Seasonal Population Structure of the Copepod Temora turbinata (Dana, 1849) in the Kuroshio Current Edge, Southeastern East China Sea

: The abundance of adult males, females, and copepodites, and sex ratio of a Temora turbinata population and seawater hydrology were studied from 2018 to 2019 in waters off northeast Taiwan, northwest Paciﬁc Ocean. The hydrological parameters showed signiﬁcant differences between sampling months caused by interactions of Kuroshio, East China Sea water, and the China Coastal Current. The highest average abundance was recorded from the June 2018 cruise with 2903.92 ± 3499.47 (inds. m − 3 ), followed by a cruise in June 2019 with an average abundance of 1990.64 ± 1401.55 (inds. m − 3 ). The sex ratio ranged between 0.25 and 2.33; the records were signiﬁcantly higher in samples of June 2018 than during other sampling cruises (one-way ANOVA). The spatiotemporal distribution of T. turbinata showed a clear pattern of seasonal changes among sampling stations and distribution zones. Abundance of females and copepodites correlated signiﬁcantly positive (r = 0.755, p < 0.001), whereas sex ratio (r = 0.247, p = 0.119) did not correlate signiﬁcantly. The present study revealed that the abundance of T. turbinate was highest in June and was positively correlated with seawater temperature; furthermore, this is the ﬁrst time the in situ sex ratio of T. turbinata in western Paciﬁc waters has been reported.


Introduction
Zooplankton provides important trophic links in aquatic and marine trophic webs by transferring energy and materials from microbes to higher trophic levels [1]. Zooplankters are the main natural food source of larval and juvenile fishes worldwide [2]. Zooplankters are valuable indicators of ecological conditions due to their intermediate position, linking primary production with consumers at higher levels of the trophic cascade, and being sensitive to environmental characteristics [1,3,4]. Zooplankters are sensitive to changing physical and chemical properties of waters. These abiotic parameters affect the spatial distribution and abundance of zooplankton [5][6][7]. As such, environmental changes could also affect the geographic distribution of certain species [3,8]. At the same time, some opportunistic species could adapt to particular environmental changes [9]. The copepod assemblage is the most dominant group of the zooplankton community and is considered as a key group in the marine pelagic environment [10].
The copepod genus Temora belongs to the family Temoridae with broadcast-spawning representatives. It is planktonic and important as it provides one of the major groups of prey for commercially shoaling fishes [11]. To date, a total of six species belong to the genus Temora worldwide. According to historical records, four species of Temora have been reported in waters around Taiwan [12][13][14][15][16][17][18][19][20][21]. Among those, Temora turbinata (Dana, 1849) and Temora discaudata (Giesbrecht, 1889) were recorded in the waters of northern Taiwan [22]. Temora turbinata has been recorded as the predominant species in mesozooplankton communities in various environments around the world, such as in Korea [23], in Taiwan [21], in Brazilian coastal waters [24], and in coastal and shelf waters [11], as well as tropical and subtropical areas in general [20,[25][26][27][28]. This species is widely adapted to a variety of environments, such as polluted and eutrophicated waters, eutrophic lagoons, outfall areas, and nuclear power plant discharge areas [17,20,27,[29][30][31]. T. turbinata could especially cooccur with the bloom-forming Karenia brevis in the Gulf of Mexico, a toxic dinoflagellate [32]. The feeding behavior of T. turbinata is opportunistic and omnivorous and depends on the food item composition in ambient waters. Previous reports found it primarily herbivorous and rarely carnivorous [33]. In addition, T. turbinata as a suspension feeder prefers microscopic and non-or slow-moving plankter, such as Thalassiosira spp. [34], and moving prey ciliates [35].
In dioecious species, the theoretical sex ratio is balanced (1:1, female:male), whereas biased sex ratios are quite common in nature [36][37][38]. The sex ratio represents an internal factor that affects population dynamics and growth [39,40]. In marine copepods, sex determination and sex ratios are governed by diverse hydrological factors, such as pH, chemical composition, temperature, depth, and hydrostatic pressure, which commonly leads to an alteration of sex ratios and reproductive timing [41][42][43]. For example, the sex ratio of Acartia tonsa increased from 0.3 ± 0.1 to 1.3 ± 0.1 with a decrease of salinity from 35 to 13 PSU in the laboratory [44]. Besides such abiotic factors, previous reports revealed that biotic factors, such as metabolism, reproductive age, and life span, also play important roles that affect the sex ratios of copepods [45][46][47]. The scarcity of data referring to in situ sex ratios is also due to the effect of differential vertical migration of sexes, and the difficulty of identifying sexes in immature copepodites. Sex ratio is, therefore, probably the most understudied population trait in copepod ecology [43].
The sampling area was next to Kueishan Island located at the I-Lan Bay in the coastal waters of northeastern Taiwan which is affected by the Kuroshio Current (KC) [55]. The Kuroshio originates near the bifurcation of the North Equatorial Current at about 13 • N, then flows northwards along the Philippine and Taiwan eastern coastlines to Japan and off Sanriku, east coast of Honshu, Japan. It affects one of the largest marine ecosystems worldwide and represents the major current in the west as a boundary in the northwest Pacific Ocean [56][57][58]. It flows over the I-Lan ridge before entering the East China Sea and transports warm water masses from the north Pacific tropical and intermediate waters [59][60][61]. Besides the Kuroshio, several freshwater discharges also affect the hydrological characteristics around Kueishan Island (KI) [62]. The southwest monsoon prevails during May to October and the northeast monsoon prevails during November to February in this region [63]. The river run-off is affected by weather changes and precipitation. The hydrology of I-Lan Bay strongly affects the aquatic biota in this area [64][65][66].
To date, we have only a limited understanding of the composition of T. turbinata populations, such as sex ratio and the proportion of copepodites and adults. At the southern coast of the ECS, T. turbinata is an important zooplankton species. Therefore, a comprehensive investigation of the issues of biology and ecology of T. turbinata is important and necessary. In the present study, the abundance of copepodites and male and female adults of T. turbinata were measured, and the spatiotemporal distribution patterns in waters off Northeastern Taiwan were compared to reveal the following issues: (1) the seasonal composition of T. turbinata populations in the zooplankton; (2) whether the geographic distribution of T. turbinata is correlated with zones of seabed depth; (3) the sex structure of T. turbinata during different monsoon periods in waters off northeastern Taiwan.

