Population Ecology of Marine Cladocerans in Tolo Harbour, Hong Kong LI, Cheuk Yan Vivian a Thesis Submitted

Population Ecology of Marine Cladocerans in Tolo Harbour, Hong Kong

LI, Cheuk Yan Vivian

A Thesis Submitted irt ~~rt~al? .P!+lfilm~pt'-r , -e.. . . ' Of the Requirements for the Degree of

Master of Philosophy

Biology

The Chinese University of Hong Kong

January 2010

Thesis/Assessment Committee

Professor Ka Hou CHU (Chair)

Professor Chong Kim WONG (Thesis Supervisor)

Professor Put 0 ANG, Jr (Committee Member)

Professor Charles RAMCHARAN (External Examiner) Abstract of thesis entitled:

Population Ecology of Marine Cladocerans in Tolo Harbour, Hong Kong

Submitted by Ll, Cheuk Yan Vivian for the degree of Master of Philosophy in Biology at The Chinese University of Hong Kong in July 2009

Abstract This thesis provides a one year study on the population ecology of four marine cladocerans (Penilia avirostris, Pseudevadne tergestina, Pleopis polyphemoides and

P. schmackeri) in Tolo Harbour, Hong Kong. Samples were obtained from November

2005 to October 2006 in Tolo Harbour and Tolo Channel. P. avirostris and P. tergestina occurred throughout most of the studied period, while P. polyphemoides and P. schmackeri occurred only from February to June 2006. Gamogenic individuals

of P. tergestina and P. polyphemoides were recorded sporadically.

Population dynamics of the marine cladocerans were studied and were used to

infer when food, predators or temperature became the limiting factor of the growth of

the populations. Birth rates were quantified based on a previous egg-ratio model of

Edmondson and Paloheimo, whereas the growth rate was determined from

successive population densities. Environmental factors such as temperature,

dissolved oxygen content and salinity were found to limit the population growth at

different times of ~he year. Fish predation played an important role in regulating the

populations during February to April. Predation by chaetognaths also helped in

regulating the marine cladocerans population.

Feeding ecology of marine cladocerans is still meager. As food could be a

limiting factor on population growth, investigation of the marine cladoceran diet would provide more information to understand their ecological role and population growth. We applied molecular technique to investigate the diet of P. tergestina. 18S

RNA genes were amplified by PCR with universal eukaryotic primers using crude

DNA extracted from P. tergestina. A total of 149 transformed colonies were assessed by restriction fragment length polymorphism using restriction enzyme Hae/11 and 23 unique RFLP patterns were obtained and sequenced on an ABI PRISM 3700 DNA

Analyzer.

Ciliates appeared to be an important prey item for P. tergestina. Picobiliphytes were also detected and had widened the size range of prey items which pod on ids can feed on. This study provides new information on what might be consumed by marine podonids apart from diatoms and dinoflagellates.

II 摘要

本論文對香港吐露港海域四種海洋枝角類(鳥喙尖頭潭、肥胖僧帽潰、多型圓囊

滋及史氏圓囊道)的生態進行了一年的研究。樣本採樣自 2005 年十一月至 2006 年十月

在吐露港內及赤門海峽的兩採樣站。採樣結果發現鳥喙尖頭海和肥胖僧帽搔皆是全年

性發生，多型圓囊搔及史氏圓囊搔則發生於 2006 年二月至六月期間.採樣期間偶爾發

現肥胖僧帽法和多型圓囊淺的有性個體。

本論文透過所計算得的四種海洋枝角類的族群資料來推論海洋枝角類族群生長的

限制因素。出生率的計算方法是依從 Edmondson 和 Paloheimo 的卵比率模型，而增長

率則由鄰近族群密度所算而得。水溫、溶氧及鹽度等環境因素於採樣年度的不同時問

內限制著族群的生長。於 2006 二月至四月期間，魚類的捕食對控制海洋枝角類族群的

生長擔當著重要角色。此外，海洋枝角類的另一捕食者一箭蟲'也協助限制海洋枝角

類族群的生長。

食物可是族群生長的限制因素之一，探討海洋枝角類的攝食資料有助進一步了解

牠們於生態環境中擔演的角色及其族群生長情況。由於海洋枝角類的覓食生態資料缺

乏，第二章以利用分子技術來對肥胖僧帽潰的覓食生態進行研究。

我們從肥胖僧帽淺中抽取出 DNA ，然後以引子對 185 RNA 進行聚合酵素鏈鎖反應擴

增。取得共 149 個殖株後，使用 HaeIII 限制晦切割進行限制晦片段度多型分析，並取

得 23 個獨特的限制晦片段長度多型性形態，再利用 ABI PRISM 3700 DNAAnalyzer 對

每個限制晦片段長度多型性形態進行基因排列分析。

研究結果顯示纖毛蟲是肥胖僧帽潰的主要食物。此外，微藻類的發現擴闊了圓囊

蚤可攝的食物大小的範圍。是次研究為海洋圓囊蚤提供了新的覓食資料。

III Acknowledgements

I would like to express my greatest gratitude to my supervisor Professor C. K. Wong

for his guidance and patience throughout my M. Phil study. I would also like to thank

Professor K.H. Chu and Professor P.O. Ang for their valuable opinions.

I appreciated the assistance from all the staff and labmates in MSL. Special thanks to

Mr. Y. H. Yung for his assistance in the weekly field samplings, Mr. M. K. Cheung

for his guidance and help in completing the molecular part, and Mr. K. M. Chau for

his support.

I would also like to thank Miss M. Fung for her support in times of frustration and

exhaustion.

IV Table of Contents

Page

Abstract (in English) I

Abstract (in Chinese) III

Acknowledgements IV

Table of contents v

List of figures VIII

List of tables XII

List of plates XIII

Chapter 1

Population Dynamics of Marine Cladocerans in Tolo Harbour

1.1 Introduction 1

1.1.1 Study site

1.1.2 Species description and distribution 2

1.1.3 Population dynamics of marine cladocerans 4

1.1.3.1 Reproduction 5

1.1.3.2 Fecundity of marine cladocerans 6

1.1.3.3 Embryonic development time 7

1.1.3.4 Food 7

1.1.3.5 Predation 8

v 1.2 Objective 9

1.3 Materials and method IO

1.3.1 Field sampling 10

1.3.2 Hydrographical parameters and chlorophyll a concentration 10

1.3.3 Zooplankton sampling II

1.3.4 Zooplankton analysis 11

1.3.5 Calculation of Population Parameters 12

1.4 Results 16

1.4.1 Hydrographical parameters I6

1.4.1.1 Temperature I6

1.4.1.2 Salinity 16

1.4.1.3 Dissolved Oxygen content 17

1.4.2 Chlorophyll a concentration I7

1.4.3 Seasonal occurrence of marine cladocerans in Tolo Harbour 26

1.4.4 Parameters of the marine cladocerans populations 32

1.4.5 Occurrence of gamogenic individuals of marine cladocerans 51

1.4.6 Occurrence of chaetognaths 51

1.5 Discussion 57

1.6 Conclusion 66

VI Chapter 2

Molecular detection of the diet of the marine cladocerans, Pseudevadne tergestina

2.1 Introduction 68

2.1.1 The importance of marine cladocerans 68

2.1.2 Previous findings on cladoceran diet 69

2.1.2.1 Sidid cladocerans 69

2.1.2.2 Podonid cladocerans 69

2.1.3 Methods to investigate the feeding habit of animals 71

2.1.4 Application of molecular detection 72

2.2 Objectives 73

2.3 Materials and method 74

2.3.1 Zooplankton sampling and preparation 74

2.3.2 DNA extraction 74

2.3.3 18S rRNA gene amplification 75

2.3.4 18S rRNA Cloning 76

2.3.5 Clone screening by RFLP 76

2.3.6 Sequencing and phylogenetic analysis 77

2.4 Results 78

2.4.1 Alveolata 78

2.4.2 Other lineages 79

2.5 Discussion and conclusion 85

2.5.1 Errors and improvements 87

Reference 89

VII List of Figures

Figure Page

Figure 1.1 14 Location of sampling stations, TH and TC, in Tolo Harbour, Hong Kong SAR.

Figure 2.1 19 Averaged water temperature at the two sampling stations, Tolo Harbour (TH) and Tolo Channel {TC).

Figure 2.2 20 Thermoclines at the two sampling stations, TH and TC, from June-August 2006.

Figure 2.3 21 Averaged salinity at the two sampling stations, Tolo Harbour (TH) and Tolo Channel (TC).

Figure 2.4 22 Haloclines at the two sampling stations, TH and TC, from June-August 2006.

Figure 2.5 23 Averaged dissolved oxygen at the two sampling station, Tolo Harbour {TH) and Tolo Channel (TC).

Figure 2.6 24 Oxyclines at the two sampling stations, TH and TC, from June-August 2006.

Figure 2.7 25 Chlorophyll a concentrations at the two sampling stations, TH and TC.

Figure 2.8 28 Seasonal abundance of Penilia avirostris at two sampling stations, TH and TC.

Figure 2.9 29 Seasonal abundance of Pseudevadne tergestina at two sampling stations, TH and TC.

VIII Figure 2.10 30 Seasonal abundance of Pleopis polyphemoides at two sampling stations, TH and TC.

Figure 2.11 31 Seasonal abundance of Pleopis schmackeri at two sampling stations, TH and TC.

Figure 2.12a 35 Size distribution of Penilia avirostris in Tolo Harbour.

Figure 2.12b 36 Size distribution of Penilia avirostris in Tolo Channel.

Figure 2.13a 37 Frequency distributions of parthenogenetic females of Penilia avirostris with different brood size in Tolo Harbour.

Figure 2.13b 38 Frequency distributions of parthenogenetic females of Penilia avirostris with different brood size in Tolo Channel.

Figure 2.14 39 Population parameters of Penilia avirostris at two sampling stations, a) birth rate, b) growth rate and c) death rate

Figure 2.15a 40 Size distribution of Pseudevadne tergestina in Tolo Harbour.

Figure 2.15b 41 Size distribution of Pseudevadne tergestina in Tolo Channel.

Figure 2.16a 42 Frequency distributions of parthenogenetic females of Pseudevadne tergestina with different brood size in Tolo Harbour.

Figure 2.16b 43 Frequency distributions of parthenogenetic females of Pseudevadne tergestina with different brood size in Tolo Channel.

IX Figure 2.17 44 Population parameters of Pseudevadne tergestina at two sampling stations, a) birth rate, b) growth rate and c) death rate.

Figure 2.18 45 Size distribution of Pleopis polyphemoides in Tolo Harbour and Tolo Channel.

Figure 2.19 46 Frequency distributions of parthenogenetic females of Pleopis polyphemoides with different brood size in Tolo Harbour and Tolo Channel.

Figure 2.20 47 Population parameters of Pleopis polyphemoides at two sampling stations, a) birth rate, b) growth rate and c) death rate.

Figure 2.21 48 Size distribution of Pleopis schmackeri in Tolo Harbour and Tolo Channel.

Figure 2.22 49 Frequency distributions of parthenogenetic females of Pleopis schmackeri with different brood size in Tolo Harbour and Tolo Channel.

Figure 2.23 50 Population parameters of Pleopis schmackeri at two sampling stations, a) birth rate, b) growth rate and c) death rate.

Figure 2.24 52 Percentage of gameogenic individuals of Pseudevadne tergestina in Tolo Harbour and Tolo Channel.

Figure 2.25 53 Percentage of gameGgenic individuals of Pleopis polyphemoides in Tolo Harbour and Tolo Channel.

Figure 2.26 56 Seasonal abundance of chaetognaths in Tolo Harbour and Tolo Channel.

X Figure 3.1 80 Gel photo showing all restriction fragment length polymorphism (RFLP) patterns in the current study.

Figure 3.2 84 Neighbor-joining phylogenetic trees based on 18s rRNA sequences.

XI List of Tables Table Page

Table 1 81 Summary of the eukaryote OTU distribution in higher taxonomic groups

Table 2 82-83 List of BLAST search result for each OTU recovered.

XII List of Plates Plate Page

Plate 1 15

Body length measurements of the four marine cladocerans. (Bar= O.lmm)

Plate 2 A male Pseudevadne tergestina 54

Plate 3 A male Pleopis polyphemoides 54

Plate 4 55 Ingestion of marine cladoceran, Pseudevadne tergestina, by a chaetognath.

XIII Chapter 1 Population Dynamics of Marine Cladocerans in Tolo Harbour

1.1 Introduction

1.1.1 Study site

Tolo Harbour is a semi enclosed estuarine bay located in the northeastern Hong

Kong. The inner harbour is a shallow basin (max.= 10 m), and it opens to the Mirs

Bay and the south China Sea through a narrow tidal channel (max.= 22 m). Tolo

Harbour has a long history of eutrophication, resulted from nutrient loading from the fish cage culture sites and the domestic sewage from the nearby urban populations.

