Upper copepod stage Std N Mean 95.0% 95.0% Mean duration of Dev 6 16.500 1.049 15.399 17.600 CLM CLM maturation time Mean number of Mean longevity 20 60.750 14.764 53.840 67.660 nauplii produced 5 16.000 3.391 11.789 20.210 in 1st clutch Mean number of size. In the hatching rate, there was a significant nauplii produced 5 24.000 7.106 15.176 32.824 in 2nd clutch similarity throughout all clutches (Table 7). Mean number of Interclutch period nauplii produced 5 42.800 6.870 34.269 51.330 in 3rd clutch The period between clutches are in Table 4. Mean number of nauplii produced 5 33.400 5.225 26.912 39.888 Time to first clutch, duration of maturation in 4th clutch time Total number of nauplii produced 116.2 E. serrulatus could produce the first clutch in in clutches 6 윤경아∙김 원

Table 7. Hatching rates of E. serrulatus in the laboratory were already used for the assessment of metal culture; Std Dev = Standard deviation, N = the pollution in the freshwater (SETAC, 1992). Be- number of observations, CLM = Confidence Li- cause E. serrulatus is suitable for the Criteria for mit of Mean. selection (ASTM, 1997), it, not previously used as Lower Upper Std a test animal, could be a candidate for the good N Mean 95.0% 95.0% Dev CLM CLM test organism to evaluate the potential hazards of toxicants. Additionally, the study about a mar- Hatching rate of 1st 0.800 clutch ine copepod has already proven that a marine copepod species (Tisbe Battagliai) is somewhat Hatching rate of 2nd 0.642 clutch more sensitive to some endocrine disrupting materials than daphnids. It shows another probabil- Hatching rate of 3rd 0.740 clutch ity of key application of E. serrulatus for assess- ing endocrine disrupting materials in freshwater Hatching rate of 4th 0.702 clutch (Hutchinson et al., 1999). Mean hatching rate 4 0.721 0.067 0.615 0.827 Suitability of Chlamydomonas reinhardii for of clutches the development of E. serrulatus’ nauplii Complete development beginning from new- the copepodid stage 6. Maturation time was the born nauplii to adult male and female was only period needed to mature enough to produce the possible on the diet of Chlamydomonas rein- first egg-sacs (Table 3). hardii. The green-flagellate Chlamydomonas reinhardii is among the most frequently used DISCUSSION food species in cultures of freshwater zooplank- The laboratory culture of E. serrulatus ton. Chlamydomonas reinhardii is a high quality food and has the advantage of being a mobile Among four cyclopoids, only E. serrulatus could organism that stays in suspension much better be cultured under the laboratory condition in the than non-mobile food organisms. It has been present study. E. serrulatus is probably the most generally accepted that algae are considered to common cyclopoid in both Korea and the world be important food sources for the juvenile stages, and is well adapted to all kinds of aqueous envi- while adults are generally thought to be primar- ronments. It is present throughout the year (Kim ily predaceous in planktonic cyclopoid copepods. and Jang, 1989). In spite of the commonness of But in some studies, additional ingestion of algae this species only few data exist on its reproduc- by the predatory stages has been reported (Toth tivity. Also, no data exists on the biology of E. and Zankai, 1985; Toth et al., 1987; Adrian, serrulatus dwelling in Korea. Therefore labora- 1991), and pure herbivory of cyclopoids has been tory culture of E. serrulatus, the most common reported in one study; Cyclops vicinus could be species in Korean freshwater, is important and is cultured with a diet consisting of phytoplankton a basic step for the study of it’s ecology. only (Santer and Bosch, 1994). A purely herbi- Under the laboratory condition, E. serrulatus vorous nutrition of E. serrulatus has not been could easily be reared, could tolerate a wide ran- described previously. No publication exists which ge of temperature (10~25�C), pH (6~8) and dis- describes the development of cyclopoid copepods solved oxygen and could reproduce itself fairly using non-motile algae as the sole food source. well (unpublished data). Because they have rela- The development of E. serrulatus tively short generation time and high fecundity, a lot of individuals could be obtained during a In this study, longevity is the mean of females. short period of time. It was found feasible to Generally, the longevity of female is longer than maintain E. serrulatus on a small scale for a long that of male (Maier, 1990). The effect of tempera- period under the experimental conditions des- ture on the development of E. serrulatus was cribed in the present study. previously studied by Maier (1990). The results The ease of maintenance may indicate that this obtained by this author are slightly different but species has the big probability of application to very similar to those given in the present work. various fields. Many freshwater invertebrates In his study, the optimal temperature of the cul- Development and Reproduction of Eucyclops serrulatus 7 ture of E. serrulatus was narrowed to the range had relatively short generation time, could pro- of 10�C to 15�C. Yet because many other studies duce lots of progenies, and could be handled ea- proved that the optimal temperature was 20�C sily, allowing to obtain many individuals during for cyclopoids in temperate regions, the culture a short period. With these characteristics, this temperature was set in 20�C in this study. These species may be a candidate for a good test orga- differences may be explained by the difference of nism for evaluating freshwater pollution. the source of E. serrulatus. In the present study, all animals studied were the pure breed of the REFERENCES laboratory culture, but Maier used animals sam- Abdullahi, B.A. 1990. The effect of temperature on pled in ponds only after acclimation. These dif- reproduction in three species of cyclopoid cope- ferences also may be due to slight differences in pods. Hydrobiol. 