Sampling Area and Field Study
Field sampling was carried out during four cruises in June (Southwest monsoon period) and September (Southwest-Northeast monsoon transition period) in 2018, and March (Northeast monsoon period) and June (Southwest monsoon period) in 2019 around Kueishan Island, in I-Lan Bay off northeastern Taiwan (Figure 1). Samples were collected from 12 selected stations distributed in three seabed depth zones [shallow depth zone (<200 m), middle depth zone (200-400 m), deep depth zone (>400 m)] during each cruise (Table 1). In the past, the research methods of copepods in this water area were to collected samples from surface zone, such as [7,55,67]. Therefore, the present study collected samples from the surface zone for do comparison with the past studies. A conical net (45 cm in diameter, 200 µm mesh size, and 180 cm length) was horizontally towed in surface waters (above the 10 m depth contour) for 10 min at each station to collect zooplankton samples. A flow-meter (Hydrobios, Germany) was fixed at the center of the net mouth to calculate the respective filtered water volumes. Zooplankton samples were immediately preserved in 5-10% neutralized seawater-formalin for identifying and counting their abundance in the laboratory. The hydrographic parameters temperature and salinity were measured by Sea-bird CTD equipment at each station.

Zooplankton Identification and Abundance Measurement
Zooplankton samples were divided by a Folsom plankton sample splitter until the subsamples contained about 300-500 individuals. Temora turbinata were identified with the help of related taxonomic references [68][69][70][71]. Male, female, and copepodites of T. turbinata were counted separately under a dissecting microscope (SMZ1500, Nikon, Tokyo, Japan). The number of each item was applied to calculate the in situ abundance of individuals per cubic meter (inds. m −3 ).

