Morphological And Ecological Study On Fish Eggs And Larvae And Ichthyoplankton Surveys In The Yellow River Estuary

Fish eggs and larvae contain much important information of fish life history and fish ecology. So they play an important role in environmental impact assessment, fishery stock analysis, fish propagation, seeding release, and fish farming. The objectives of this study were to establish a standard procedure for identifying fish eggs, understand the biological characteristics of early development of fishes and the factors that affect its development, carry out ecological investigation of ichthyoplankton. First, this paper reviews the research advances at home and abroad on the identification methods of fish eggs, the main developmental events occurred at each stage of early development, factors that have been proposed to alter early development, and the ecological investigation on fish eggs and larvae. Section A, identification methods of fish eggs. The shape and surface structures of unfertilized mature and fertilized developing pelagic eggs of four flounders were studied, characters differences observed that would be useful for species identification and phylogenetic inference were summarized; and Japanese halfbeak Hyporhamphus sajori eggs, which collected in field, were used to establish a standard procedure for identifying fish eggs by using the light microscope, scanning electronic microscopy, and genetic analysis simultaneously. Section B, biological and experimental ecology studies of early development of fish. The embryo and yolk sac larvae of Pacific cod Gadus macrocephalus, one of the key species in the subarctic Pacific, were studied. We focused on the main developmental events occurred at each stage and the effects of temperature or temperature and salinity combinations on early stage of Pacific cod. Based on these studies, we aim at exploring the possibility causes of mortality; the using of developmental characters and their implication in phylogenic determination of the Gudas species; bio-ecological implications of the dynamic process of the adhesive characteristics, the diameters and mass density; also the ecological implications of hatching asynchronously were determined. Section C, ichthyoplankton surveys. The categories composition and distributional patterns of ichthyoplankton surveys were carried out in waters over the Yellow River estuary during spring and summer 2007, the preliminary analysis of environmental factors on category composition and distribution patterns of ichthyoplankton were discussed.Section A, identification methods of fish eggs1. Morphology of the unfertilized mature and fertilized developing marine pelagic eggs in four multiple spawning floundersStarry flounder Platichthys stellatus, spotted halibut Verasper variegates, turbot Scophthalmus maximus, and Japanese flounder Paralichthys olivaceus are four commercially cultivated multiple spawning flounders that spawn pelagic eggs. Through appropriate light and scanning electron microscope processing, the shape and surface structures (such as micropyle, pores, pore density, and paten) of unfertilized mature and fertilized developing eggs of the four species were observed and measured. First, individual or intraspecific comparisons in the surface structures of eggs at different developmental stages were made. Second, interspecific differences in the four species at the same developmental stage of unfertilized mature eggs were statistically computed and analyzed through one-way analysis of variance and hierarchical cluster. Eggs of the same species collected at different stages of development tend to be different in morphology. Smoothing of the convoluted egg envelope surface and closure of the micropyle to serve as a final step of the polyspermy-preventing reaction are common after fertilization. Based on detailed morphology of micropyle of just-mature fertilizable eggs, turbot, starry flounder, and Japanese flounder each has a micropyle with a long canal but no distinct micropylar vestibule, type III of Riehl and Gotting (1974). In contrast, spotted halibut owns a micropyle with a distinct flat micropylar vestibule and a long canal, type II. Ultrastructure of the envelope surface, size and distribution density of pores, and size of eggs are also useful characters for distinguishing and phylogenetic analysis among the four species. However, ultrastructural features of the micropyle are the most important envelope surface characters for egg identification.2. Morphological and genetic identification of Japanese halfbeak Hyporhamphus sajori eggsA larger number of fertilized eggs with attaching filaments attached to some seaweeds were collected in the southern Yellow Sea (33°49'N,122°10'E).Inorder to make an accurate identification of the fish eggs, light microscope, scanning electronic microscopy (SEM), and genetic analysis