Though domestic sewage disposal has been stopped in 1996/7 with the implementation of the 'effluent export scheme', the nutrient levels are still higher in

Tolo Harbour than in Mirs Bay. The flushing rate in the basin is low, mean annual water residence time was estimated around 35 days (Oakley and Cripps, 1972), and previous studies had publicized the environmental stresses acting upon the marine ecosystem in the captioned area (Chan and Wong, 1993; Leung, 1997).

Copepods and cladocerans are important and frequent members in the plankton community (Chen, 1982), they act as mediators between primary producers and higher trophic levels. Alterations of marine zooplankton abundance and composition may alter the structure of the pelagic food webs (Katechakis and Stibor, 2004).

Marine cladocerans are tiny crustaceans that occur mostly in coastal pelagic waters (Bosch and Taylor, 1973; Della Croce and Venugopal, 1972; Grahame, 1976;

Tang et al., 1995). Unlike their freshwater counterparts, marine cladocerans are always considered as a subordinate component of the zooplankton, coming behind the copepods (Egloff et al., 1997). However, Kim et al. (1989) noted that marine cladocerans may play a significant role in the trophodynamic pathways of the plankton community during periods of high abundance, where their densities could reach up to 50,000 (Bosch and Taylor, 1973; Onbe, 1974; Grahame, 1976) and

100,000 individuals m-3 (Platt, 1977; Bryan and Grant, 1979). Chen (1982) found marine cladocerans as the second most abundant group of the zooplankton community in Tolo Harbour. Despite their numerical abundance in plankton communities in temperate and warm waters, marine cladocerans have received very little attention in the past as compared with the massive work on marine copepods

(Onbe, 1985).

1.1.2 Species description and distribution

Of the approximately 600 described species of cladocerans (Schram, 1986),

only 8 species are truly marine (Onbe, 1977). Among them, four could be found in

Tolo Habour, which includes Penilia avirostris, Pseudevadne tergestina, Pleopis polyphemoides and P schmackeri.

Penilia avirostris

P avirostris is the sole representative from the Family Sididae. P avirostris is

easily recognized by its six pairs of filter-feeding phyllopodial limbs, enclosed by a

carapace (Dolgopolskaya, 1958; Gore, 1980). It has a worldwide distribution

inhabiting most of the coastal and oceanic waters. Lochhead (1954) suggested that

surface water temperature is the factor restricting the distribution of P.avirostris in

coastal water, and a minimum temperature of 21 °C is required for permanent

establishment of P. avirostris population.

2 Previous records of P. avirostris population include the coastal waters of China

(Cheng and Chen, 1966; Cai, 1990; Chen and Huang, 1992; Tang eta/., 1995; Ji,

2001), coastal water of Korea (Yoo and Kim, 1987), coastal water and inland sea of

Japan (Onbe, 1977; Mullin and Onbe, 1992; Onbe and Ikeda, 1995), Indian Ocean

(Della Croce and Venugopal, 1972), Jamaica (Webber eta/., 1996), Nigeria (Egborge et a/., 1994 ), Chile (Mujica and Espinoze, 1994 ), the Black sea (Baloga et a/., 1995), northwestern Mediterranean (Pagano eta/., 1993; Atienza eta/., 2008), southeastern

Brazil (Rocha, 1985; Marazzo and Valentin, 2003), Chesapeake Bay and Westport

River Estuary in the United States (Bosch and Taylor, 1967; Conley and Turner,

1991 ), Gulf of Mexico (Checkley et al., 1992), the North Sea (Johns et a/., 2005), and the Canadian water (Bernier and Locke, 2006).

The remaining seven spec.ies of marine cladocerans belong to the Family

Podonidae. Podonids are characterized by a reduced carapace into a dorsal pouch and

4 pairs of elongated, prehensile trunk appendages. Each of the seven species could be differentiated by noting the number of setae on the exopods of the thoracic appendages (Egloff et al., 1997).

Pseudevadne tergestina

P. tergestina, formerly known as Evadne tergestina, is mostly limited to tropical and subtropical waters within 40° latitude from equator (Cheng and Chen, 1966).

Population of P. tergestina were found along the coastal waters of China (Cheng and

Chen, 1966; Chen and Huang, 1992; Tang eta/., 1995; Ji, 2001), coastal water of

Korea (Yoo and Kim, 1987), inland sea of Japan (Onbe, 1983), Indian Ocean (Della

Croce and Venugopal, 1972), Chile (Mujica and Espinoze, 1994), southeastern Brazil

(Rocha, 1985; Marazzo and Valentin, 2004), Chesapeake Bay (Bosch and Taylor,

3 1967), and Mexico (Della Croce and Angelino, 1987; Checkley et al., 1992;

Castellanos-Osorio and Elias-Gutierrez, 1999).

Pleopis polyphemoides

P. polyphemoides is a neritic epizooplankter occurring mainly in surface waters of coastal regions and embayments. It was regarded as an estuarine species by many investigators and was poorly studied in tropical waters (Onbe, 1983).

P. polyphemoides was recorded in coastal waters of China (Cheng and Chen,

1966; Ji, 2001 ), coastal water of Korea (Yoo and Kim, 1987), inland sea of Japan

(Onbe, 1983), west coast of Sweden (Eriksson, 1974), Mediterranean Sea (Thiriot,

1972), Narragansett Bay (Fofonoff, 1994), South African waters (Della Croce and

Venugopal, 1972) and southeastern Brazil (Rocha, 1985; Marazzo and Valentin,

2003).

Pleopis schmackeri

P. schamckeri is the least known species among the eight marine cladocerans

(Onbe, 1977). Occurrence of P. schmackeri was described in the coastal water and inland sea of Japan (Onbe, 1983), Korea (Kim and Onbe, 1989), coastal waters of

China (Cheng and Chen, 1966), Brazilian coast (Rocha, 1985; Marazzo, 2002) and

Indian Ocean (Frontier, 1973).

1.1.3 Population dynamics of marine cladocerans

Annual cycles of abundance have been reported for many populations of marine cladocerans, and the pattern of occurrence is believed to be dependent primarily on water temperature (Onbe, 1977), However, the underlying biological and/or

4 physiochemical factors governing the population of marine cladocerans are not fully considered.

Marazzo et al. (2003, 2004) quantified birth rates of marine cladocerans based on the egg-ratio model of Edmondson ( 1968) and Paloheimo ( 1974 ). Fluctuations observed in the birth rate of P avirostris suggested that the population density variations were influenced by both water temperature and fluctuations in brood size.

Besides, species birth rates, death rates and embryonic developmental time are also important factors affecting the population size.

1.1.3.1 Reproduction

The reproductive cycle of manne cladocerans alternates between parthenogenesis and gameogenesis (Egloff et al., 1997). Under favorable conditions, marked increase in the population is achieved due to high reproductive potential of parthenogenetic females. Parthenogenetic females retain their brood in a pouch under the carapaces and give birth to free-swimming miniature parthenogenic females

(Platt and Yamamura, 1986; Egloff et al., 1997). When conditions for growth become unfavourable, transition from parthenogenetic to gameogenic reproduction occurs and marine cladocerans reproduce sexually and produce resting eggs which remains dormant over long periods of time.

Percentage of gameogenic individuals in a population ranges from less than

10% to 86% (On be, 1978; Fofonoff, 1994; On be et al., 1996). Study on the population dynamics of Pseudevadne tergestina in a coastal bay in Brazil had demonstrated a steady increase, from 7% to 70%, of gamogenic individuals in the population towards the unfavorable time of the year, together with an increase in

5 resting eggs production (Marazzo and Valentin, 2004 ). The abundance of resting eggs has also been put forward to be closely correlated with the occurrence of the planktonic population for Penilia avirostris and Pleopis polyphemoides in the Inland

Sea of Japan (On be, 1985).

Emergence of gameogenic generation is related to environmental conditions as well as to endogenous factors. Environmental factors may include deterioration of feeding conditions, decrease in water temperature, changes in population density and changes in photoperiod (Onbe, 1985). Despite the numerous experimental studies, the mechanisms of environmental sex determination in cladocerans remains unclear.

1.1.3.2 Fecundity of marine cladocerans

Fecundity of female cladocerans contributes to affecting the size of a population, and it is mainly determined by the number of embryos a female can carry at a time.

Brood size is determined by various factors, and is different from species to species.

F ofonoff ( 1994) reported that Evadne nordmanni has a mean brood size of 9 to 12 per female during the spring maximum abundances and could carry as many as 26

embryos per female. P. polyphemoides has a mean brood size of 7 to 10 embryos per

female, with individuals bearing up to 19 embryos (Onbe, 1974; Fofonoff, 1994).

Previous studies revealed that there was a direct relationship between body size

and egg number with larger females carrying more eggs. The brood size of P.

avirostris increased from 2 to 12 per female as body length increased from 0.5 to 1.0

mm (Della Croce and Venugopal, 1973 ). And Cheng ( 194 7) documented an increase

in mean fecundity as a positive function of body size in E. nordmanni.

6 Several workers observed that brood sizes of marine cladocerans are generally highest during the initial phases of population growth and decrease rapidly as populations increase (Bainbridge, 1958; Della Croce and Bettanin, 1965; Onbe, 1974;

Platt and Yamamura, 1986; Fofonoff, 1994). Brood sizes could reduce to 1 to 2 embryos per female during declining phases (Bainbridge, 1958; Onbe, 1974; Platt and Yamamura, 1985b).

P avirostris carries a larger brood in warmer season than in cooler periods in the

Inland Sea of Japan (Onbe, 1974), suggesting that there might be a slight relationship between temperature and variations in brood size. However, similar pattern was not observed in other P avirostris populations (Egloff eta!., 1997).

1.1.3.3 Embryonic developmen·t time

Embryonic development time is an important factor in determining the rate of population growth and is dependent primarily on temperature. Bottrell ( 1975) established a second-degree logarithmic polynomial equation to calculate the development time of embryos of different freshwater cladocerans as a function of water temperature. Egloff et a!. (1995) summarized the relationship between embryonic development time and temperature ranging from 10 to 30°C. In general, the lower the temperature, the longer the development time. At I 0°C, the development time may take as long as 12 days for P polyphemoides. It only takes about 2 days for embryos of P tergestina to develop at 28°C.

1.1.3.4 Food

P avirostris is a filter feeder, while other marine podonids are regarded as raptorial herbivorous feeders (Russell-Hunter, 1979), and they feed mainly on

7 diatoms (Gore, 1980; Kim et a!., 1988; Wong et a!., 2006). Previous studies had reported there was an increasing dominance of dinoflagellates over diatoms species in Tolo Harbour, causing an increase in incidences of red tides and oxygen depletion

(Wear et al., 1983; Hodgkiss and Chan, 1987; Lam and Ho, 1987). Sauders et al.

(1999) suggested that poor food quality may reduce the brood sizes of Daphnia.

Accordingly, it is believed that decrease in food availability and condition would also limit the growth of marine cladocerans, thus affecting the population density.

1.1.3.4 Predation

Predation by larger zooplanktons and juvenile fish exert certain pressure on and help regulating the population of marine cladocerans. Several workers claimed that marine cladocerans exhibit diel vertical migration and diel reproductive cycle to

prevent visual predation (Onbt\ 1974; Tang et al., 1994; Wong et al., 2007). Wong et

al. (2004) had showed that planktivorous fish exhibited strong selection on female

marine cladoceran with mature embryos, and also on large size individuals. Nip et al.

(2003) also reported that copepods and cladocerans were the dominant food item for

larvae and juveniles of Black seabream, Acanthopagrus schlegeli, and Japanese

seaperch, Lateolabrax japonicus, in Tolo Harbour.

8 1.2 Objective

Marine cladocerans are the second most abundant group of the zooplankton community in Tolo Harbor. Seasonal and spatial patterns of marine cladocerans population had been observed in previous studies. Penilia avirostris and

Pseudevadne tergestina occur sporadically throughout the year, while the other two

Podonids, Pleopis polyphemoides and P schmackeri, appear during the winter time

(Ji, 2001 ). However, the underlying biological and/or physiochemical factors controlling the population of marine cladocerans are not clear.

Analysis of population parameters such as the birth rate, death rate, population growth and fecundity of marine cladocerans could be an efficient method for evaluating population regulation, allowing us to determine whether the cladoceran populations are limited by food, predators, and/or temperature. In this study, we are going to investigate the influence of major environmental factors have on the seasonal variations of the four marine cladocerans found in Tolo Harbour.