Data Analysis
Spatial and temporal variations of T. turbinata populations were studied by collecting them from different monsoon throughout 4 cruises, and the PAST (Paleontological statistics [72]) software package was used to analyze the abundance and proportion of T. turbinata in each sample. A total of 48 samples were used to calculate similarities before clustering and nonmetric multidimensional scaling (NMDS) analyses. Euclidean distance was used to evaluate the relative similarity of populations between samples. In order to reduce the bias of samples, the functional test for transformation of data was applied prior to conducting a similarity analysis by Box and Cox [73]. The value (λ) was 0.919, therefore, the original abundance data of T. turbinata were using log10 (x + 1) for all samples. The value of sex ratio represents all male individuals divided by females in each sample.
One-way analysis of variance (ANOVA) with the post hoc Tukey's Honestly Significance Difference (HSD) test provided differences in population structure among sampling months and sampling zones. Pearson's correlation analysis estimated the relationship between abundance of copepods and hydrographic parameters. Statistical analysis made use of the statistical software package SPSS v24.

Hydrological Conditions
The salinity profiles showed high variation during the four sampling cruises (Figure 2a   shows at 5 m depth the correlation between salinity and temperature. The averaged surface seawater temperature (SST) was highest in September 2018 with values 27.87 ± 0.65 • C (mean ± standard deviation). During this cruise, SST varied from 26.39 • C to 28.55 • C, while the average sea surface salinity (SSS) was only 33.53 ± 0.16 PSU which was the lowest of all sampling cruises. During the cruises in June 2018 and June 2019, the average SST was 26.35 ± 0.48 • C and 27.14 ± 0.39 • C and the average SSS was 34.47 ± 0.03 PSU and 33.88 ± 0.10 PSU, respectively. The lowest average SST was 24.14 ± 0.57 • C, recorded during the cruise in March 2019, with the highest average SSS 34.61 ± 0.03 PSU. The SST limit was 23.61 to 25.21 • C in March 2019. The average SST and salinity were significantly different among the four sampling cruises (p < 0.01, one-way ANOVA).

Variation and Distribution of the Temora turbinata Population
The average abundance of T. turbinata showed a seasonal variation (

Sex Ratio Differences of Temora turbinata
The  and that of females was 606.34 ± 394.21 (inds. m −3 ); the highest average abundance of copepodites was 1751.09 ± 2080.13 (inds. m −3 ), which was recorded during the cruise in June 2018. SST and salinity were 26.41 • C and 34.43 PSU at this station. The lowest abundance was recorded at station S5, being located in the most eastern area far away from KI. The SST and salinity were 26.82 • C and 34.56 PSU at station S5. The abundance at station S4-1 was 155.71 (inds. m −3 ), which is near to station S5. The highest SST (27.50 • C) was recorded at station S4-1 during this cruise, where the salinity of the surface waters was 34.48 PSU. Taken together, the horizontal copepod distribution showed that the total abundance of the western inshore area was higher than in the eastern offshore area of KI.
During the cruise in September 2018, the average abundance of males and females was 34.04 ± 48.18 (inds. m −3 ) and 54.60 ± 75.56 (inds.m −3 ), and the copepodite average abundance was 76.18 ± 21.99 (inds. m −3 ). The lowest total abundance was recorded at station S4 (9.80 inds. m −3 ), which was located at the eastern offshore area with an SST of 28.12 • C. The highest abundance was recorded at station Ex2 (518.16 ind. m −3 ), near to the east of KI with an SST of 28.07 • C. The abundance center appeared in the region of the southeastern coast of KI and the far eastern offshore area (Figure 3b). This was due to a relatively higher abundance at the offshore station S5 (483.34 inds. m −3 ), where the SST was 26.39 • C.
A low abundance of the T. turbinata population appeared in March 2019 at station 12 which is located at the northwest of KI and at station S4-1 which is at the eastern offshore area of KI. During the spring cruise, the male average abundance was 27.14 ± 27.49    (Table 2). From the composition of the two groups, it can clearly be seen that the densities of adult males, adult females and copepodites in the samples of group A were higher than those of group B. A similar result is indicated by the proportion of copepodites in both groups that accounts for a relatively higher proportion of adult males compared to adult females. In contrast, the proportion of adult males in group A (29.72%) is higher than that in group B (20.19%), and the proportion of adult females in group B (26.23%) is higher than that of group A (23.76%). This phenomenon of switching proportions of adult individuals matches with the analysis of sex ratios (Figure 4).

