Proliferative zones in the brain of the Amur sturgeon fry. Interactions with neuromeres and migration of secondary matrix zones

Neurogenesis in the forebrain region was studied in the Amur sturgeon Acipenser schrenki fry using immunocytochemical marking of the proliferative nuclear antigen. The brain zones with high proliferative activities were located at the brain ventricle surface facing the periventricular cavity. In addition to the periventricular zone of primary proliferation, several secondary proliferative zones were found in the forebrain region of the Amur sturgeon Acipenser schrenki.     

Processes of Proliferation and Apoptosis in the Brain of the Amur Sturgeon

Using techniques of immunoperoxidase staining of proliferating cell nuclear antigen (PCNA) and TUNEL labeling of fragmented DNA, we studied sites of proliferation and apoptosis in the myelencephalon, cerebellum, tectum opticum, thalamus, and hypothalamus of the Amur sturgeon (Acipenser schrenckii). We found that the processes of proliferation and apoptosis are maintained in the brain of 3-year-old sturgeon individuals; the ratio of these processes in different cerebral regions varied significantly. The maximum intensity of proliferative activity was found in the periventricular zone of the myelencephalon (proliferation index, on average, 21.0 ± 1.3%). This fact allows us to consider this cerebral region a most important zone were adult neurogenesis occurs in the sturgeon. In the medial reticular formation, dorsal thalamic nuclei, inner fibrous layer of the tectum, and lateral hypothalamus, the maximum numbers of apoptotic elements were found. Therefore, these zones in the brain of the sturgeon correspond, apparently, to the regions where postmitotic neuroblasts are localized. In sensory centers (tectum and nuclei of the V, VII, and X nerves), significantly varying ratios of intensities of proliferation and apoptosis were found; this is indicative of dissimilar rates of growth and differentiation in visual and chemosensory centers of the sturgeon brain. The high proliferative activity in sensory and motor cerebral centers of the sturgeon allows us to hypothesize that a neotenic pattern is preserved in these CNS regions of adult sturgeons over a long period after the embryogenesis has been completed.     

Immunoglobulin heavy chain constant region of five Acipenseridae: cDNA sequence and evolutionary relationship

The immunoglobulin M (IgM) heavy chain constant region genes of Russian sturgeon (Acipenser gueldenstaedtii), Sterlte sturgeon (Acipenser ruthenus), Amur sturgeon (Acipenser schrenckii), Chinese sturgeon (Acipenser sinensis) and Great sturgeon (Huso huso) were cloned and analyzed with molecular biology and bioinformatics methods. We cloned IGH nucleic acid sequences by RT-PCR using the specific primer, then determined the characteristics and functions of the amino acid sequences. The gene contains four constant region domain-encoding exons (CH1-4), of which CH4 sub-regions were the most conserved in IgM heavy chain constant region domain and had the highest identity within all the experimental species. According to the analysis of the phylogenetic tree, the variation expectation value (K(aa)), and species differentiation time (T) in the CH4 sub-region, we found that Chinese sturgeon and the other five sturgeon form one whole bifurcation of the tree, while, among the five left, Amur sturgeon and Huso sturgeon, Russian sturgeon and Siberian sturgeon (data from GenBank), Sterlte itself forms three other bifurcations. This result can clearly explain the relations of taxonomic status, geographical distribution and evolution among the species studied.     

Seasonal variation in diet of juvenile Amur sturgeon Acipenser schrenckii in the lower reaches of the Songhua River,Northeast China

We investigated seasonal variation in the diet of hatchery-reared juvenile Amur sturgeon Acipenser schrenckii in the Tongjiang reach of the Songhua River, Northeast China. The results indicated that Amur sturgeon fed mainly on Chironomidae and Ephemeroptera and secondarily on unspecified bony fish, Odonata, and Trichoptera. Diet changed throughout the year, with autumn 2017 and spring 2018 dominated by Ephemeroptera larvae, while summer 2018 prey comprised primarily Chironomidae larvae. Unspecified bony fish were observed at greater frequency in autumn 2017. Amur sturgeon captured in summer contained a greater quantity of Trichoptera larvae compared to those from autumn and spring sampling. Results were consistent with previous reports of Amur sturgeon diet in other areas of its native distribution and suggested that they may be opportunistic feeders throughout the year, although larval Chironomidae and Ephemeroptera appear to be important prey. These results represent the first reported diet composition data for juvenile Amur sturgeon in the lower Songhua River. Results of the study form part of a comprehensive investigation of feeding habits of juvenile Amur sturgeon that provides basic data for study of the biological characteristics of the species.     