were used. Under light microscope, the egg is an oblated and agglutinated demersal with a narrow perivitelline space; eggs ranged in diameter from 1.95 to 2.38 mm with a mean of 2.18±0.03 mm; their membrane are smooth and they have 5 to 7 cannular keratose egg-filaments; the oil globules are multiple, ranged in diameter from 0.05 to 0.50 mm and in number from 7 to 145 with a mean of 28±2, the position of them migrate during embryonic development; the yolks are ivory-white with no segments, ranged in diameter from 1.00 to 2.30 mm with a mean of 1.90±0.03 mm; the development phases of the embryo are variable. So they were identified as the eggs of one Beloniformes. Under SEM:the egg's micropyle is apparent, it lines in the middle of the attaching filaments and the outer diameter of it is about 12.3μm; the pores on the envelope are unapparent, but there are many grain substances on it, with the density was about 50 pieces/100μm2. The result of genetic analysis conveyed that in partial sequences of mtDNA Cyt b gene there were no variable sites between the eggs and Japanese halfbeak, and the genetic distance between them was 0, however, many variable sites between the eggs and the other fishes of Beloniformes were exised, the variable rate was 18.72-21.3%, and the genetic distance between them were 21.9-26.4%. The result of Neighbor-joining (NJ) molecular phylogenetic tree also indicated that the eggs and the Japanese halfbeak were assembled at the same embranchment, they shared one haplotype, but the eggs were assembled at different embranchments with the other species of Beloniformes so the fish eggs were alienated from them. The validity of test results by using mtDNA Cyt b gene has been examined with a 443 bp segment of mtDNA control region, the result conveyed that only 0-0.7% in the genetic distance between the fish eggs and Japanese halfbeak. It is also the diversity within specie. Based on the results above, it is suggested that the fish eggs to be Japanese halfbeak.Section B, biological and experimental ecology studies of early development of fish1. The biological study on the embryonic development of Pacific cod Gadus macrocephalusIn order to know the embryonic development pattern of Pacific cod Gadus macrocephalus, the morphological characteristics of fertilized eggs and larvae were observed and recorded by Nikon SMZ1500 photomicroscope equipped with amicrometer ocular lens at the laboratory from January to March,2008. Moreover the mass density of Pacific cod embryos at each day of development was determined by using the Stokes'law equation and the fine structures of both the micropyle and the egg envelope surface of 7 development stages were studied with scanning electron microscopy. Eggs of Pacific cod were spherical, slightly adhesive, and semidemersal with none of oil globule in diameter of 0.95-1.08mm, mass density of 1.0422±0.001g/cm3 at the time spawning, with weak striations under light micropyle. With the incubated temperature 6.30±1.24℃, salinity 34psu, the blastoderm formed at 2 hours post fertilization (hpf), blastula stage at 23hpf, blastodermal cap stage with the adhesive characteristics lost at 38hpf, early germ ring stage at 52hpf; the segmentation period began at 196hpf, the tail separated at 228hpf, heart beat and body shrink at 252hpf. Hatching occurred at 330.56hpf, after which most of the individuals remained motionless at the water's bottom. The diameter and the mass density of the Pacific cod eggs were in dynamic process over embryonic development. The diameters of the eggs rise during the embryonic development; however, the mass density of eggs was downtrend. The larvae were incubated until full use of the yolk sac (20 days post hatching). Under SEM, the micropyles of Pacific cod eggs belong to type I with deep micropylar vestibule and short canal. In unfertilized ones, the micropylar canal is in diameter of 3.28±0.55μm, the outer diameter of the micropylar vestibule is 18.13±2.03μm and the inner one is 10.22±3.07μm; the outer envelope surface has numerous hexagonal reticulated patterns and is characterized by a crisscross pattern of depressions in distance 1.95±0.29μm between the pore canals on the envelope. After fertilization the micropylar canal was blocked by secretions of the perivitelline, the wrinkles on the outer surface of the envelope were indistinct and the whole surface becomes roughness. The shape of the micropyle and the distance between the pore canals on the envelope were in the dynamic process along with the development stage forward. The maximum diameter of the micropylar vestibule and the maximum distance between the pore canals on the envelope occurred at the 2 cell stage. Numerous bacilli deposited at the micropyle and the outer surface of envelope at the blastopore closure and embryo 5/8 around yolk stages; the micropyle was whole deformed at embryo 5/8 around yolk stage. The dynamic process of the adhesive characteristics, the diameters and mass density were associated with the development process of the embryo and it has bio-ecological significance for increasing the fertilization rate and influencing the dispersal potential of the eggs in the field. So does the dynamic process of the micropyle, the ultrastructure changes on the envelope surface and it has bio-ecological significance for protecting the Pacific cod embryo in the long incubation period from microorganism infections. 