Statistical Analysis of the Spatiotemporal Distribution of Temora turbinata
Among four sampling cruises, multiple comparisons of mean values for abundance of all adult males and females, as well as the copepodites of T. turbinata, were conducted using ANOVA, followed by a Tukey post hoc test. First, the temporal patterns of total T. turbinata abundances showed variations among sampling cruises ( Figure 6). The abundance of all adult T. turbinata was significantly higher in June 2019 (1447. 37 (Figure 6d). In contrast, the abundance of all adult, male, female, and copepodite of T. turbinata were not significantly different to the location of stations across the three sampling depth zones (p > 0.05, one-way ANOVA) (Figure 7). The average abundance of T. turbinata showed no significant differences among the 12 selected stations (p > 0.05, one-way ANOVA). Similarly, there was no significant distribution pattern among sampling stations and depth zone during each sampling cruise (p > 0.05, one-way ANOVA).  Abundance of all adult males and females, the copepodites, and the sex ratio (male/female) of T. turbinata were not significantly correlated with salinity and seawater temperature (p > 0.05) ( Table 3). The abundance of all adult T. turbinata was significantly positive correlated with sex ratio (r = 0.405, p = 0.009). It is worth noting that there was a significantly positive correlation between the abundance of females and the abundance of copepodites (r = 0.755, p < 0.001), whereas this was not significantly correlated with sex ratio (r = 0.247, p = 0.119) ( Table 3). Table 3. Correlation results of all adult abundance (individuals m −3 ), male abundance, female abundance, copepodite abundance, and sex ratio (male/female) according to Pearson's correlation analysis. The value represents the correlation (r), and the value in parentheses represents p.

Hydrography off Northeastern Taiwan Waters
In the northwestern Pacific, the seasonal monsoon, impinging eddies, and typhoons affect the Kuroshio Current (KC) and shift its path [74,75]. During the northeast monsoon in winter, the southward cold current (such as the China Coast Current marked with blue arrows in Figure 1) increased and the Kuroshio Current (the pathway was marked with black arrows in Figure 1) shifted closer to the eastern shore of Taiwan, whereas during the southwest monsoon in summer, fall and spring, the KC moved seawards [74,76]. Where the KC encountered the continental shelf of China in the ECS, the I-Lan ridge northeast of Taiwan, its mainstream was forced to turn northeastwards [77]. A small branch of the KC maintained its momentum towards the shelf, mixing the waters of the ECS and the KC, providing a complex oceanographic environment [78,79] which subsequently greatly affected the biota [16,17,55,66,80]. The Kuroshio waters transport tropical and subtropical fauna, and transport neritic fauna towards offshore, this way increasing the diversity of the mesozooplankton assemblage in I-Lan Bay [55,80]. The present study investigated the seasonal distribution and variation of T. turbinata populations in the area around KI, revealing remarkable differences in spatiotemporal variability throughout the developmental cycles of its populations.