Moreover, males play an important role in sexual reproduction through hard time. Emergence of gameogenic generation is related to environmental conditions.

Presence of gameogenic females was reported, but there was no record for the presence of male cladocerans in the population. The present study will look into greater detail of the occurrence of male cladoceran in the population throughout the study period.

9 1.3 Materials and method

1.3.1 Field sam piing

Population dynamics of four marine cladocerans were studied in Tolo Harbour and Mirs Bay from November 2005 to October 2006 (Figure 1.1 ). Water depth at the sampling site in Tolo Harbour (TH, 22°25 '034"N, 114 °13' 164"E) was 10 m. The sampling site in Tolo Channel (TC, 22°28' 539"N, 114 °18'034"£) has a water depth of21 m.

Hydrographical parameters and zooplankton samples were obtained at intervals of about 7 days during the studied period. The sampling interval correspond to about

1-2x the in situ egg development time for Pleopis polyphemoides and Pseudevadne tergestina (Egloff et al., 1997): All field sampling trips occurred between 1030 h and

1230 h to minimize diel variations.

1.3.2 Hydrographical parameters and chlorophyll a concentration

Vertical profiles for temperature, dissolved oxygen content and salinity were measured at 2 m depth intervals from surface to bottom with a HydroLab sensor.

Water samples collected at 1 m for chlorophyll a measurement. Water samples were immediately put into brown bottles and stored in ice to prevent algal growth and chlorophyll degradation. In the laboratory, algae were separated into 3 different size fractions: <125 J.Lm, 20-80 J.Lm and <20 J.Lm. For each size fraction, duplicate

10-mL water samples were filtered through 0.45 J.Lm Millipore nitrogenous membrane filters. Each filter was put into a labeled glass culture tube containing 5 mL of 90% acetone. Chlorophyll was extracted in darkness at 4°C overnight to

10 ensure thorough extraction of the chlorophyll a (Strickland and Parson, 1972).

Concentration of chlorophyll a was measured fluorometrically with Turner Designs

10 fluorometer. Fluorescence is measured before and after acidification with 60 fll of

1Oo/o HCl (Parson et al., 1984 ). Chlorophyll a concentration was calculated according to the equation ofDagg and Wyman (1983):

1 [Chlorophyll a] flg L- = k (Rb- Ra) X (Vacetonel Ysample)

where k is the machine calibration constant, Rb and Ra are the fluorescence readings obtained before and after acidification, Yacetone is the volume of acetone used in extraction and V sample is the volume of filtered sample.

1.3.3 Zooplankton sampling·

Duplicate zooplankton samples were collected by hauling a conical net (0.5 m mouth diameter, 125 IJ.m mesh) vertically from 8 m to surface at TH and from 10m to surface at TC. Zooplankton collected were immediately stored into 250 mL plastic bottles and preserved in 5% sugar-buffered formaldehyde to prevent egg loss (Haney and Hall, 1973).

1.3.4 Zooplankton analysis

In the laboratory, the volume of each sample was adjusted to 100 mL.

Zooplankton samples were observed under a Nikon SMZ645 dissection microscope.

All cladocerans were identified to species and abundances of gameogenic and parthenogenic individuals were enumerated from subsamples using 5%-30% of the original sample. Individuals were carefully examined for sex determination.

Generally, the densities were determined by the average of two counts. A third count

11 was made when 1) the densities in the first two counts differed by more than 30%, or

2) the total number of the cladocerans in the first two counts was <50.

Fifty individuals of each species were randomly sorted out from each sample for body size measurements. Body length was measured to the nearest 20 Jlm using a calibrated ocular micrometer on a Nikon SMZ645 dissecting microscope at X50. For

P. avirostris, body length was measured from the tip of the head to the end of the carapace before the mucro. For Pseudevadne tergestina, Pleoplis polyphemoides and

P. schmackeri, body length was recorded as the gross length (Onbe, 1983) from the tip of the head to the dorsa-posterior edge of the brood pouch (Plate 1). Egg- and embryo-bearing parthenogenetic females were then dissected to determine the number of eggs or embryos. The size of a randomly selected egg or embryo from each clutch was measured. Abundance of chaetognath was also recorded.

1.3.5 Calculation of Population Parameters

Calculations of the population dynamics of cladocerans are generally based on the egg ratio model of Edmondson ( 1968) and Paloheimo ( 1974) which yields an estimate for the instantaneous birth rate (b):

b = [ln(Ep + 1)] IDe (1)

where Ep is the population egg ratio (average number of eggs per individual in the population at time t), and De is the temperature-dependent egg development time

(days) estimated based on water temperature (T °C) by the second-degree

logarithmic polynomial ofBottrell (1975):

12 2 log De= 0.847(log1) - 3.609logT+ 3.796 (2)

The rate of increase (r) is determined from successive population densities by

r = (lnNt2- lnNti) I llt (3)

where Nu and Nt2 are the population densities at times t1 and t2, respectively. The instantaneous death rates (d) is then obtained from the equation:

d=b-r (4)

13 N

TaiPo

~ TH

Shatin

Figure 1.1 Location of sampling stations, TH and TC, in Tolo Harbour, Hong Kong SAR.

14 -· IE Rl . . I I Hl ~ )I I I I I . I 1..

Pet,ilia rtJ.•iroslris J:l/eopis polJ~pllelttoides

I~ BL >I I.P- DL . I I I i I I ..- 1 ·I

P.~e11devt1tlne lerge~-.tina .Plet~pis ~· cit ltltlckeri

Plate 1. Body length measurements of the four marine cladocerans. (Bar= 0.1 mm)

15 1.4 Results

1.4.1 Hydrographical parameters

1.4.1.1 Temperature

Figure 2.1 showed the averaged water column temperature recorded at the two sampling sites, Tolo Harbour (TH) and Tolo Channel (TC), from November 2005 to

October 2006. Averaged water temperature ranged from 16.5 to 29.4 °C, and did not differ between the two sampling sites (Student's t-test, P = 0.673). Lowest water temperature of 16.5 °C was recorded in January and highest water temperature of

31.9°C was recorded on August 17, 2006 at TH and 30.6°C on July 6, 2006 at TC.

From November 2005 to April 2006 were classified as the cooler months of the year, with an average temperature of 19.61 °C, while from May to October 2006 were classified as warmer months, with an average temperature of 26.89 °C. Thermal stratification occurs in summer months from mid-June to August at both sites, where the surface and bottom water temperature differed by more than 2 degree Celsius

(Figure 2.2).

1.4.1.2 Salinity

Figure 2.3 showed the averaged salinity recorded at the two sampling sites from November 2005 to October 2006. Salinity ranged from 10.3 to 34.7 %o.

Salinity followed a seasonal pattern, where it was higher and more stable during the

dry winter, lower and fluctuated greatly during the rainy months in summer. Mean

values were 31.32 and 32.31 %o at TH and TC respectively. Domestic sewage

discharge from Ma On Shan may have lowered the salinity at TH, resulting an

overall lower salinity than at TC (Student's t-test, P = 0.011 ). Haloclines (Figure 2.4)

were observed during the summer months at TH.

16 1.4.1.3 Dissolved Oxygen content

Figure 2.5 showed the averaged dissolved oxygen content recorded at the two sampling sites from November 2005 to October 2006. Averaged dissolved oxygen

1 ranged from 1.80 to 8.27 mg L- • Dissolved oxygen content was generally higher at

TC than TH (Student's t-test, P = 0.004), and higher in cooler period than warmer season. Oxyclines (Figure 2.6) were observed during the summer time at TH.

Dissolved Oxygen content dropped below 3 mg L- 1 for most of the time in summer.

In addition, there was a significant difference in dissolved oxygen content between the two sampling stations in September and October (Student's t-test, P = 0.001 ).

1.4.2 Chlorophyll a concentration

Chlorophyll a concentration was used to indicate the levels of phytoplankton biomass. Chlorophyll a concentration was higher at TH than TC (Student's t-test, P

1 = 0.000). Mean chlorophyll a concentrations were 13.32 and 4.61 J.tg L- at TH and

TC respectively and both stations showed similar patterns (Figure 2. 7a). The highest chlorophyll a concentration was 71.43 and 28.74 J.tg L- 1 on December 9, 2005 at TH and TC respectively. The lowest concentration recorded was 1.66 Jig L- 1 at TH in

November 2005, and 0.52 J.tg L- 1 at TC in April 2006.

Water samples collected after February 2006 were only filtered through

different meshes to obtain chlorophyll a concentrations in phytoplankton of different

size fractions. Figure 2. 7 b & c show the chlorophyll a concentration in

phytoplankton of different size fractions. For the 20-80 J.tm size fraction,

chlorophyll a concentration ranged from 0.04-10.57 and 0.22-4.29 Jig L- 1 at TH and

TC respectively. In the <20J.tm size fractions, chlorophyll a concentration ranged

from 1.11-16.59 and 0.22-7.14 J.tg L- 1 at TH and TC respectively. Results suggested

17 that micro phytoplankton is a major component of the phytoplankton biomass in the study sites.

18 32

26 o · ,-.,u 0 ·.. o ·o '--" o · (l) 24 '- .....:J ro '- (l) 0. 22 E (l) E- 20

18 16 I · · · ~ ·· ~~ I 14 Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006

Figure 2.1 Averaged water temperature at the two sampling stations, Tolo Harbour (TH) and Tolo Channel (TC)

19 a)TH

0 ...,------~------( )------1 ~------{[ ~'----.,r,~---.

----8 8 -'---~---~ ~~)------{'tll~----.----r-Q------..l-r------r-----r-_____J ~ ..c -+--> 0.. Q) 0 b) TC 1-c Q) ~ 0 ~ 2

6 05/06/2006 8 15/06/2006 ----T--- 23/06/2006 - ·· --6·- ·· - 10 29/06/2006 06/07/2006 - ·-D- ·- 14/07/2006 12 ---- 20/07/2006 --+------o- 25/07/2006 14 ·· ···· ·· • ··· ··· ·· 14/08/2006 --....sv--- 17/08/2006 21/08/2006 16 ---o-·---·-··- 31/08/2006 18 22 24 26 28 30 32

Temperature (C)

Figure 2.2 Thermoclines at the two sampling stations, TH and TC, from June-August 2006

20 36 ~------~

34 0 .00 0 · · · rn--v o .· · 0 Cbo- · Oo · o 0 ... . · o ·· ··· .o a . Q

.o . .o·O ··O 32 0 Q ,.-.... 0 ...... 0 0 '-._;' 0 30 :§ ~ r:/)

26 1-· · ~ .. ~~I

241_--~--~--~--~--~--~--~--~~~~~=-~~~:Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.3 Averaged salinity at the two sampling stations, Tolo Harbour (TH) and Tolo Channel (TC)

21 2

\ \ \ \ 6 +9 \ i ~ '\ "

6 • 05/06/2006 8 ...... o ...... 15/06/2006 ---T--- 23/06/2006 - · ·~ · - ·· - 29/06/2006 10 06/07/2006 - ·- D-· - 14/07/2006 ---- 20/07/2006 12 --+-- ---o- 25/07/2006 ...... A ...... 14/08/2006 14 ---v--- 17/08/2006 - ··--·- ··- 21/08/2006 16 - -o - 31/08/2006

18 20 22 24 26 28 30 32 34 36

Figure 2.4 Haloclines at the two sampling stations, TH and TC, from June-August 2006

22 9

q 8 o0 .o . .· 0 o ... o I ··· ~ · · ~~ I 0 o o ...... 0 0 7

0. ? 0 0. 6 .._....0. 0 0. c:: Q) 0 00 :> _.o·.o·· 0 Cl) 0 0 aCl) 4

Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.5 Averaged dissolved oxygen at the two sampling stations, Tolo Harbour (TH) and Tolo Channel (TC)

23 a)TH

0.------~~--~------£~~--~~~r----~--~~--~

~ . · I I 6 f I I

b) TC 0

6 05/06/2006 8 ...... o• ...... 15/06/2006 --~-- 23/06/2006 - ·· ---6·- ·· - 10 29/06/2006 06/07/2006 - ·-D- ·- 14/07/2006 12 ---- 20/07/2006 --+-- --<>-- 25/07/2006 ...... 14 14/08/2006 ---v--- 17/08/2006 .. .. _ _ _.._ 21/08/2006 16 --o- 31/08/2006

18 0 2 4 6 8 10 12 Dissolved oxygen (ppt)

Figure 2.6 Oxyclines at the two sampling stations, TH and TC, from June-August 2006

24 a) Unfiltered water samples

80 ~------~

b) Filtered samples (20 - 80 urn)

-~ ...t:: 0.. 0 $..., :a0 u

c) Filtered samples (<20 urn)

20 ~------~

0+---~----~----~--~------~--~~--~~~--~~~~--~~--~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.7 Chlorophyll a concentrations at the two sampling stations, TH and TC.

