草鱼仔鱼耳石茜素络合物标记特征研究

草鱼(Ctenopharyngodon idellus)是我国主要增殖放流经济鱼种之一,探索有效的标志方法对其增殖放流的技术保障和效果评价无疑至关重要。本研究对草鱼仔鱼耳石进行了1 d茜素络合物(ALC)荧光标记,以确认该方法的可行性及适宜条件。结果显示,微耳石、矢耳石和星耳石均能被标记。标记环在可见光及荧光下均能被观察到。其中,后者使用蓝色激发光(WBS)观察效果最佳。总体上,微耳石的标记效果最好,矢耳石次之。ALC浸泡对鱼体的生存及生长无显著影响,耳石上荧光信号出现和消失与鱼体开始浸泡和结束均存在约12 h的时滞。浸泡标记过的实验鱼在经过长达60 d的恢复续养后,标记环仍清晰可见,表明ALC标记效果明显且可长期存在。该方法对草鱼仔鱼的增殖放流标记具有较强的可行性。     

茜素络合物在青鱼幼鱼耳石上的标记效果评价

放流标记技术研究是支撑增殖放流效果评估的重要内容。该研究用质量浓度为100 mg·L-1的茜素络合物(ALC)对约2月龄的青鱼(Mylopharyngodon piceus)幼鱼进行48 h浸泡,随后移入正常水体中恢复续养20 d。对其3种耳石在可见光和不同的激发光下进行比较分析,评价了茜素络合物在不同耳石上的沉积情况与标记效果。结果表明,通过在可见光和不同的激发光下进行观察,其3种耳石上均可见明显的标记区,标记率达100%。在对标记鱼恢复续养20 d后发现,耳石上的荧光标记区强度仍很高,表现出了较好的标记效果。此外,相关标记的最佳检测光源为蓝色激发光,绿色激发光次之。微耳石是用于青鱼幼鱼耳石荧光标记观察的最适类型,同时也是用于时滞检测的最适类型,其次是星耳石。此外,将鱼体浸泡入茜素络合物溶液中至其在耳石上形成标记沉积,以及将鱼体移出浸泡溶液至其耳石上标记区域消失,均存在1 d时滞。     

月鳢仔鱼耳石的荧光标记及其日轮确证

利用茜素络合物对体长(9.99±0.56)mm的月鳢(Channa asiatica)仔鱼耳石进行浸泡标记试验。结果表明,经荧光显微镜检测,80 mg/L、100 mg/L和120 mg/L的溶液浸泡24 h后均可以在月鳢仔鱼的耳石上形成橘红色标记环,星耳石、微耳石和矢耳石的标记率均为100%;其中,以星耳石标记环荧光强度最大,且在普通光学显微镜下也可观察到紫红色标记环。经100 mg/L和120 mg/L溶液浸泡后,月鳢仔鱼在其后饲养阶段出现较高的死亡率,显示高浓度茜素络合物溶液对月鳢仔鱼有一定毒性。标记后星耳石上的新增轮纹数(N)和饲养天数(D)密切相关,其线性方程为:N=0.9291D-0.2974(R2=0.9793,n=64,P<0.01),斜率0.9291与1无显著差异(P>0.05),表明星耳石上的生长轮即为日轮。研究表明,在追踪月鳢仔鱼迁移行为时,可采用80 mg/L的茜素络合物溶液对其星耳石进行染色标记。     

淡水中心在淡水鱼增殖放流标记技术研究上取得新进展

<正>目前，有效的标记手段和增殖放流评价方法，仍是制约增殖放流应用工作的“瓶颈”问题。淡水渔业研究中心渔业微化学实验室选取鲢鱼和鲫鱼，通过鱼体茜素络合物（ALC）荧光标记和锶元素浸泡标记，探索相关标记方法在鱼类多类硬组织标记和效果评价上的适用性特征和优化途径。研究发现，ALC不仅能够成功标记鲢鱼幼鱼广受关注的耳石（矢耳石、星耳石、微耳石），     

茜素络合物对半滑舌鳎(Cynoglossus semilaevis)苗种耳石的染色标记效果

采用茜素络合物(Alizarin Complexone,ALC)分别浸泡标记70 d和170 d半滑舌鳎(Cynoglossus semilaevis)苗种耳石,设置了5个浓度组和3个时间梯度组.结果显示,ALC对半滑舌鳎70-170 d苗种的耳石染色效果明显.综合考虑存活率和标记效果,ALC浸泡标记半滑舌鳎苗种耳石的适宜浓度为100 mg/L (36 h浸泡处理)和150 mg/L(24 h浸泡处理).相同浸泡时间下,随着ALC浓度的增加,耳石上标记信号强度增加,但实验鱼死亡率升高;相同浸泡浓度下,随着时间的增加,耳石上标记信号强度增加,实验鱼死亡率也会升高.对标记的170d苗种进行养殖实验,每30 d取样矢耳石进行观察,发现4个月后实验鱼生长良好,且耳石荧光信号仍可清晰检测到,表明ALC在半滑舌鳎矢耳石上形成的荧光标记可长期存续,标记效果好.研究结果可为半滑舌鳎大规模标志放流及早期生活史研究提供群体身份识别技术支持.     