Biology and Ecology of Temora turbinata
Temora turbinata is a warm water epipelagic, neritic species. It can tolerate wide ranges of temperature (5-35 • C) and salinity (20-45 ppt). The ideal temperature and salinity for its culture was 25-28 • C and 30-35 ppt, respectively [81]. It is more frequent and more abundantly recorded in the northern hemisphere compared to the southern hemisphere [82]. Temora turbinata was more dominant at low salinity waters in the Indus Delta of Pakistan [81]. Ara reported that the most dominant Temora stylifera was substituted by T. turbinata along the southern border of São Paulo State [27,83].
Temora turbinata was reported to be associated with the KBC in the Taiwan Strait [3,21,84,85] and was also commonly found around coastal waters of Taiwan [7,20,[85][86][87]. Hwang et al. [3] and Dur et al. [88] found that T. turbinata occurred in waters of >28 • C temperature and was considered as a warm-water indicator species in the northwest of Taiwan. Tseng et al. [20] reported about the decadal data of T. turbinata and found that it was an important copepod species, abundant in the southwestern monsoon prevailing period from waters adjacent to nuclear power plants along the northeast coast of Taiwan. In their research, peak of abundance was recorded in summer, indicating a possible temperature effect, and was followed by a second peak in autumn. The lowest record of abundance was 96.35 ± 77.91 (inds. m −3 ) in spring when the northeastern monsoon prevails. Previous studies confirmed that T. turbinata has strong adaptability and could survive in various environments. Therefore, it represents the most dominant copepod species and is representative of the mesozooplankton in coastal Taiwan waters.
Copepods account for the majority of mesozooplankton abundance in the open seas of the tropics and can comprise 70% or more of the zooplankton community in abundance and about 30% of its biomass [89]. However, copepod populations in the field are dominated by juvenile stages. The abundance of copepodites usually outnumbers those of adults [90]. For example, copepodites account for 27-41% of total zooplankton abundance in the southeastern Baltic Sea [91]. McKinnon and Duggan [90] reported copepodite percentages ranging between 59.77% and 90.82% of the total copepod numbers in Bathurst Island, Indonesia, Darwin Harbour, the Arafura Sea, Kimberley, the Great Barrier Reef, Northwest Cape, and Scott Reef. In this research, the percentage of copepodite limits was 27.29% to 60.30%, which is relatively lower than reported from other areas. The copepodite percentage may be closely related to the differences in life strategies and life spans of different species. For example, T. turbinata spawned about 15-30 eggs per day almost without diapause or dormant eggs. At optimal temperature conditions, about 80% eggs hatched after 15-20 h and it would take about 15 days from the naupliar phase to metamorphose into adults.
The life span of the adults was reported to be about 20-25 days [81], while the longevity of A. tonsa, which belongs to the Acartiidae Family, was about (29.31 ± 5.9) to (32.52 ± 6.1) days in laboratory cultures under different salinity conditions [44]. The longevity of T. turbinata was obviously longer than A. tonsa.

Sex Ratio of Copepods
Unbalanced sex ratios were discovered at the beginning of the 20th century by Sewell [92]. Kiørboe [40] reviewed the sex ratio of 12 copepod families and pointed out that the values varied between 0.15 and 1.1. The general sex ratios were higher and had a wider range of variation in broadcast spawners than in sac spawners (Table 4). Hirst and Kiørboe [93] reported that the sex ratio of Temora longicornis was lowest with 0.75 during February to October in 1947 and the highest number was recorded with 1.63 during March to September in 1993. The sex ratio of Temora stylifera ranged from 0.32 (from September 1971 to August 1973) to 1.27 (during September 1986 to June in 1988). The present study found that T. turbinata and the other two congener species (T. longicornis and T. stylifera) show similar changes in sex ratio.   The sex ratio of pelagic copepods is typically female biased. Species of the copepod families Oithonidae and Paracalanidae were often recorded with extremely female-skewed sex ratios [40,111,112]. Hirst et al. [112] estimated that predation contributed to more than 69% of the field mortality rate in adult males, whereas in adult females it was about 36%.
This suggests that the male to female ratio skews in pelagic copepods were primarily caused by differential predation mortality of the sexes in the adult stage [112]. Lack of food can cause female-skewed sex ratios and if females have low physical fitness, this would result in a decrease of population size [113]. In contrast, the low survival rate of males in an environment lacking food leads to a change in sex ratios [114]. A female-biased sex ratio due to the higher mortality of males to females was found during cyanobacteria blooms in an open sea area near to Storfjärden, Baltic Sea [115]. Moreover, a harmful dinoflagellate bloom was reported to decrease the abundance of male copepods [116,117]. Male life span is shortened [38]. Further investigations should be carried out to clearly understand the interaction of sex ratio with the dynamics of populations.

Conclusions
The hydrographic environment across our survey area was dominated by the mixing of water masses of the Kuroshio Current with near shore waters. The water masses showed an obvious seasonal successional variation indicating possible temperature effect during our research. The abundance of T. turbinata was mainly linked to hydro-parameters such as seawater temperature. The results confirmed that T. turbinata showed significant seasonal changes as indicated by previous studies in coastal areas of Taiwan. The present study revealed for the first time the in situ sex ratio of T. turbinata in western Pacific waters. Population dynamics are as yet very little studied in the area, and they are worth investigating in more detail in the future. Furthermore, information about food supply, mortality, and high spatiotemportal sampling resolution will be needed to comprehensively understand the population dynamics of T. turbinata in these understudied waters.