196: 101-109. experimental conditions or genetical differences Abdullahi, B.A. and J. Laybourn-Parry. 1985. The among populations. effect of temperature on size and development in In copepods, the developmental times were three species of benthic copepod. Oecologia. 67: longer at lower temperature and were shorter at 295-297. higher temperature (Maier, 1990). And they were Adrian, R. 1991. Filtering and feeding rates of cyclo- also affected by food preference (Santer and poid copepods feeding on phytoplankton. Hydro- biol. 210: 217-223. Bosh, 1994; Amarasinghe et al., 1997; Hopp et Amarasinghe, P.B., M. Boersma and J. Vijverberg. al., 1997), and pH (Hansen et al., 1991). 1997. The effect of temperature, and food quantity The reproduction of E. serrulatus and quality on the growth and development rates in laboratory-cultured copepods and cladocerans No literature data on the reproduction of E. from a Sri Lankan reservoir. Hydrobiol. 350: 131 serrulatus is found. When compared with other -144. littoral cyclopoid species, for example with Mega- ASTM. 1997. Standard Guide for Selection of Resi- cyclops viridis, Macrocyclops albidus, and Acan- dent Species as Test Organisms for Aquatic and Sediment Toxicity Tests. thocyclops vernalis (Abdullahi, 1990), the general Hansen, A., J.V. Christensen and O. Sortkjaer. 1991. efficiencies of E. serrulatus in reproduction are Effect of high pH on zooplankton and nutrients in higher. Abdullahi’s study (1990) shows that fish-free enclosure. Arch. Hydrobiol. 123: 143- among three cyclopoids, the largest one has the 164. smallest clutch size and longest interclutch peri- Hoffman, D.J., B. Rattner, G.A. Burton Jr. and J. od, but in the smallest one vice versa. The fact Cairns Jr. 1995. Handbook of Ecotoxicology. CRC that E. serrulatus is smaller than other three press, Inc. Florida. cyclopoids, could explain it’s higher efficiency of Hopp, U., G. Maier and R. Bleher. 1997. Reproduc- tion and adult longevity of five species of plank- reproduction. E. serrulatus may adopt to laying tonic cyclopoid copepods reared on different diets; much more eggs during short time as its ecolog- a comparative study. Freshwater Biology. 38: 289 ical strategy for survival. The reproduction of -300. copepods is dependent on temperature and fem- Hutchinson, T.H., N.A. Pounds, M. Hampel and T.D. ale size (Abdullahi and Laybourn-Parry, 1985; Williams. 1999. Life-cycle studies with marine Abdullahi, 1990; Maier, 1990). copepods (Tisbe Battagliai) exposed to 20-hadro- xyecdysone and diethylstilbestrol. Environmental ABSTRACT Toxicology and Chemistry. 18: 2914-2920. Huys, R. and G.A. Boxshall. 1991. p. 9-13. In: Cope- pod Evolution. The Ray Society. London. Four cyclopoids were tested for laboratory cul- Kim, H.S. and C.Y. Chang. 1989. Freshwater Cyclo- ture. Among these animals, only Eucyclops ser- poid Copepods (Cyclopoida: Cyclopidae) of Korea. rulatus was successfully cultured in the labora- The Korean Journal of Systematic Zoology. 2: 225 tory. Under the laboratory culture condition, -256. nine kinds of diets were tested for the suitability Maier, G. 1990. The effect of temperature on the of nauplius development. Development and re- development, reproduction, and longevity of two production of E. serrulatus were also investigat- common cyclopoid copepods- Eucyclops serrulatus (Fisher) and Cyclops strenuus (Fisher). Hydrobiol. ed. Chlamydomonas reinhardii was the only one 203: 165-175. which could induce complete development from Mayer, F.L.Jr. and M.R. Ellersieck. 1986. Manual of nauplii to adults. It was found that E. serrulatus Acute Toxicity: Interpretation and Data Base for 8 윤경아∙김 원

410 Chemicals and 66 Species of Freshwater Ani- vicinus (Uljanin) (Copepoda: Cyclopoida) in Lake, mals. United States Department of the Interior Balaton on the basis of gut content analysis. Hyd- Fish and Wildlife Service/ Resource Publication robiol. 122: 251-260. 160. Toth, L.G., N.P. Zankai and O.M. Messner. 1987. Santer, B. and F. Van den Boshch. 1994. Herbivorous Alga consumption of four dominant planktonic nutrition of Cyclops vicinus: the effect of a pure crustaceans in Lake Balaton (Hungary). Hydro- algal diet on feeding, development, reproduction biol. 145: 323-332. and life cycle. J. Plankton Res. 16: 171-195. Vijerberg, J. 1989. Culture techniques for studies on SETAC. 1992. Ecotoxicology of metals in invertebra- the growth, development and reproduction of tes (Dallinger eds.), Reinhard and S.R., Philip. copepods and cladocerans under laboratory and in Lewis publisher. situ conditions: a review. Freshwater Bio. 21: 317 Toth, L.G. and N.P. Zankai. 1985. Feeding of Cyclops -373.