25 1.4.3 Seasonal occurrence of marine cladocerans in Toto Harbour

Four species of marine cladocerans were found in Tolo Harbour. While Penilia avirostris and Pseudevadne tergesting_ occurred all year round, Pleopis polyphemoides and Pleopis schmackeri appeared only in the cooler season.

Penilia avirostris

Figure 2.8 shows the abundance of P. avirostris at the study sties. P. avirostris were found throughout the studied period, except a sudden disappearance on

3 October 5, 2006 at TH. Average densities were 991 in d. m -J and 2, 359 in d. m - at

TH and TC respectively. TC generally had higher P. avirostris abundance

throughout the study period (Student's t-test, P = 0.02). Low density of P. avirostris

was recorded in July and August in Tolo Harbour. Highest population densities of

> 12,000 ind. m-3 were recorded· at both sampling stations in mid-January 2006,

where water temperature was around 18 °C.

Pseudevadne tergestina

Figure 2.9 shows the abundance of P. tergestina at the study sties. Like P.

avirostris, P. tergestina were found all year round. Average densities were 1,213 in d.

m-3 and 1,450 ind. m-3 at TH and TC respectively. Highest population density

recorded at TH was 8230 ind. m-3 in mid-January, and 7395 ind. m-3 was recorded at

TC in late September 2006.

Pleopis polyphemoides

Figure 2.10 shows the abundance of P. polyphemoides at the study sties. P.

polyphemoides appeared from February till early June, where temperature ranged

from 17-25 °C. P. polyphemoides obtained a bell shape occurrence at TH, the

26 population appeared in early February, increased steadily and attained its highest density· of 1298 in d. m -3 in mid-April, 2006. Then the population started to collapse and disappeared in the water column in early-June.

Average densities were 154 ind. m-3 and 62 ind. m-3 at TH and TC respectively.

Highest population density recorded at TH was 1,299 in d. m -3 in mid-April, and 93 7 ind. m-3 was recorded when the population first popped up at TC in February 2006.

Pleopis schmackeri

Similar to P. polyphemoides, P. schmackeri appeared in the water column from

February till June 2006 (Figure 2.11 ). The population in TH appeared in early

February, attained its highest density of 404 ind. m-3 in April and gradually disappeared in the water column· before July. Density of P.schmackeri fluctuated between 0 and 326 in d. m-3 in TC. Average densities were 39 in d. m-3 and 36 in d. m-3 at TH and TC respectively.

27 a) TH

14000 • 12000

10000 ,...-._ <'( 8 8000 • "'Cj ·-.._,~ (1) () ~ clj "'Cj ~ ~ .£) < • 2000 • • • • • • •••• 0 • • Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006

b)TC

14000.------~

• 12000 ,...-._ • <'( 8 "'Cj .s.._, (1) () ~ clj "'Cj ~ 6000 ~ • .£) • < • • • • 4000 • • • • • 2000 • • • • • • • • • • 0 Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.8 Seasonal abundance of Penilia avirostris at two sampling stations, TH and TC.

28 a) TH

10000 ~------.

8000 •

,-._ <";' E "0 6000 vt:: (!) (.) t:: ro "0 t:: 4000 :::3 ..n •

b)TC

10000

8000

,-._ • <";' E • "0 6000 .5 "-" (!) (.) t:: • ro • "0 t:: 4000 :::3 ..n •

Figure 2.9 Seasonal abundance of Pseudevadne tergestina at two sampling stations, TH and TC. a)TH

1600

1400 • 1200 ,..-..... • <";l E 1000 "'d ·-...... _,t:: Q) u 800 • t:: ~ "'d • t::::s 600 • .£; ~ 400 • • 200 • • •

0 ~---.~.-.-~~----.----.----.---~--~~--~~~.. ~~NH~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

b)TC

1600

1400

1200 ,..-..... <";l E 1000 "'d .5...... _, Q) u 800 t:: ~ "'d t:: ::s 600 .£; ~ 400

200

0 Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.10 Seasonal abundance of Pleopis polyphemoides at two sampling stations, TH and TC. a)TH

600 ~------~

500

~ I(e 400 • ."'ds '_,/ • Q) u 300 0 c!j "'d 0::s ..D 200 • • ~

100 • •• • 0 ~----~---.~--~~~~.-~----~--~--~--~~~--~~~~--~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

b)TC

600

500

~ I(e 400 .s"'d '_,/ Q) u 300 0 c!j "'d ::s0 ..D 200 ~

100

0 Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.11 Seasonal abundance of Pleopis schmackeri at two sampling stations, TH and TC. jl 1.4.4 Parameters of the marine cladocerans populations

Penilia ·avirostris

Body length of P. avirostris ranged from 0.34 to 1.12 mm (Figure 2.12 a & b).

Large individuals of > 1 mm in size were observed at both stations in July and

August where densities were low, but there was no significant difference in mean body length between cooler months, from November to April, and warmer months, from May-October (Student's t-test, P = 0.171).

Figure 2.13 a & b show the frequency distributions of parthenogenetic females of P. avirostris with different brood size. Maximum fecundity of females observed was 8. There was no significant difference in mean fecundity between cooler months and warmer months (Student's t-test, P = 0.176).

Development time ranged from 2.11 to 4.48 days, and differed significantly between November to April and May to October (Student's t-test, P = 0.000). Figure

2.14 shows the changes in birth rate, growth rate and death rate of the P. avirostris population. Birth rate varied from 0.35 ind. day- 1 in January to 0.83 ind. day- 1 in

August. Birth rate was positively correlated with temperature (TH: r=0.923, p=O.OO;

TC: r=0.970, p=O.OO at the 0.01 level), which lower birth rate was observed during cooler months and increased as temperature rose. Population growth rate ranged

1 from -0.4 7 to 0. 75 in d. day- , negative growth rate were observed more frequently during cooler months. The instantaneous death rate varied from -0.12 to 1.19 ind.

1 day- •

Pseudevadne tergestina

Body length of P. tergestina ranged from 0.38 to 0.86 mm (Figure 2.15 a & b).

32 There were no significant differences in mean body length between cooler and warmer·months at both stations (Student's t-test, P > 0.1 ).

Figure 2.16 a & b show the frequency distributions of parthenogenetic females of P. tergestina with different brood size. Maximum fecundity of females observed was 8. There was significant difference in mean fecundity between cooler months and warmer months at both stations (Student's t-test, P < 0.05). Most females carried

4 embryos.

Figure 2.17 shows the changes in birth rate, growth rate and death rate of the P. tergestina population. Birth rate varied from 0.35 ind. day- 1 in January to 0.81 ind. day- 1 in July. Birth rate was positively correlated with temperature (TH: r=0.988, p=O.OO; TC: r=0.995, p=O.OO at the 0.0 I level). Population growth rate ranged from

1 -0.73 to 0. 78 in d. day- , negative growth rate were observed more frequently during

1 cooler months. The instantaneous death rate varied from -0.16 to 1.14 ind. day- •

Pleopis polyphemoides

Body length of P. polyphemoides ranged from 0.20 to 0.60 mm (Figure 2.18).

Maximum fecundity of females observed was 12 (Figure 2.19). Most females

1 carried 4 to 8 embryos. Birth rate ranged from 0.43 to 0.83 ind. day- • The

1 instantaneous population growth rate varied from -0.71 to 0.37 ind. day- • Death rate

1 varied from 0.14 to 1.18 ind. day- • High death rate was observed before the population disappeared from the water column (Figure 2.20).

33 Pleopis schmackeri

Body length of P. schmackeri ranged from 0.30 to 0.62 mm (Figure 2.21 ).

Maximum fecundity of females observed was I 0 (Figure 2.22). Most females

1 carried 4 to 8 embryos. Birth rate ranged from 0.45 to 0.86 ind. day- • The

1 instantaneous population growth rate varied from -0.28 to 0.21 ind. day- • Death rate

1 varied from 0.32 to 0.92 ind. day- • Similar toP. polyphemoides, high death rate was

observed before the population disappeared from the water column (Figure 2.23).

34 25 ,------~

20 2005 November May

0 .J...... _---.--....t::l..-!.i.l..ll.I::I..U..LJ..

25 .------~

20 December June

0 ..I._____.WL._r------":l..llJ..!:J..~L....ll.l;ll;

25 ~------~

20 2006 January July

5 -~ Q) ~ 5 25.------~ 2 Q) ~ 20 ·February August

OL_~~~~~illillrnill~~~~-----J

25 -...------~

20 March September

0 -'---.------"'~~

25 ~------~

20 April October

400 600 800 1000 1200 400 600 800 1000 1200 Body length (urn)

Figure 2.12a Size distribution of Penilia avirostris in Tolo Harbour. 35 25 .------~

20 2005 November May

0 ...__--..----""'-""-'"""

25 .------~

20 December June

0 ..______,_....!;.;L_..L;U::l....~~ ;l.l;u:J..~;JJW::J.J:;.u;u::LJ.J..l;;.L_ ____...------1

25 ~------~

20 2006 January July

,-.. ?]( 5 '-" ~ o L----.-_.JllWUlJUJllill ...... ro ~ 25 ~------~ 2 ~ 20 February August

0 ~--~-=~==~~==~~==~~------~ 25 .------~

20 March September

0 ..L...-...1.'1.....--.,..-.J;i;l.C..I~

25 ~------~

20 April October

400 600 800 1000 1200 400 600 800 1000 1200 Body length (urn)

Figure 2.12b Size distribution of Penilia avirostris in Tolo Channel. 36 50 November May 40

0 50 December June 40

0 50 January Jul y 40

20 -e 10 (!) 0 ~s:: (!) (.) ...... 50 (!) ~ February August 40

0 50 March September 40

0 50

40 April October

0 0 2 4 6 8 10 0 2 4 6 8 10 Brood size

Figure 2.13a Frequency distributions of parthenogenetic females of Penilia avirostris with di fferent brood size in Tolo Harbour. 37 50 2005 November May 40

0 50 December June 40

0 50 2006 January July 40

10 -~ 11) OJ) 5 0 s:: 11)e 50 11) p.. February August 40

0 50 March September 40

0 50

40 April October

0 0 2 4 6 8 10 0 2 4 6 8 10 Brood size

Figure 2.13b Frequency distributions of parthenogenetic females of Peni/ia avirostris with different brood size in Tolo Channel. 38 a) Birth rate ~ Tolo Ha rbour ___. __ Tolo Channel

0.8 ,.-..., ~ ""0 0.6 '-' (!)

~;...... c :E 0.4 co 0.2

0.0 ~----~----~----~----~----~----~----~----~----~----~----~.---~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006 b) Growth rate

1.0 ~------,

,.-..., 0.5 ;;;...... ro ""0 '-' ...... (!) ro;..... 0.0 ...c ~ 0;..... 0 -0.5

-1.0 -+------.------.-----~----~----~----~----~----~----~------r----~------i Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006 c) Death rate

1.5

,.-..., 1.0 ~ ""0 '-' (!) 0.5 ~;...... c ~ (!) Q 0.0

-0.5

Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006

Figure 2.14. Population parameters of Penilia avirostris at two sampling stations, a) birth rate, b) growth rate and c) death rate. 39 25

20 2005 November May

0 25

20 December June

0 25

20 2006 January July

5 -~ Q) 01) 0 g t: Q) 25 (.).... Q) p... 20 February August

0 25

20 March September

0 25

20 April October

0 300 400 500 600 700 800 900 300 400 500 600 700 800 900 Body length (urn)

Figure 2.15a Size distribution of Pseudevadne tergestina in Tolo Harbour. 40 25~------.

20 2005 November May

o~---4~~~~~~~~~~~~~

25~------~

20 December June

o~--~~~~~~¥=~~~~~~~

25~------~

20 2006 January July

February August

o~--~~~~~~~==~~~~~~

25~------~

20 March September

o~--~~~~~~~==~~~~~~

25~------~

20 April October

o~---¥~~~~~~~~~~~~~ 300 400 500 600 700 800 900 300 400 500 600 700 800 900 Body length (urn)

Figure 2.15b Size distribution of Pseudevadne tergestina in Tolo Channel.