Age structure of juvenile Amur sturgeon Acipenser schrenckii and kaluga Huso dauricus in the Fuyuan reach of the Amur River,Northeast China

Amur sturgeon Acipenser schrenckiii Brandt 1869 and kaluga Huso dauricus (Georgi 1775) are critically endangered species with populations showing significant decline from historical levels due to overexploitation, yet little is known about their population structure. Adults are not often captured in the Fuyuan reach of the Amur River, Heilongjiang Province, Northeast China, and the government prioritizes juvenile sturgeon management. This study was conducted to determine the age and length/weight relationships of juvenile Amur sturgeon and kaluga in the Fuyuan reach of the Amur River. We estimated age using pectoral fin spine sections obtained from 65 juvenile Amur sturgeon and 50 juvenile kaluga. We compared the age estimates from two readers, and found 100% between‐reader agreement in 67.7% of the Amur sturgeon and 64.0% of the kaluga. The majority of differences in estimated age were within 1 year. Length/weight relationships (LWR) were calculated, and the LWR of the Amur sturgeon and kaluga were W = 0.0025L3.106 (r² = 0.966）and W = 0.0022L3.175 (r² = 0.989), respectively. Our study provides the age structure and LWR in juveniles of two sturgeon species.     

三种鲟科鱼类和两种鲑科鱼类卵黄免疫球蛋白性质的比较

从施氏鲟(Acipenser schrencki)、小体鲟(Acipenser ruthenus)、西伯利亚鲟(Acipenser baeri)、哲罗鲑(Hucho taimen)和金鳟(Oncorhynchus mykiss)五种鱼类卵黄中分离、纯化Ig,并对其分子结构进行了初步研究。结果表明:五种鱼类卵中Ig的分子量分别为施氏鲟524kD、小体鲟468kD、西伯利亚鲟475kD、哲罗鲑为490kD,金鳟498kD,其Ig重链的相对分子量相同,约为97kD。轻链的相对分子量各不相同,施氏鲟为27kD,小体鲟28.5kD,西伯利亚鲟30kD,哲罗鲑28.5kD和金鳟16kD。其中西伯利亚鲟、施氏鲟和小体鲟Ig等电点约为5.85,哲罗鲑和金鳟的Ig等电点约为6.55,其蛋白质结构上具有一定的相似性,且均为糖蛋白     

史氏鲟免疫球蛋白重链可变区序列及多样性

为了研究史氏鲟免疫球蛋白重链可变区基因的组织结构和多样性,采用RTPCR技术从史氏鲟(Acipenserschrenckii)脾脏总RNA中获得了免疫球蛋白重链可变区cDNA克隆,随机挑取31个阳性克隆进行测序。结果表明:所有序列相同率高于75%,前导肽相同率高于90%,应属于同1个VH家族。其变异主要存在于互补性决定区,特别是CDR3区。在D片段序列中发现大量保守的基因序列(motif)。并发现多个VH基因片段可以共用一个J片段的现象。在基因组DNA重排过程中,VH片段可以与任意的D和J片段结合。此外,史氏鲟免疫球蛋白重链可变区的VH,D和J片段的随机重排外,外切核酸酶作用,以及在重排位点大量N,P片段的插入现象,都大大增加了鲟鱼免疫球蛋白的多样性。     

Unidirectional hybridization of kaluga <Emphasis Type="Italic">Acipenser dauricus</Emphasis> Georgi, 1775 and Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis> Brandt, 1869, inferred from the mitochondrial DNA genotyping of their natural hybrids

In 2009 through 2011, among 730 individuals of kaluga and Amur sturgeon collected in the lower reaches of the Amur River and the Amursky Liman, 17 morphologically intermediate individuals (hybrids) with the body length of 56 to 202 cm (median, 81 cm) were identified, including 11 individuals (4.6%) found in 2009, three individuals (1.6%) found in 2010, and three individuals (1.1%), in 2011. In 16 hybrids, 819 bp of the mtDNA control regions were sequences and 11 haplotypes were identified. Since all these haplotypes were from the mtDNA lineages of kaluga, it was concluded that hybridization occurred in one direction, kaluga (♀) × Amur sturgeon (♂). This asymmetry could be caused by the large size differences between these species. Since the earlier examined morphologically typical Amur sturgeons showed the absence of alien haplotypes (Shedko et al., 2015), the absence of the mtDNA introgression is claimed. This can be caused by low viability or sterility of the backcross females (kaluga (♀) × Amur sturgeon (♂)) × Amur sturgeon (♂). The samples of hybrids and typical kaluga individuals demonstrated no differences in the frequency spectra of the mtDNA haplotypes. However, haplotype and nucleotide diversity in the first sample was somewhat higher than in the second one (0.950 versus 0.927 and 0.0054 versus 0.0044, respectively). The data obtained will be useful for population monitoring of kaluga and Amur sturgeon, Amur River endemics, which are classified as critically endangered by the IUCN Red List of Threatened Species.     