2. Effects of temperature on embryonic development of the Pacific cod Gadus macrocephalusLaboratory experiments were conducted to determine the effect of water temperature on development, survival and hatching of Pacific cod Gadus macrocephalus eggs from the Mutsu Bay. The eggs were reared at 0℃,1℃,2℃,4℃,6℃,8℃,10℃,12℃and under a diel light cycle (13 h light,11 h dark) at the salinity of 34psu (normal sea water). A piece-wise least squares nonlinear regression model with midpoints of each stage describes the relation between time to each stage of development and incubation temperatures. Development was normal for all temperatures except 0℃, with no eggs hatched. Eggs survived to hatching and produced viable embryos at the highest temperature range 4-8℃, reflecting this species'winter spawning season. "Critical periods" of high mortality during the egg development did occur through the gastrula stage to blastopore closure at each temperature checked. The developmental rate was sequential with and directly proportional to incubation temperature while the time spent in each developmental stage was inversely proportional to temperature. An exponential relationship between times, over which initiated hatched,50% hatched and eggs all hatched, and temperature were found. Time to 50% hatch ranged from 817.87 h at 1℃to 217.9h at 12℃. Fish larvae hatch asynchronously from egg batches despite experiencing a common environment during their development. The combined observations of recent studies suggest a dome-shaped relationship between size at hatch, yolk storage and incubation temperature. At all temperatures, early hatching larvae were smaller but had more yolk storage; late-hatching larvae were generally larger and had smaller yolk sacs than early hatched larvae. Together these data suggest that variations in water temperatures within an ecological range can markedly influence development rates, survival and hatching of the eggs also the stage at hatch larvae of Pacific cod. Hence, temporal and spatial heterogeneity in sea temperatures should be considered along with hydrodynamic conditions in estimating chances for survival and hatching of demersal Pacific cod eggs and the dispersal potential of Pacific cod larval in the field. Hatching asynchronously is considered to be an evolutionary strategy of "bet hedging" against such dynamic variation in the Pacific cod.3. Some effects of temperature and salinity on laboratory-reared eggs and yolk sac larvae of Pacific cod Gadus macrocephalusThe combined effects of temperature and salinity on eggs and yolk sac larvae of Pacific cod Gadus macrocephalus were examined under controlled laboratory conditions. Two developmental phases were investigated:1) fertilization to hatch. Artificially fertilized eggs, obtained by induced spawning of captive broodstock at 6℃and 34psu, salinity, were stocked (100 eggs/piece) into one hundred and thirty five shallow sterile plastic petri dishes at combinational of constant temperatures (0,1,2,4, 6,8,10,12,14℃) and salinities(15,20,25,30,34psu). Photoperiod was 11 h light:13 h dark. Responses of Pacific cod eggs were measured in terms of incubation period, hatchability and morphological characters at stage of hatching larvae.2) hatch through yolk sac absorption. Time to 50% survival of the newly hatching unfed larvae (incubation at 6℃,34psu) were studied in a 9×5 (temperature×salinity, the same as the egg incubation experiment) array of treatments to determine how environment will influence the survival rate when they were emergence and dispersed into various water mass and the fluctuate sea surface at the time of hatching. The results indicated that temperature 14℃were beyond the tolerance limits for normal embryonic development; hatching rate was moderate to high (53.67-73.66%) at 4℃and 6℃in all salinities. Viable hatch was significantly influenced by the effect of salinity as the upper and lower thermal limits were approached. Though there was no eggs hatching at salinity of 30psu and 34 psu in 0℃, when the salinity was lower (15,20,25psu) there would still be some hatching; hatching success was moderate under temperature range 8-10℃at the high salinity but the hatching rate was markedly reduced at low salinity 15psu. This indicated that low-temperature-low-salinity synergism, as well as high-temperature-low-salinity inhibitory