41 100 2005 November May 80

0 100 December June 80

0 100 2006 January July 80

20 -~ 11)on $3 0 t: 11) (.)..... 100 11) 0.. . February August 80

0 100 March September 80

0 100

80 April October

0 0 2 4 6 8 10 0 2 4 6 8 10 Brood size

Figure 2.16a Frequency distributions of parthenogenetic females of Pseudevadne tergestina with different brood size in Tolo Harbour. 42 100 2005 November May 80

0 100 December June 80

0 100 2006 January July 80

20 -~ Q) bJ) cC'::S 0 Q) u..... 100 Q) ~ February August 80

0 100 March September 80

0 100

80 April October

0 0 2 4 6 8 10 0 2 4 6 8 10 Brood size Figure 2.16b Frequency distributions of parthenogenetic females of Pseudevadne tergestina with different brood size in Tolo Channel. 43 -e-- Tolo Harbour a) Birth rate --··• .. ···· Tolo Channel

0.8

'"0~ 0.6 "-"' Q) ~,_. ...c 0.4 .E co

0.2

0.0 ~------.------.------.------.------,.------,------,------,------,------~------~~-----~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006 b) Growth rate

--. 0.5 ;;...... ro '"0 "-"' Q) ~,_. 0.0 ...c ~ 0,_. 0 -0.5

-1. 0 -i------~------,.------r------.------,.------.,------..------r------~------r------..------1 Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006

c) Death rate 1.5

--. 1.0 ~ '"0 "-"' ...... Q) ro,_. 0.5 ...c...... ro Q) Cl 0.0

-0.5

Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006

Figure 2.17 Population parameters of Pseudevadne tergestina at two sampling stations, a) birth rate, b) growth rate and c) death rate. Tolo Harbour Tolo Channel.

40.------~ 2006 February 2006 February 30

o~--~~~~~~~~~~~--~

40~------~ March March 30

o~~~~~~~~~~--~----~

40~------~ April April 30

10 -~ Q) OJ) £3 §c 40~------~ Q) t:l. May May 30

o~~~~~~~~~~~~----~

40~------. June June 30

0~--~~~~~~~~--~----~ 200 300 400 500 600 700 200 300 400 500 600 700

Body length (urn)

Figure 2.18 Size distribution of Pleopis polyphemoides in Tolo Harbour and Tolo Channel.

45 Tolo Harbour Tolo Channel.

2006 February 2006 February 60

0 80 March March 60

,-., 20 ~ 0 00 5 0 s::: 0 (.) 80 p..."""0 April April 60

0 80 May May 60

0 80 June June 60

0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Brood size

Figure 2.19 Frequency distributions of parthenogenetic females of Pleopis polyphemoides with different brood size in Tolo Harbour and Tolo Channel.

46 a) Birth rate -e- Tolo Harbour ·-• - Tolo Channel 1.0 .,------======:;

0.0~----~--~----~--~----~----~--~~--~----~----~--~----~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov

2005 2006 b) Growth rate

1.0.------.

,-._ 0.5 ~ "'0 '-" a) d 0.0 1-o I ..t:: 1\~I ~ \I 0 1-o \ ~ 0 -0.5 \ ~

-1.0 -t-----~----r-----.---~------.-----.----~----~----.,.----~----~----1 Nov Dec Jan Feb Mar Apr May Jun Jut Aug Sep Oct Nov

2005 2006

c) Death rate 1.5

,-._ 1.0 ~ "'0 '-" .....a) ro 0.5 1-o ..t::..... ro a) Cl 0.0

-0.5

Nov Dec Jan Feb Mar Apr May Jun Jut Aug Sep Oct Nov

2005 2006

Figure 2.20 Population parameters of Pleopis polyphemoides at two sampling stations, a) birth rate, b) growth rate and c) death rate. '"t/ Tolo Harbour Tolo Channel.

40,------~ 2006 February 2006 February 30

o~----~~~~~~~~~~--~

40~------~ March March 30

o~----~~~~~~~~~----~

40~------~ April April 30

10 ~ ~ Q) & ~ 40~------~ ~ Q) 0.. May May 30

o~----~~~~~~~~~~--~

40~------~

June June 30

0~----~--m_~~~~~~----~ 200 300 400 500 600 700 200 300 400 500 600 700

Body length (urn)

Figure 2.21 Size distribution of Pleopis schmackeri in Tolo Harbour and Tolo Channel.

48 Tolo Harbour Tolo Channel.

2006 February 2006 February 60

0 80 March March 60

40 e,-.. 20 Q) OJ) g 0 s:: Q) ....(.) 80 Q) t:l.. April April 60

0 80 May May 60

0 80 June June 60

0 0 2 4 6 8 lO 12 0 2 4 6 8 10 12 Brood size

Figure 2.22 Frequency distributions of parthenogenetic females of Pleopis schmackeri with different brood size in Tolo Harbour and Tolo Channel.

49 a) Birth rate ---- Toto Harbour ---• --· Toto Channel 1.0

0.8 ,-.., ~ "'0 0.6 "-" (!) ...... ro ~ ..s:: 0.4 .~ =" co 0.2

0.0 ~----.----.----~----~----.---~----~----~----r----.-----.--~ Nov Dec Jan Feb Mar Apr May Jun Jut Aug Sep Oct Nov

2005 2006 b) Growth rate

1.0 ~------~

,-.., 0.5 - ~ "'0 "-" ...... (!) ro ~ 0.0- ..s:: ~ ~ Jt0 0 ~ 0 -0.5 -

-1.0 -+-----.------.----~-----.------.,.-----..------.-----.----.----~----.,...------i Nov Dec Jan Feb Mar Apr May Jun Jut Aug Sep Oct Nov

2005 2006

c) Death rate 1.5 -

1.0 -

0.5 -

0.0-

-0.5 -

Nov Dec Jan Feb Mar Apr May Jun Jut Aug Sep Oct Nov

2005 2006

Figure 2.23 Population parameters of Pleopis schmackeri at two sampling stations, a) birth rate, b) growth rate and c) death rate. 1.4.5 Occurrence of gamogenic individuals of marine cladocerans

Gamogenic females and males of P tergestina and P polyphemoides appeared sporadically throughout the study period (Figure 2.24 and Figure 2.25).

Three peaks, where gamogenic females of P tergestina made up more than

15o/o of the population, were observed in February, late July and October 2006 at TH.

Percentage of gamogenic females of P tergestina at TC was lower than that at TH, but appeared at similar times of the year. Males of P tergestina were recorded in the sampling sites (Plate 2) and were observed at low percentage, usually not exceeding

5o/o of the population, at both stations at similar times.

Gamogenic females of P polyphemoides were detected at both sampling stations. A maximum of 30o/o of gamogenic females occurred when the population dropped sharply in late February at TC. Males were rarely observed and only a few

individuals were recorded in March at TH and in February at TC (Plate 3).

1.4.6 Occurrence of chaetognaths

Ingestion of marine cladocerans by chaetognath was often observed in the

samples (Plate 3). Figure 2.26 shows the abundance of chaetognaths during the

sampling period. Average abundance of chaetognaths were 18.16 and 7.58 in d. m -3

in Tolo Harbour and Tolo Channel respectively, and were more abundant during

summer.

51 ~ Tolo Harbour a) Gameog.enic females --··• ····- Tolo Channel

30 .------~

,-.. 20 .....__,'t( (!)on .....ro 15 s:: (!) (.) (!)""" ~ 10

-~ Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

b) Gameogenic males

30 ~------~

,-.. 20 .....__,'t( (!)on .....ro 15 s:: (!) (.) (!)""" ~ 10

0 Nov 2005 2006

Figure 2.24 Percentage of gameogenic individuals of Pseudevadne tergestina in Tolo Harbour and Tolo Channel.

52 -e- Tolo Harbour a) Gameogenic females ·--··•···-· Tolo Channel

~ ~ '-' Q) 30 on .....C'd s:: Q) () 20 Q)""'" ~

0-t---.-----.-----rl'la-- Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

b) Gameogenic males

10~------~

8 -

~ ~ '-' 6 - Q) .....~ s:: Q) () 4- ""'"Q) ~

o -+---..-----..----rl~~Ji. ~\ ~P~~ ---~:HI; :~--r---..-----.----.------~ Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.25 Percentage of gameogenic individuals of Pleopis polyphemoides in Tolo Harbour and Tolo Channel.

53 200 micron

Plate 2. A male Pseudevadne tergestina

200 micron

Plate 3. A male Pleopis polyphemoides

54 Plate 4. Ingestion of marine cladoceran, Pseudevadne tergestina, by a chaetognath.

55 a) Tolo Harbour 100

~ 80 - C( 8 • ""Cj .5 60 - • ~ (!) u • • ~ <:\$ 40 - • ""Cj • ::s~ .r:J • • ~ 20- • • • • • • • • .. • ....• .,. • • • • 1- I I I -·I I I I I I I 0 .. - ... •• • Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

b) Tolo Channel 100

~ 80 C( 8 "'Ci .5 60 ~ (!) u ~ <:\$ 40 ""Cj • ::s~ .r:J • ~ 20 • •• • • • • • • • • 0 Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov 2005 2006

Figure 2.26 Seasonal abundance of chaetognaths in Tolo Harbour and Tolo Channel.

56 1.5 Discussion

The seasonal distribution, size composition and fecundity of the four marine cladocerans were studied in Tolo Harbor and Tolo Channel from November 2005 to

October 2006. The occurrence of the marine cladocerans followed similar patterns as observed in previous studies (Tang et a/., 1995; Ji, 2001 ). Penilia avirostris and

Pseudevadne tergestina occurred throughout the year, whereas Pleopis polyphemoides and P. schmackeri occurred only in cooler months of the year.

Previous studies recorded annual cycles in population abundance of marine cladocerans in different regions (Cheng and Chao, 1982; Egloff et a/., 1997; Onbe,

1977). Unlike their freshwater counterparts, there were few studies on the population dynamics of marine cladocerans, not until Marazzo et a/. (2003, 2004) attempted to quantify birth rates of marine · cladocerans based on the egg-ratio model of

Edmondson (1968) and Paloheimo (1974). By studying the population dynamics help to understand more on the short- and long-term changes in the marine cladocerans population size and age composition, and the biological and environmental processes that influences those changes.

The observed birth rates of all four cladocerans in this study ranged roughly

1 from 0.4 to 0.8 ind. day- , and were positively correlated with temperature. Yet, many values of the population growth rate were near zero, high negative values occurred in low temperature months for P. tergestina and P. avirostis, whereas high negative growth rate values were observed in Pleopis spp. population as temperature

increased. The low growth rate, with a doubling time of around 2 days, may help

explain the difference in population abundance among studies in the Pacific Ocean.

In the inland sea of Japan, On be (1974) recorded a maximum abundance of 58,800 P.

57 3 avirostris in d. m - , whereas Yoo and Kim ( 1987) reported a density of 21,491 P. avirostris in d. m -3 in Chinhae Bay of Korea. Maximum abundance of marine cladoceran species recorded in Hong Kong seldom passed ten thousand individuals per cubic meter, this could be a result of the poor water quality in Tolo Harbour.

However, growth rate records were lack in previous studies for good comparison.

Effects of temperature

On be ( 1977) suggested that the occurrence of marine cladocerans depend primarily on water temperature. Elgoff et al. (1999) added that each species has its own unique niche in relation to salinity and temperature. P. avirostris and

P. tergestina have been classified as warm water species because their peak occurrence are confined to summer months in temperate waters of northwestern

Pacific (Onbe, 1974; Onbe and -Ikeda, 1995). Johns et a!. (2005) reported that an increase in sea surface tetnperature provided favorable environment to aid P. avirostris colonization in the North Sea. Nevertheless, Yoo and Kim ( 1987) reported that both P. avirostris and P. tergestina were found to survive in the plankton community at 10 °C in Chinhae Bay and the coastal water of Korea.

Although the abundance of P. avirostris and P. tergestina in this study varied

3 roughly with temperature, the unexpected high occurrences (>8,000 ind. m- ) of these warm-water species during periods of low water temperature (18°C) and relatively low abundance during the summer period suggested that water temperature

is not the only major factor controlling the seasonal distribution of these marine

cladocerans. One possibility for the observed peak during winter could be the

emergence of neonates from the resting eggs. Although the data for resting egg

abundance is lacking, it was known that all the four marine cladocerans in Tolo

58 Harbour produce resting eggs to survive through difficult times in the year (Ji, 2001 ;

Li, unpublished data 2005). The conditions leading to the hatchment of the resting eggs are still unclear, but it was suggested that salinity, temperature and duration of dark incubation may be the stimulating factors (Egloff et al., 1997). Valenin and

Marazzo (2003) reported that resting eggs of P. avirostris hatch in pulses rather than continuously, this also explain why sudden peak in abundance may be observe in the population.

P. polyphemoides and P. schmackeri, however, showed that they are more temperature-regulated. Presence of P. polyphemoides and P. schmackeri were detected in late winter and early spring, when temperature dropped below 20°C.