Endemic sturgeons of the Amur River: kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii

General biological characteristics and the contemporary status of the kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii, are described. Both inhabit the Amur River basin. Kaluga is the largest freshwater fish in this river system reaching more than 5.6 m in length and more than 1000 kg in weight. We recognize four populations of kaluga: the first is from the estuary of the Amur River and coastal brackish waters of the Sea of Okhotsk and Sea of Japan, the second is from the lower Amur River, the third is from the middle-Amur, and the fourth occurs in lower reaches of the Zeya and Bureya rivers. Freshwater and brackish water morphs exist in the estuary population, with the freshwater morph predominating in number. The number of individuals in the lower Amur River population at age 2 or greater was recently estimated to be 40 000, and in the middle Amur, 30 000. The population will continue to decline because of rampant overfishing. The Amur sturgeon is represented in the Amur River basin by two morphs: brown and gray. Brown morphs occur in the middle and lower parts of the Amur River; they grow more slowly than the gray ones. Today, the lower Amur River population of Amur sturgeon is made up of 95 000 fish at age 2 or greater and is approximately half as large as the population in the middle Amur River. Populations of kaluga and Amur sturgeon in the Zeya and Bureya rivers are extremely small and on the verge of extinction.     

Comparison of biology of the Sakhalin sturgeon, Amur sturgeon, and kaluga from the Amur River, Sea of Okhotsk, and Sea of Japan biogeographic Province

Three Acipenseridae species live in the rivers and marine waters of the Khabarovsk Territory, Russia: Sakhalin sturgeon, Acipenser mikadoi, Amur sturgeon, A. schrenckii, and kaluga, Huso dauricus. We review the general biology and life history of each species, including their historic and current distributions, and examine current paleogeographic theories to outline the possible origin and evolution of these three species in the Amur River, Sea of Okhotsk, and Sea of Japan biogeographic province. Apparently, these species have evolved during distinct geologic time periods, which has reinforced the reproductive isolation of these species although hybridization does occur. They have convergently adapted to the unique environmental conditions of the Amur River and Russian Maritime regions, and yet developed behavioral adaptations to reduce competition between species. Sakhalin sturgeon is the least studied species among anadromous sturgeon in the world. This species is highly migratory and spends the majority of its life in the ocean only returning to natal rivers to spawn. Amur sturgeon and kaluga are distributed throughout the Amur River basin and the estuary and share many life history traits. They are both represented by distinct morphs. Additionally, we present size and weight relationships to estimate the growth of Amur sturgeon and kaluga. All three species have suffered declines in abundance due to over fishing and their contemporary distributions have contracted compared to their historic ranges. We identify gaps in knowledge and suggest further research useful for guiding management of each species.     

中华鲟(Acipenser sinensis)及相关种类的mtDNA控制区串联重复序列及其进化意义

采用 PCR技术和 DNA测序技术 ,发现了我国一级珍稀保护动物中华鲟 ( Acipensersinensis)线粒体 DNA( mt DNA)的控制区 ( D- loop)存在数目不等的串联重复序列 ,该重复序列造成了中华鲟广泛的异质性现象 .从分子水平进行了不同类型重复序列变化规律的研究 ,同时还初探了重复序列在我国其它几种鲟鱼类的存在情况 ,发现在白鲟 ( Psephurus gladius)、达氏鲟 ( A.dabryanus)和史氏鲟 ( A.schrenckii)均存在类似的重复序列结构 .序列比较分析表明 ,不同鲟鱼类重复序列在鲟鱼类进化过程中扮演着一定的角色 ,很有可能碱基差异大小与它们的亲缘关系的远近呈正相关 .     