effects. While data on developmental rates and as influenced by temperature are presented, no significant differences in temperature influences on developmental times between the tested salinities were found. As expected, dome-shaped relationship between size of stage at hatch larvae and incubation temperature at each salinity, no such affecting existed when concerned to the affecting of salinity at each tested temperature. Larvae were more tolerant to extreme high temperatures than were newly fertilized eggs, most larval could survival when they were dispersed into various temperature and salinity combination till the yolksac is exhausted. Average time from hatching to 50% survival in unfed larvae was markedly reduced along with the increased temperature, ranged from 39 days in 0℃to 6 days in 14℃, and the influence could be described in all cases by a power function. The temperature of 4℃and 6℃, possibly associated with peak abundance of eggs and larvae in nature, is optimal for culture of Mutsu Bay embryos and yolk sac larvae. At this two temperatures, hatchability, size of stage of hatch larvae, tolerance to reduced salinities are maximized and time to 50% survival of the unfed larvae was moderate.Section C, ichthyoplankton surveys1. Category composition and distribution patterns of ichthyoplankton in the Yellow River estuary during spring and summer 2007The categories composition and distributional patterns of ichthyoplankton surveys were carried out in waters over the Yellow River estuary during spring and summer 2007. In the investigated area where were 119°05'-119°31'E,37°35'-37°57'N,19 fixed sampling stations (6 transects) were set up respectively. The distributional patterns of ichthyoplankton were investigated by horizontal tows associated with vertical tows with a zooplankton net (mouth diameter 50 cm,140 cm in length, mesh size 0.50 mm). The horizontal tows were of 5 min duration at a speed of 2.0 nmile/h on the sea surface, while the vertical tows were from surface to near-bottom at a speed of 0.5 m/s at each sampling station. According to the investigation results,7661 eggs and 70 larvae of fishes of 18 taxa were collected during spring and summer 2007, of which,17 taxa were identified as species, belonging to 16 genera,12 families and 5 orders,1 taxa of gobiidae fishes as only family level. The preponderant species were Konosirus punctatus, Sphyraena pinguis, Cynoglossus joyneri. During late spring, a total of 6943 eggs and 6 larvae fishes were captured respectively. In the horizontal tows,6932 eggs and 6 larvae were captured, and in the vertical tows,11 eggs and 0 larvae were captured. The occurrence frequencies of eggs and larvae were 84.21% and 10.53% in the horizontal tows,26.32% and 0% in the vertical tows, respectively. The average density of eggs and larvae were 364.84 ind/net,0.32 ind/net respectively in the horizontal tows, and 0.59 ind/m3 and 0 ind/m3 respectively in the vertical tows. During summer, total of 718 eggs and 64 larvae of fishes were captured respectively. In the horizontal tows 658 eggs and 57 larvae were captured, and in the vertical tows,60 eggs and 7 larvae were captured, respectively. The occurrence frequencies of eggs and larvae were 84.21% and 63.16% respectively in the horizontal tows,31.58% and 15.79% in the vertical tows. The average density of eggs and larvae were 34.63ind/net and 3ind/net respectively in the horizontal tows,1.85 ind/m3 and 0.22 ind/m3 respectively in vertical tows. Further analyzing showed that two seasonal ecological groups could be distinguished. A pronounced seasonal succession from spring-summer group to summer group regarding the category composition of ichthyoplankton can be observed throughout the two cruises. The succession rate was about 83.33%. The distribution patterns of species were different on account of different waters environmental conditions. In waters of southern Yellow River estuary the community structure of ichthyoplankton was relatively stable, the dominant species was evident and the dominance value was high. The diversity index of species and the uniformity of interspecific distribution in waters of southern Yellow River estuary was higher than that of in waters of northern Yellow River estuary. The variability in the spatio-temporal distribution patterns of ichthyoplankton may result from the interaction of hydrological and biological factors at different scales. The equivalency density distribution of ichthyoplankton and fish eggs of preponderant species, such as Konosirus punctatus, Sphyraena pinguis, Cynoglossus joyneri were drawn. The category composition and distribution patterns of ichthyoplankton in the Yellow River estuary changed evidently from 1982 to 2007, it was in correspondence with the alternation of fishery resources strongly affected by double disturbances of overfishing and ecological environment changes.