Their populations disappeared from the water column as temperature began to rise towards summer. High temperature (>22°C) is a limiting factor for P. polyphemoides and P. schmackeri. Yet, P. schmackeri has also been termed as warm water species because its peak occurrence is limited to summer months in temperate regions (Onbe,

1974, Onbe and Ikeda, 1995). P. schmackeri was found in the plankton community with temperature ranging from 19.71 to 30.39 °C (On be and Kim, 1989), while P schmackeri are more likely to occur during the cooler months in Tolo Harbour with water temperature around 16 to 22 °C. In addition, On be et al. ( 1996b) stated that P. polyphemoides has a high affinity to estuarine water and prefer less saline water.

However, population of P. polyphemoides occurred in period where salinity was

32-34%o in Tolo Harbour. It is obvious that most species are not strictly limited to

warm or cold water, and they could have adapted to local regime of temperature and

salinity.

59 Effects of oxygen

Dissolved oxygen concentration (DOC) in the water column is a crucial environmental parameter for the successful development of many pelagic organisms

(Ekau et a/., 2009). Previous research had shown that cladocerans were one of the

dominant food items detected in the gut content of larvae and juveniles of Black

seabream and Japanese seaperch in Tolo Harbour (Nip et al. 2003). Low oxygen

levels make it impossible for the animals to migrate to the dark bottom layers in

daytime to avoid predators.

DOC at 3 mg L- 1 is set as a reference for hypoxia in the sea (Miller eta/., 2002).

During the summer stratified period, hypoxia conditions were observed below 4 m in

TH and 8 m in TC. Mean dissolved oxygen concentrations below 3 mg L- 1 were

observed several times from July to October in TH.

As we compare the results from the two sampling stations, DOC was apparently

a major limiting factor on populations of P. avirostris and P. tergestina from July to

October. We observed a higher abundance of the two marine cladocerans in TC than

in TH in September and October. While temperature and salinity were similar at both

sampling stations, mean dissolved oxygen level differed significantly, causing the

differences in the cladocerans abundances at the two stations.

Effects of salinity

Unsteady weather and heavy rainfall during summer time had led to vast

fluctuations on salinity. We believed that great fluctuations on salinity over short

period of time could make the environment unfavourable for the marine cladocerans,

and this was supported by the presence of gameogenic individuals of P. tergestina

60 recorded in mid-June till September, 2006. However, we cannot conclude the effects of unstable salinity had on the claodceran population.

The low abundance of marine cladocerans during mid-late summer may be resulted from the combined effects of poor tidal flush in the embayment and the presence of thermocline, halocline and oxycline. Acting together, these factors limited the mixing of nutrients in the water column and limited the growth of marine cladocerans. Combining results from Ji (200 1), there is a promising rise in population for P avirostris and P tergestina in September, which suggested that the population growth was supported by the redistribution of nutrients after the breakdown of stratification.

Effect of trophic factors

Chlorophyll a concentration

Tolo Harbour has a long history of eutrophication (Wear et a/., 1984; Hodgkiss and Chan, 1987), although the discharge of domestic sewage has stopped since the

late 1990s, phytoplankton biomass is still high (HKEPD, 2003). We recorded a mean annual primary biomass of 13.31 and 4.61 Jlg chlorophyll L- 1 in TH and TC

respectively. Several workers reported a gradual increase in dominance of

dinoflagellates and decrease in diatoms in the 1980s (Hodgkiss and Chan, I 987; Wu,

1988; Lam and Ho, 1989), suggesting that marine cladocerans may become food

limited as dinoflagellates are considered as unsuitable food for zooplantkers (Kim et

a!., 1988; Huntley eta!., 1987).

Chlorophyll a was measured at three different stze fractions, including

unfiltered water sample and filtered water samples of 20-80 J.lm and <20 J.lm. These

61 were chosen according to the feeding selectivity of marine cladocerans. Katechakis and Stibor (2004) showed that P avirostris and Podon intermedius preferred prey items ranged from 15-70 J.tm , while Evadne nordmanni had higher grazing coefficients for organisms of 10 J.tm and 125 J.tm, but not of intermediate size.

However, we did not detect a relationship between cladocerans population or fecundity and chlorophyll concentrations. Also, we could not use chlorophyll a concentrations to explain the variations in cladocerans populations, it may only suggest that grazing pressure was strong in times of increasing marine cladocerans abundance.

Predation

Another important factor that may help regulating the manne cladocerans population is predation. Predation by fish larvae tended to be the main factor controlling the populations of P polyphemoides and P schmackeri from February to

April. Although fish larvae were not investigated in this study, our results were in agreement with previous findings. Nip eta!. (2003) reported the feeding selectivities of larval and juvenile black seabream and Japanese seaperch in Tolo Harbour.

Juvenile black seabream showed a strong preference on Pleopis spp. to other

cladocerans species from February to early April. After that, we observed an increase

in the Pleopis populations in April and a noted decrease in death rates. On the other

hand, P avirostris and P. tergestina were preyed upon by juvenile Japanese seaperch

from March to April (Nip eta!., 2003), a decrease in the abundance of P. avirostris

and P. tergestina were observed.

Chaetognath is considered as another important predator of cladocerans (Canino

and Grant, 1985; Marazzo and Valentin, 2003b). Although we did not examine the

62 gut contents and feeding rate of chaetognaths, we observed ingestion of cladocerans in the gut (as shown in Plate 4) and also an increase in death rates of P. avirostris and

P. tergestina when chaetognath population increased in late-January and mid-July.

While dissolved oxygen content was the limiting factor on marine cladocerans population from July to October, a further increase in the death rates of P. avirostris and P. tergestina was observed when the abundance of chaetognaths increased in mid-July. These observations suggested that chaetognath also plays an important role in regulating the cladoceran population.

Gameogenic reproduction

Cladocerans are known to have an environmental sex determination system with cyclic parthenogenesis. Environmental factors affect cladoceran developmental time and survival rate, as well as its mode of reproduction. Marine cladocerans

perform two types of reproduction: parthenogenic (asexual) gameogenic (sexual).

During unfavorable times, a portion of the cladocerans population will switch to

reproduce sexually and produce resting eggs to survive the hard time. Percentage of

gam eo genic individuals in the population may vary from less than 10% to 86%

(Onbe, 1978; Fofonoff, 1994; Onbe et al., 1996). The study in Brazil (Marazzo and

Valentin, 2004) demonstrated a steady increase of P. tergestina gameogenic

individuals towards the unfavorable time of the year. Gameogenic females of P

avirostris, P. tergestina and P polyphemoides have been observed in Tolo Harbour

(Ji, 2001; Li, unpublished data). Gameogenic females of P tergestina occurred

during the peak occurrence in summer and those of P polyphemoides co-occurred

with the parthenogenic population. Ji (200 1) did not record any male in the

population.

63 Gameogenic individuals, both female and male, of P. tergestina and P. polyphemoides were observed in the current study. Gameogenic individuals of P. tergestina were found to co-occur sporadically with the parthenogenic population, and at times gameogenic females were composed up to 20%, while males usually took up 5o/o of the planktonic population. Gameogenic individuals of P. polyphemoides co-occurred with the population.

Interestingly, the pattern of occurrence of males was not totally parallel to the

occurrence of females with resting eggs. There were times when males are detected

in the planktonic community, but no gameogenic females were seen and vice versa.

Marazzo and Valentin (2004) hypothesized the concept of "constant population sex

ratio" in Penilia avirostris, suggesting the percentage of male in the population

remains constant and low over time, and that the appearance of gameogenic females

with resting eggs is a function of the probability of sexual reproduction. Our results

may help support this hypothesis, while the constant percentage of males of P.

tergestina may only contribute around 5% of the population, and the percentage of

male P. polyphemoides is less than 2%.

Onbe (1985) reported that the fluctuation of the number of resting egg was

closely correlated with the occurrence of the planktonic population, where the egg

number is highest just before the population disappeared from the water column. We

observed similar pattern in P. tergestina, where gameogenic individuals happened to

appear before population declination.

Since the gameogenic population only comprises a small portion, we did not

detect a major influence of gameogenic population had on the caldoceran population.

64 Nevertheless, the switching from parthenogenesis to gameogenesis should cause a further reduction in the population birth rate and density, as each gameogenic female could only carry one resting egg at a time and a portion of individuals are males.

Although the underlying environmental conditions that caused sexual reproduction in cladocerans is still unrevealed, it is known that juvenile hormones could induce the production of male neonates. Olmstead and LeBlanc (2002) reported that the juvenile hormone in insects and crustaceans is a sex determinant in

Daphnia magna, which when exposed could induce the production of male neonates and reduce reproduction rates. Oda et al. (2005) had shown similar results in four other freshwater cladoceran species by exposing the animals to a juvenile hormone analog. However, the effect of juvenile hormone and their analogs had on marine cladocerans had not been studied. From our study, there was no direct relationship between the occurrence of male and the occurrence of juvenile in the planktonic community.

65 1.6 Conclusion

General trend of marine cladoceran populations in Tolo Harbour

Penilia avirostirs and Pseudevadne tergestina

P. avirostris and P. tergestina are the only two marine cladocerans that occurr in the plankton community throughout the year in Tolo Harbour. Their populations

increase during the summer months, but summer mid-way decline is also observed.

Population increase due to a rise in temperature that speeds up their developmental time. However, the population growth is then limited by certain factors, such as the

unstable weather during summer time causes fluctuations in salinity, stratification

established in the water column limits the mixing of nutrients in the water column,

dissolved oxygen was found below 3 mg L- 1 and predation by chaetognaths. As the

temperature start to cool down in September, there is a promising rise in population

abundance as de-stratification causes the redistribution of nutrients in the water

column.

The population declines during winter as lower temperature slows down the

developmental time and there is a reduction in brood size. The population is also

regulated by predation. Predation by chaetognaths starts from mid-January to March,

and followed by predation by juvenile Japanese sea perch from March to May (Nip

et al., 2003).

Pleopis polyphemoides and P schmackeri

P. polyphemoides and P schmackeri appear only in the cooler months of the

year, usually from late January to early June. Temperature seems to be a major

regulator of the Pleopis populations. The population starts in late January, but it is

66 suppressed by strong fish predation (Nip et al., 2003). They populate fast during

March to May, and as temperature rises, the population declines and disappears from the water column.

The pattern in abundance in one place, however, could not be seen as a universal pattern in other places. Although the pattern of occurrence is quite similar in the study carried out in Tolo Harbour by Ji (200 1) and the current study, we observed an exceptional high occurrence of P. avirostris and P. tergestina during the cold period. Differences in annual occurrence and abundance are due to the fact that biotic and abiotic factors vary from year to year. All in all, marine cladocerans are environmental-sensitive, the combined effect of environmental factors could have great impact on the population development. Future research could, therefore, focus on how global warming has affected the marine cladocerans population.

67 Chapter 2 Molecular detection of the diet of the marine cladocerans, Pseudevadne tergestina

2.1 Introduction

2.1.1 The importance of marine cladocerans

Zooplankton play an important role in the marine food web as mediators of energy transfer (Riegman et al., 1993). Mesozooplankton grazers distribute organic matters synthesized by phytoplankton towards higher trophic levels (Katechakis and

Stibor, 2004). Marine cladocerans play an important role in the pelagic food web as grazers of phytoplankton and as food of zooplanktivores.

Unlike their freshwater counterparts, which are major components of freshwater e~osystems, marine cladocerans are often considered as a minor component of the marine zooplankton because of their low abundance. Marine cladocerans generally play a less important role than copepods in marine pelagic systems (Egloff et al., 1997), but

Kim et al. (1989) noted that marine cladocerans may influence the trophodynamic pathways of the plankton community during periods of high abundance.

Understanding the feeding relationships between organisms is essential for disclosing the trophic structures of complex ecosystems. Compared to the feeding ecology of planktonic copepods which has been the subject of considerable research, information on the feeding habits of marine cladocerans is still insufficient for defining their natural diets and for analyzing their feeding impact on variation of food organisms.

68 Besides, published information is sometimes contradictory, giving little details on their trophic impacts on microbial communities (Turner, 1984).

2.1.2 Previous findings on cladoceran diet

The eight true marine cladocerans belong to two families. Penilia avirostris is the only member of the family Sididae, while the remaining seven species belong to the family Podonidae.

2.1.2.1 Sidid cladocerans

More information is available on the feeding habit of Penilia avirostris. P. avirostris is a suspension feeder which uses feather-like feeding appendages to entrain and transport food items towards the mouth. P. avirostris often prefers small particles such as small diatoms, microflagellates and bacteria (Gore, 1980; Paffenhofer and

Orcutt, 1986; Kim et al., 1989), although the presence of bacterivory in P. avirostirs was rejected by Turner et al. (1988).