Organization of diencephalon of the sturgeons. Posterior tubercular area. Periventricular and rostral parts

Cytoarchitectonics of periventricular and rostral parts of the posterior tubercular area of diencephalon was studied in four species of the cartilaginous ganoids by using routine Nissl staining and Bielschowski impregnation technique in Viktorov’s modification. The posterior tubercular area in the giant sturgeon Huso huso L., the Kura sturgeon Acipenser güldenstädtii persicus n. Kurensis Belyaeff, the stellate sturgeon Ac. stellatus Pall., and the barbel sturgeon Ac. nudiventris Lov. was shown to have similar structure. Six structures were identified in these areas: the periventricular nucleus, paraventricular organ, nucleus of the paraventricular organ, and posterior tuberal nucleus in the periventricular region and preglomerular medial and lateral nuclei in the rostral region. Both nuclei of the rostral zone are migrated nuclei. Out of nuclei of the posterior tubercle regions, the posterior tuberal and medial preglomerular nuclei are characterized by polymorphism of cellular elements. A conclusion is made that these parts of the posterior tubercular area in the sturgeons considered to be the lower ray-finned fish are comparable with those of the higher teleosts, and even are more differentiated according to some parameters.     

Ecological characteristics of the hemoglobinograms of 3 species of sturgeons

Studies have been made on fractional composition of hemoglobin in the Russian Acipenser güldenst?dti, Siberian A. baeri and Amur A. shrencki sturgeons by polyacrylamide gel disc-electrophoresis. High electrophoretic heterogeneity (6--7 components) of hemoglobin in each of the species was found together with a distinct species specificity of hemoglobinograms. The observed differences in fractional composition of hemoglobin are of an adaptive character which makes it possible to consider the Amur sturgeon as a perspective object for acclimatization in the seas of the Southern region of the USSR with lowered salinity.     

Morphofunctional Characteristics of Gonadotropin-Releasing Hormone Immunoreactive Structures in the Brain of the Sturgeon Acipenser güldenstädti before Spawning

Light-microscopic immunohistochemical study of the brain with use of the unlabeled antibody to gonadotropin-releasing hormone (GnRH) was carried out on sexually mature individuals of the sturgeon of stage IV of gonadal maturity (before spawning). The brain was examined as a whole; the GnRH-immunoreactive (GnRH-IR) structures were revealed in the olfactory bulb, forebrain, hypothalamus, and neuropituitary. In the fish studied at prespawning period, the highest density of GnRH-IR structure was noticed in the ventral region of forebrain, preoptic region, and anterior neuropituitary. The GnRH-IR cells of the forebrain ventral region, preoptic, and tuberal nuclei send their axons to the region of anterior neuropituitary, in which they contact vessels of the primary portal pituitary system. Thereby, the gonadotropin secretion regulation is performed by corresponding cells of adenopituitary. Rare GnRH-IR fibers in the posterior pituitary lobe contact the general circulation vessels. The dendrites of the GnRH-IR cells that we have revealed in the preoptic region and in the region of tuberal nucleus are located very close to the preoptic bay cavity and to the cerebral III ventricle, respectively. This indicates a possibility of secretion of the neurohormone into the cerebrospinal fluid.     

Comparative Ontogenetic Behavior and Migration of Kaluga, Huso Dauricus, and Amur Sturgeon, Acipenser Schrenckii, from the Amur River

We conducted laboratory experiments with kaluga, Huso dauricus, and Amur sturgeon, Acipenser schrenckii, to develop a conceptual model of early behavior. We daily observed embryos (first life phase after hatching) and larvae (period initiating exogenous feeding) to day-30 (late larvae) for preference of bright habitat and cover, swimming distance above the bottom, up- and downstream movement, and diel activity. Day-0 embryos of both species strongly preferred bright, open habitat and initiated a strong, downstream migration that lasted 4 days (3 day peak) for kaluga and 3 days (2 day peak) for Amur sturgeon. Kaluga migrants swam far above the bottom (150cm) on only 1 day and moved day and night; Amur sturgeon migrants swam far above the bottom (median 130cm) during 3 days and were more nocturnal than kaluga. Post-migrant embryos of both species moved day and night, but Amur sturgeon used dark, cover habitat and swam closer to the bottom than kaluga. The larva period of both species began on day 7 (cumulative temperature degree-days, 192.0 for kaluga and 171.5 for Amur sturgeon). Larvae of both species preferred open habitat. Kaluga larvae strongly preferred bright habitat, initially swam far above the bottom (median 50–105cm), and migrated downstream at night during days 10–16 (7-day migration). Amur sturgeon larvae strongly avoided illumination, had a mixed response to white substrate, swam 20–30cm above the bottom during most days, and during days 12–34 (most of the larva period) moved downstream mostly at night (23-day migration). The embryo–larva migration style of the two species likely shows convergence of non-related species for a common style in response to environmental selection in the Amur River. The embryo–larva migration style of Amur sturgeon is unique among Acipenser yet studied.     