Gore ( 1980) investigated the feeding behaviour of P. avirostris using plastic micronic beads and found that individuals ingested particles up to 50um, but preferred

particles smaller than 20~m. Turner et al. (1989) found that the filtering appendages of

P. avirostris formed a fine mesh for filtering particles as small as 2 ~m.

2.1.2.2 Podonid cladocerans

The seven species of marine podonids belong to the genera Evadne,

Pseudevadne, Pleopis and Podon. Very little is known about the feeding habits of

69 marine podonids.

Ceratium sp. was considered as an important food item for podonids by several early studies (Bainbridge, 1958; Morey-Gaines, 1979). However, later work suggested that centric diatoms are the most important food for marine cladocerans. The diatoms

Rhizosolenia sp., Amphora sp. and Entornoneis sp. were found in the faecal pellets of

Podon intermedius (Jagger et al., 1988). Kim et al. (1989) examined the gut contents of five species of marine cladocerans (Evadne nordmanni, Pseudevadne tergestina, Penilia avirostris, Podon leuckarii and P. polyphemoides) with scanning electron microscope.

Centric diatoms including Skeletonema costatum, Chaetoceros spp., Thalassiosira allenii, Cyclotella meneghiniana and Coscinodiscus spp. were found to be the most dominant food type, and food particles smaller than 35um were preferred.

Contrary to P. avirostris, podonids cannot handle small particles efficiently

(Jagger et al., 1988). The structure of the feeding appendages (Nival and Ravera, 1981) and the presence of a prominent compound eye (Russell-Hunter, 1979) suggested that podoniids are raptorial feeders and visual predators. Nival and Ravera (1981) reported that the mouthparts of podonids are capable of consuming food particles ranging from

20 to 170 J.!m in diameter. Microzooplankton and large phytoplankton are within this size range. The freshwater cladoceran Leptodora kindtii is known to be a raptorial carnivore (Branstrator, 2005). However, since no animal remains have ever found in the guts and fecal pellets of marine podonids, they are regarded as raptorially-feeding herbivores (Jagger et al., 1988).

70 Several workers agreed that the weak swimming capability of manne cladocerans limit them to feed more on immotile organisms and materials, such as diatoms or detritus, than on motile organisms like dinoflagellates and microzooplankton

(Freyer, 1968; Conover, 1978; Jagger et al., 1988; Kim et al. 1989).

2.1.3 Methods to investigate the feeding habit of animals

Previous researches on the feeding ecology of marine cladocerans have been based on in vitro feeding experiments, gut pigment analyses, gut content and fecal pellet analyses (Gore, 1980; Jagger et al., 1988; Turner et al., 1988; Kim et al., 1989;

Katechakis and Stibor, 2004; Wong et al, 2006). However, each approach has its own experimental limitations.

In vitro grazing experiments are widely used to study the feeding selectivity of

organisms, but results are limited to the prey items provided in the environment and

deviations may arise when laboratory handling and artificial setting influence the

feeding behaviour of the organisms. Marine cladocerans are not easy to rear under

laboratory conditions (Turner, 1984).

Analysis of gut contents and fecal materials are problematic as digested

organisms often become unrecognizable while organisms with hard remains are more

easily detectable. Minute organisms are often difficult to be recognized by light

microscospy and are sometimes too fragile to be handled and retained for SEM

examination.

71 Lebour (1922) and Bainbridge (1958) found brownish remains with no apparent cell structure in Evadne nordmanni guts, and concluded that the animal discarded the hard frames and ingested only the cell contents. While Jagger et al. (1988) and Kim et al.

( 1989) also reported the presence of unidentified materials in the cladoceran guts.

Gut pigment analysis is limited to the detection of feeding on pigmented organisms. Although high performance liquid chromatography (HPLC) pigment analysis allows rapid separation and quantification of a broad range of chlorophyll and

carotenoid pigments (Jeffrey et al., 1999), chemotaxonomic marker pigments may not be

present in every member of the same class and the same pigments may be found in more

than one class. Breakdown of pigments during gut passage could also have an effect on

the outcome of pigment analysis (Pandolfini et al., 2000).

Nejstgaard et al. (2003) noted the importance of direct investigation of copepod

feeding on non-pigmented organisms. Since podonid cladocerans are long presumed, but

not yet proven, to be raptorial feeder that can catch and ingest animal prey or large algal

cells (Nival and Ravera, 1979; Russell-Hunter, 1979; Nival and Ravera, 1981; Jagger et

al., 1988; Kim et al., 1989), the possibility of cladoceran consuming non-pigmented prey

should not be ignored.

2.1.4 Application of molecular detection

Molecular techniques based on PCR amplification of DNA have been widely

applied to detect prey items of various invertebrates, including copepods (Symondson,

2002; Nejstgaard et al., 2003).

72 Advantages of using genetic sequence-based approach are that DNA is more prey-specific and less easily oxidized than plant pigments. Prey DNA should have a much larger potential as a quantitative prey tracer in zooplankton guts and fecal pellets than plant pigments as DNA is not completely degraded during digestion. Also, one can easily develop and apply alternative genetic markers for different target groups

(Nejstgaard et al., 2003; Blankenship and Yayanos, 2005).

Utilization of group- or species-specific primers to target a narrow group of closely related prey items requires prior knowledge on the potentially consumed taxa, which is still meagerly known for marine cladocerans. Alternatively, universal primer can be used to target a wider range of eukaryotic prey items, and avoid missing any untargeted dietary taxa (Blankenship and Yayanos, 2005).

2.2 Objectives

This study a1ms to use molecular detection method to study the dietary composition of the marine cladocerans, Pseudevadne tergestina.

73 2.3 Materials and method

2.3.1 Zooplankton sampling and preparation

Live zooplankton samples were obtained by duplicate vertical hauls of a conical plankton net (mouth diameter 0.5 m, mesh size 125 J..Lm) from 18 m to surface in Tolo

Channel on 22 March 2007. Live planktons were filtered onto a mesh and were immediately frozen in liquid nitrogen. In the laboratory, samples were defrosted with autoclaved seawater before being observed under a Nikon SMZ645 dissection microscope. A total of seven Pseudevadne tergestina specimens were sorted from the sample and rinsed in autoclaved seawater for 15 minutes. The animals were then transferred to a new autoclaved seawater bath and rinsed for 10 more minutes. The animals were placed on a GF/F filter paper (Whatman, pore size 0.7 J..Lm) and immersed in DNA lysis buffer in a 15 mL centrifuge tube (Coming). Samples were stored at -80°C until use.

2.3.2 DNA extraction

DNA was extracted according to the cetyltrimethylammonium bromide (CTAB)

extraction method described by Doyle and Doyle (1990). After defrost, the filter paper

was placed inside a mortar and ground into powder with the addition of liquid nitrogen.

The grindate was then added to 3o/o CTAB isolation buffer in a 50 mL centrifuge tube

(Coming) and incubated at 60°C water bath for 30 minutes. DNA was extracted once

with an equal volume of 24:1 mix of chloroform/isoamyl alcohol. Tube was mixed

gently but thoroughly and then centrifuged at 12,000 rpm for 10 minutes. The aqueous

phase was gently pull off to a new 50 mL centrifuge tube. Two-third volume of cold

isopropanol was added to the tube and allowed to stand overnight at room temperature to

74 precipitate the nucleic acids. Nucleic acids were harvested by centrifugation at 8000 rpm for 2 minutes and washed once by resuspension in 200 f.lL 70% ethanol. The whole content was transferred to a clean eppendorf, let stand for 20 minutes and spinned down at 6000 rpm for 10 minutes. The pellet was dried with a vacuum dryer and re-suspended in 200 f.lL milli-Q water. The DNA extracts were purified following the manufacturer's instructions of Geneclean II Kit (BIO 101) and stored at -20°C until use.

2.3.3 188 rRNA gene amplification

The 18S rRNA gene was amplified by PCR using universal eukaryotic primers

Euk328f ( 5 '-ACCTGGTTGATCCTGCCAG-3 ') and Euk329r (5'-

TGATCCTTCYGCAGGTTCAC-3'), which were complementary to regions of conserved sequences proximal to 5' and 3' termini of the 18S rRNA gene as described by Moon-Van der Staay et al. (2000).

The PCRs were prepared under UV sterilized flow hoods with DNA-free equipments. The 50 f.lL PCRs included 5 f.lL of 1Ox PCR buffer (50 mM KCl; 10 mM

Tris-HCl; 1.5 mM MgCh), 200 f..tM of dNTP, 0.2 f..tM of each primer, 2.5 units of Taq polymerase (Promega) and 2 f.lL of the purified DNA template. The PCR cycle consisted of an initial step of 3 minutes at 94 °C; followed by 33 cycles of 45 seconds at 94 °C, 30 seconds at 60 °C and 2 minutes at 72 °C; and a final cycle of 10 min at 72 °C.

5 f.lL tracking dye (0.25% Xylene cyanol and 0.25% Bromophenol blue) was added to the 50 f.lL PCR products. The mixture was divided into 2 replicates of 27 f.lL and were loaded onto a 1.5% agarose, 1x T AE, 2 f.lL ethidium bromide gel and ran at

80V for 40 minutes. Target PCR products ('"'"' 1,800 bp) were resolved in the agarose gel

75 and were purified following the manufacturer's instructions of MEGA-spin Agarose Gel

Extraction Kit (Intron).

2.3.4 18S rRNA Cloning

The purified PCR products were cloned into pMD18-T vector (TaKaRa

Biotechnology) with several modifications made to the TA cloning procedures. We reduced the volume of the ligation mix into half and incubated at 16°C overnight in a

PCR machine. XL-1 blue competent E. coli cells were used for transformation. The cells were heat-shocked at 42°C for 90 seconds and placed on ice for 2 minutes. The cells were then cultured in 250 J.!L LB medium at 37°C, 200 rpm for 30 minutes. Two 50 J.!L and a 100 J.!L- transformation mix were spread onto separate LB agar plates with added ampicillin (LB/AMP) aseptically and the culture plates were incubated at 3 7°C for 18 hours. One hundred and ninty-two positive clones were randomly picked out. Individual

.clone was cultured in 500 J.!L LB/AMP medium in the 96-wells plate at 37°C, 250 rpm overnight. Presence of the 18S rRNA gene insert in colonies was verified by PCR reamplification with the described primer set and PCR protocol by using 1 J.!L of a culture as the template. Gel electrophoresis was done by adding 1 J.!L tracking dye to 5

J.!L PCR product.

2.3.5 Clone screening by RFLP

5 J.!L PCR product were digested with 2.5 units of restriction enzyme Haeiii

(Promega) at 3 7°C for 2 hours. Digested fragments were separated on a 2.5% agarose gel at 90 V for 50 minutes. A 50-bp DNA ladder (Invitrogen) was included in each gel to aid visual comparisons of the Restriction Fragment Length Polymorphism (RFLP)

76 patterns of clones. Clones with the same RFLP pattern (DNA fragments of same size) were grouped together and considered members of the same operational taxonomic unit

(OTU).

2.3.6 Sequencing and phylogenetic analysis

At least one PCR product of each RFLP type was purified using PCRquick-spin

PCR Product Purification Kit (Intron). The purified PCR products were partially sequenced using the internal primer Euk528f (5'-GCGGTAATTCCAGCTCCAA-3')

(Elwood et al., 1985) and ABI PRISM BigDye Terminators V 3.1 Kit (Applied

Biosystems) on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems). Euk528f binds to the conserved upstream region of the hyper-variable V 4 region of the 18S rRNA gene, which is sufficient for phylogenetic identification at roughly the genus level for eukaryotes.

Determination of phylogenetic affiliation of all operational taxonomic units

(OTUs) was performed by BLAST 2.2 search on NCBI web server

(http://www.ncbi.nlm.nih.gov/). Known sequences with the maximum identity in the

GenBank nucleotide database were retrieved. Phylogenetic analyses were conducted

using MEGA version 4 (Tamura, Dudley, Nei and Kumar, 2007). All nucleotide

sequences were first aligned using Clustal W multiple alignment tool included in

MEGA4 and minor adjustment were then made. Neighbor-joining (NJ) tree with K2P

model was constructed with MEGA4. Bootstrap analyses of the neighbor-joining data

were conducted based on 1,000 samplings to assess the stability of the phylogenetic

relationships.

77 2.4 Results

Presences of the 18S rRNA gene insert were detected in 149 of 192 transformed colonies. Restriction digests of plasmids showed 23 unique RFLP patterns (Figure 3.1 ), representing 23 OTUs (Table 1). The average length of sequences generated was 660 bp.

Eighty-seven percent of the sequences obtained in this study were of 2:95% identity to

GenBank sequences (Table 2).