Discrimination of interspecific hybrids in natural populations of Amur sturgeon fish by means of multilocus RAPD-PCR markers

A RAPD-PCR analysis of a sample of Amur sturgeon fish (46 individuals) is performed. An estimate of the genetic state of native populations of Amur sturgeon Acipenser schrenckii (Brandt, 1869) and kaluga fish Huso dauricus (Georgi, 1775) is given. Genetic evidence for the hybrid origin of two phenotypical hybrids is obtained. Estimates of the genetic distances between the species and the hybrids proved to be at the level of interspecific distances. It is shown that an Exact Test for differentiation of the populations is the most effective means of discriminating the two species, while multidimensional scaling (MDS) is the most effective means of discriminating the hybrids. It is concluded that the populations of sturgeon fish in the Amur River have maintained a substantial level of genetic diversity; the presence of hybrids in these populations is judged to be one of the risk factors. Multilocus RAPD-PCR markers are acknowledged as a convenient and reliable tool for conducting genetic monitoring of populations of Amur sturgeon fish for the purpose of preserving the gene pool of the fish.     

5种鲟鱼免疫球蛋白重链恒定区序列研究

为了探讨几种鲟鱼免疫球蛋白(IgM)所包含的信息与其亲缘和进化之间的关系, 分别对俄罗斯鲟(Acipenser. gueldenstaedtii)、小体鲟(A. ruthenus)、施氏鲟(A. schrenckii)、中华鲟(A. sinensis)和欧鳇(Huso huso)的IgM重链(IgH)恒定区进行了研究。采用RT-PCR的方法对IgH核酸序列进行了克隆, 通过软件获得了相应的IgH氨基酸序列。在分别对这5种鲟鱼免疫球蛋白重链恒定区4个区(CH1～CH4)进行研究后发现, 其CH4区氨基酸序列相似性最高。通过对CH4区序列氨基酸变异期望值(Kaa), 物种分化时间(T)及物种间系统进化树(Phylogenetic Tree)等参数的分析, 将克隆的5种鲟鱼IgH恒定区序列与已发表的西伯利亚鲟同源序列比对(源于NCBI序列)后发现: 西伯利亚鲟与俄罗斯鲟、施氏鲟与欧鳇、小体鲟各构成一个分支, 并与中华鲟相对。实验结果从体液免疫系统的演化这个角度, 反映了被研究的鲟鱼物种间的分类地位、地理分布及进化关系之间的联系。     

Periventircular and central nuclei of the diencephalon pretectal area of the sturgeons

Cytoarchitectonics of periventricular and central nuclei of the pretectal area was studied in four species of the sturgeons: the great sturgeon Huso huso, L., the Russian sturgeon Acipenser güldenst?dti persicus n. kurensis, Belyaeff, the starred sturgeon Ac. stellatus, Pall., and the barbel sturgeon Ac. nudiventris, Lov.; this pretectum part has a similar structure. Study of these parts of the pretectal area was carried out by methods of Nissl and Bielshowskii modified by Viktorov. In this part of the pretectal area, nine nuclear structures were described, eight of them--nuclear; these are ventral periventricular pretectal nucleus and its dorsal component, dorsal periventricular pretectal nucleus, nucleus of medial longitudinal bundle, subcomissural organ, medial and lateral intercalate nuclei, and central and posterior pretectal nuclei. The main attention has been paid to the issue of the evolutional progression of this part of the pretectal area in the sturgeons as compared with other Actinopterygii.     

几种鲟鱼基因组大小、倍体的特性及鲟形目细胞进化的探讨

采用Ｆｅｕｌｇｅｎ－显微分光光度计方法,以鸡红细胞为标准ＤＮＡ（３．２２ｐｇ／２Ｃ）测定了长江白鲟、达氏鲟、中华鲟、史氏鲟和北美匙吻鲟的体细胞基因组大小（ＤＮＡ含量）。结果表明,上述五种鲟鱼ＤＮＡ含量分别为４．１１、８．２６、９．０７、６．０７和３．９６ｐｇ。长江白鲟和北美匙吻鲟均属于四倍体鱼类,分布在长江水系的中华鲟和达氏鲟两种鲟属鱼类为八倍体类型。史氏鲟初步判断为八倍体,据分析可能存在四倍体的