Result from BLAST search indicated that the insert of 99 clones had 18S rRNA sequence similar to the cladoceran Leptodora kindtii (93% nucleotide identity), and were assumed as host sequences belonging to Pseudevadne tergestina.

Of the remaining fifty potential prey clones, ciliates, in 32 clones with 13 OTUs, appeared to be most dominant prey type. Twelve clones with 5 OTUs belonged to

.alveolates group II, while 3 OTUs showed high identities to Amoebophrya species.

Members from Picobiliphyta, Dinophyceae, Prasinophyceae and stramenopiles were also detected at low frequencies, each represented by only 1 OTU in 1-2 clones.

An NJ tree was constructed to affiliate the 18s rRNA sequences obtained. Most of the taxonomic groups were assembled with high bootstrap probabilities (Figure 3.2).

2.4.1 Alveolata

Of the twenty three OTUs recovered, nineteen belonged to the group Alveolata, which includes the ciliates, dinoflagellates and alveolate group II (NAGII).

78 We detected thirteen OTUs affiliated with the Ciliophora lineages: 85% belonged to Spirotrichea, 7.5% belonged to Colpodea and 7.5% belonged to Prostomatea.

Six OTU s (08-528f, 09-528f, 10-528f , 11-528f, 14-528f and 34-528f) in the Spirotrichea lineage were identified with Strombidium sp.; two OTUs (13-528f and 15-528f) were

94% similar to the phytosynthesizing ciliate Laboea strobila; others were closely related to identified species like Eutintinnus pectinis, Parastrombidinopsis shimi and

Strobilidium caudatum. 27 -528f was >95% similar to a Colpodea environmental

sequence TH10.53; whereas 07-528fwas 99% associated with the Prostomatea sequence

MB01.26

Five OTUs (02-528f, 04-528-f, 17-528f, 19-528f and 29-528f) were affiliated

with the alveolate group II. Two of which showed >95o/o similarity to Amoebophyra sp.

One (16-528f) of the 149 prey-clones was 97% similar to the dinoflagellate

frorocentrum dentatum. It was the only dinoflagellate detected in this study.

2.4.2 Other lineages

05-528f and 12-528f belonged to 2 different lineages with 99% bootstrap support.

The former was 99% similar to the photosynthetic Bathycoccus prasinos and the later

was 99% similar to a Picobiliphyte environmental sequence MB04.45. A clone similar

to the stramenopile was also detected.

79 Figure 3.1 Gel photo showing all restriction fragment length polymorphism (RFLP) patterns in the current study.

80 Taxonomic group Number ofOTUs (clones) Alveolata; Ciliophora 13 (32) Alveolates group II 5 (12) Picobiliphyta 1 (2) Stramenopiles 1 (2) Alveolata; Dinophyceae 1 (1) Chlorophyta; Prasinophyceae 1 (1) Metazoa; Cladocera 1 (99) Total 23 (149)

Table 1. Summary of the eukaryote OTU distribution in higher taxonomic groups

81 OTUs Gene Bank % Similarity BLAST score Taxon Clone Closest relatives Accession# f Uncultured marine eukaryote clone MBO 1.21 EF539027 99 1349 06-528f 3 Parastrombidinopsis shimi AJ786648 92 1038 Uncultured marine eukaryote clone MBO 1.26 EF539028 99 1061 07-528f 2 Ciliate sp. NCMS060 1 AM412525 91 785 Uncultured marine eukaryote clone MB01.12 EF539024 97 1101 08-528f 7 Strombidium sp. SNB99-2 AY143564 95 1029 Uncultured marine eukaryote clone MB01.12 EF539024 97 1267 09-528f 1 Laboea strobila _ AY302563 96 1221 Uncultured marine eukaryote clone TH04.16 EF539036 99 1242 10-528f 5 Strombidium sp. SNB99-2 AY143564 96 1125 Uncultured marine eukaryote clone TH10.34 EF539053 99 1310 Alveolata; 11-528f 1 Strombidium sp. SNB99-2 AY143564 99 1310 Ciliophora 13-528f Laboea strobila AY302563 94 935 1 Uncultured eukaryote clone ENI42482.00244 AY938119 99 918 14-528f 4 Strombidium sp. SNB99-2 AY143564 96 824 15-528f Laboea strobila AY302563 95 1155 4 Uncultured marine eukaryote clone TH10.58 EF539056 97 1197 25-528f 1 Eutintinnus pectinis clone Epec99ssu 2 AF399170 94 1079 27-528f Uncultured marine eukaryote clone TH1 0.53 EF539055 98 1210 1 Uncultured eukaryote clone ENI4 7296.00253 AY938360 98 1123 31-528f 1 Strobilidium caudatum AY143573 93 998 Uncultured marine eukaryote clone MBO 1.18 EF539026 99 1236 34-528f 1 Strombidium sp. SNB99-2 AY143564 98 1210

82 Gene Bank Closest relatives % Similarity BLAST score Taxon Clone Accession# f Uncultured marine picoplankton AP-picoclone8 DQ386744 97 992 02-528f 1 Amoebophrya sp. ex Karlodinium micrum AF472553 94 893 04-528f Uncultured marine picoplankton clone He000803 3 7 AJ965142 92 734 3 Alveolates 17-528f Uncultured marine eukaryote clone BLOO 1221.40 AY426886 96 1133 1 group II Uncultured marine eukaryote clone MB04.25 EF538981 98 1221 19-528f 5 Amoebophrya sp. ex Scrippsiella sp. AF472555 97 1192 Uncultured eukaryote clone F11N10 EF172968 96 869 29-528f 2 Amoebophrya sp. 'Dinophysis' AF239260 96 865 Picobiliphyta 12-528f Uncultured marine eukaryote clone MB04.45 - EF539140 99 1221 2 Alveolata; Uncultured eukaryote clone SCM16C75 AY664939 99 1234 16-528f 1 Dinophyceae Prorocentrum dentatum strain CCMP 151 7 DQ336057 97 1181 Uncultured eukaryote clone SSRPD78 EF172962 99 1304 Stramenopiles 21-528f 2 Papiliocellulus elegans X85388 90 933 Chlorophyta; Uncultured prasinophyte clone NW414.29 DQ055172 99 1301 05-528f 1 Prasinophyceae Bathycoccus prasinos AY425315 99 1295 Metazoa; 35-528f Leptodora kindtii AF144214 93 1013 99 Cladocera 36-528f Leptodora kindtii AF144214 93 1090 Total 149 Table 2. List of BLAST search result for each OTU recovered.

83 82 14-528f ENI42482.00244 (AY938119) MB01 .12 (EF539024) 10-528f

Strombidium

99 34-528f MB01.18 (EF539026) 99 100 Strombidium sp. SNB99-2 (AY143564) 11-528f TH1 0.34 (EF539053) Spirotrichea ....------Laboea strobila (AY302563) 13-528f 100 '------1 100 15-528f Ciliates 25-528f TH1 0.58 (EF539056) '---- Eutintinnus pectinis (AF399170) 100 06-528f 63 .------1 99 MB01 .21 (EF539027) .----- Parastrombidinopsis shimi (AJ786648) ....------Strobilidium caudatum (AY143573) 31-528f '-----1 100 ENI47296.00253 (AY938360) 1 00 27 -528f J ....------1 TH1 0.53 (EF539055) Colpodea '------i 99 Ciliate sp. NCMS0601 (AM412525) l 07 -528f Prostomatea 1 00 '------1 100 MB01.26 (EF539028) Papiliocellulus elegans (X85388) ] 21-528f Stramenopiles 99 '------1 1 oo SSRPD78 (EF172962) 16-528f ] 100 SCM16C75 (AY664939) Dinoflagellates Prorocentrum dentatum (00336057) 100 04-528f ..------1 98 '------He000803 37 (AJ965142) 100 17-528f .------i BL001221.40 (AY426886)

Amoebophrya sp. (AF239260) 29-528f NAG II 02-528f AP-pi coclone8 (00386744) Amoebophyra '------Amoebophrya sp. (AF472553) 57 Amoebophrya sp. (AF472555)

100 19-528f 99 MB04.25 (EF538981) 1 00 12-528f J ..------1 MB04.45 (EF539140) Picobiliphytes

Bathycoccus prasinos (AY425315) ]

1 00 05-528f Prasinophyceae 68 NV\1414.29 (00055172) ..------Euryte sp. (AY626996) ] Copepoda ..------Oiaphanosoma sp. (AF144210) 100 L------1 .----- Bythotrephes cederstroemi (AF144207) 100 Metazoan ....------Leptodora kindtii (AF144214) Cladocera 35host-528f 36host-528f

0.05 Figure 3.2 Neighbor-joining phylogenetic trees based on 18s rRNA sequences. 84 2.5 Discussion and conclusion

The diet of marine cladoceran has long been an area of interest to planktologists.

Unfortunately, results of many studies were sometimes contradictory (Turner et al., 1984;

Kim et al., 1989) and the natural diet of marine cladocerans remains poorly known.

Traditional methods involve a great deal of laboratory handling and filtration where small-size potential prey items may be lost. This study presents an initial attempt to use a universal primer and PCR to reveal the diet of the marine cladocerans

Pseudevadne tergestina.

While diatoms and dinoflagellates were frequently found in the diet of marine podonids (Bainbridge, 1958; Morey-Gaines, 1979; Kim et al., 1989; Onbe and Iketa,

1995; Ji, 2001), they did not form the major food of P. tergestina . The absence of evidence for predation on other zooplankton further confirms that marine podonids are raptorially-feeding herbivores (Jagger et al., 1988; Kim et al., 1989).

Ji (2001) examined the gut content P. tergestina collected from Tolo Harbour using scanning electron microscopy and fluorescence microscopy. While he reported that centric diatoms (Skeletonem coastatum, Thalassiosira sp. and Chaetoceros sp.) were the most common food items, these centric diatoms were not detected in the present study even though P. teregestina examined were collected from a site nearby. Only one of our clones was found to be distantly related to the centric diatom Papiliocellulus elegans.

85 Nival and Ravera ( 1981) reported that the mouthparts of podonids are capable of consuming food particles of 20-170 J.lm in diameter. However, Kim et al. (1989) reported prey size ranging from 4-115 J.lm. Although the molecular technique does not allow us to measure prey size, protistan organisms such as those identified in the current study normally range from 2-200 J.lm, which is within the size range of food for P. tergestina. Also, the detection of picobiliphytes, extremely small unicellular seaweed of just a few microns in diameter (Not et al., 2007), may further widen the food range of podonid food items. Whereas the Amoebophyra-related clones detected may arise from the parasites commonly found in dinoflagellates.

Yet, phytoplankton assemblages in the water column were not determined simultaneously in the current study, and we obtained no information on the feeding selectivity of the marine cladocerans over the detected dietary items.

Jagger et al. (1988) suggested that manne cladocerans feed less on fast swimming organisms due to their weak mobility. Surprisingly, ciliates appeared to be an important prey item for P. tergestina. Almost 60o/o of the recovered items were closely or distantly related to ciliates. Kim et al. (1989) found unidentified materials were reported in gut and fecal content of marine podonids and suggested that these materials may come from digested food. Unlike diatoms and dinoflagellates, ciliates are not

encased with hard cell covering and may easily be fully or partially digested.

86 2.5.1 Errors and improvements

A common problem associated with PCR-based assays is the possibility of false positive amplification caused by contamination, thus, a negative control should be included in every PCR reaction to ensure the amplification result is not from contamination of samples.

Digesting the whole animal for DNA extraction raised the problem of missing

out potential prey items in a greater chance. Although the positive clones were randomly

picked out from the agar plate, the probability of obtaining host DNA clones is much

higher than that of the dietary items. The current study could be improved by first

isolating the gut from the animal before DNA extraction. Removing the gut from P.

tergestina requires careful examination of the animal and good handling technique, but it

trims down the amount of host DNA being extracted, and increase the chance of picking

more clones of the potential prey items. Furthermore, by removing most of the host part,

it also reduces the chance of extracting DNA from epiphytes that stay on the host even

after a series of washing.

One of the advantages of using molecular technique over traditional microscopic

method is that prey items could be detected even it is partially digested or unidentified

under the microscope. However, it is hard to quantify prey consumption by using

molecular techniques. Although, Witter et al. (2003) has conducted preliminary studies

to quantify prey consumption by copepods by using a general SYBR Green-based

quantitative PCR assay, more research have to be done under controlled laboratory and

field conditions to test the reliability of this approach.

87 The current study provides new information on what might be consumed by marine podonids apart from diatoms and dinoflagellates. However, more could be revealed in the feeding habits of marine cladocerans. With the advancement of modem technology, future insights on the feeding habits and trophic role of marine cladocerans could be obtained by combining the application of molecular technique with traditional microscopic analysis.

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