茜素络合物对半滑舌鳎(Cynoglossus semilaevis)苗种耳石的染色标记效果

采用茜素络合物(Alizarin Complexone,ALC)分别浸泡标记70 d和170 d半滑舌鳎(Cynoglossus semilaevis)苗种耳石,设置了5个浓度组和3个时间梯度组。结果显示,ALC对半滑舌鳎70–170 d苗种的耳石染色效果明显。综合考虑存活率和标记效果,ALC浸泡标记半滑舌鳎苗种耳石的适宜浓度为100 mg/L(36 h浸泡处理)和150 mg/L(24 h浸泡处理)。相同浸泡时间下,随着ALC浓度的增加,耳石上标记信号强度增加,但实验鱼死亡率升高;相同浸泡浓度下,随着时间的增加,耳石上标记信号强度增加,实验鱼死亡率也会升高。对标记的170 d苗种进行养殖实验,每30 d取样矢耳石进行观察,发现4个月后实验鱼生长良好,且耳石荧光信号仍可清晰检测到,表明ALC在半滑舌鳎矢耳石上形成的荧光标记可长期存续,标记效果好。研究结果可为半滑舌鳎大规模标志放流及早期生活史研究提供群体身份识别技术支持。     

茜素络合物浸泡标记秦岭细鳞鲑发眼卵及仔鱼耳石

采用茜素络合物(ALC)在秦岭细鳞鲑(Brachymystax lenok tsinlingensis)发眼卵和仔鱼阶段进行浸泡标记试验,为确定合适浸泡浓度和持续浸泡时间,试验设置了6个的浓度组和4个时间梯度组.结果显示:在荧光显微镜下观察被浸泡的仔鱼和经浸泡处理的发眼卵而出膜后的仔鱼,其耳石都出现橘红色荧光标记.浸泡...     

叉尾斗鱼仔鱼耳石形态发育与日轮形成特征

对实验室人工繁殖的叉尾斗鱼(Macropodus opercularis)仔鱼耳石形态发育与日轮进行了观察研究.结果显示,叉尾斗鱼微耳石和矢耳石在胚胎时已出现.微耳石在仔鱼刚孵化时为圆盘状,随仔鱼发育转变为近椭圆形,孵出后19 d转变为中部圆凸两端较尖的菱形;矢耳石在仔鱼刚孵出时为圆盘状,随仔鱼发育转变为椭圆形,孵出后19 d转变为一端略尖的桃形;星耳石在仔鱼孵出后第19天才出现,呈中部略凹的椭圆形.叉尾斗鱼仔鱼耳石长径(包括微耳石和矢耳石)与鱼体全长(TL,mm)呈线性相关.仔鱼耳石上第一个轮纹在孵出后第2天形成,其后每日形成1个新轮纹,生长轮数目与仔鱼日龄(D)呈线性相关,且线性方程斜率接近于1.结论认为,叉尾斗鱼仔鱼星耳石出现时间晚,矢耳石形态在后期出现较大变化,而微耳石在胚胎时即形成,形态稳定;日轮可读性较好,故更适合做日轮研究的材料.本研究旨在为叉尾斗鱼自然种群年龄结构调查及其资源保护提供基础数据.     

荧光染料对短须裂腹鱼早期鱼苗的浸泡标记研究

为探究短须裂腹鱼(Schizothorax wangchiachii)早期鱼苗浸泡标记的合适荧光染料及标记条件,为短须裂腹鱼大规模标记放流及其效果评估提供参考,实验使用茜素络合物(AC)、茜素红S(ARS)、盐酸四环素(TC)、酸性茜素蓝B(AAB)、茜素黄GG(AYG)和钙黄绿素(CAL)溶液分别浸泡标记短须裂腹鱼早期鱼苗(仔、稚鱼),实验结束后摘取耳石检测荧光标记。结果显示:AC、ARS均可在短须裂腹鱼耳石上形成荧光标记,标记在绿光下最明显,呈橘红色;TC、AAB、AYG、CAL均未形成有效的荧光标记;AC标记造成的死亡率和标记质量随着溶液浓度、浸泡时间的增加而升高,随着鱼体全长的增加而降低;AC标记20日龄短须裂腹鱼结束16 d后,与空白对照组相比,除浓度300 mg/L、浸泡时间24 h的这组全长显著较低外,其他各组无显著差异;AC及ARS重复标记均可形成两个橘红色标记环,首次标记时AC造成的死亡率低于ARS,再次标记时前者高于后者。综合考虑标记质量、死亡率及对生长影响等多方面因素,本研究推荐使用AC或ARS浸泡标记短须裂腹鱼早期鱼苗,具体条件为:溶液浓度50～100 mg/L,浸泡时间12～24 h。     

乌江中华倒刺鲃仔、稚鱼耳石的形态发育与生长

研究中华倒刺鲃(Spinibarbus sinensis)耳石的发育和生长特征,补充早期生物学资料,为进一步研究该鱼野生资源的早期生活史特征提供基础数据。在实验室养殖条件下观察了乌江中华倒刺鲃仔、稚鱼及3种耳石的形态发育过程和生长特点。结果表明,在水温(26.0±2.1)℃下,初孵仔鱼微耳石和矢耳石已经形成,星耳石在仔鱼出膜后第6天出现。在形态发育过程中,微耳石由出膜时的近圆形经卵圆形发育成贻贝形,且中心核位置偏移到前端靠近背侧;矢耳石由近圆形经卵圆形发育成箭矢状;星耳石由逗号形发育成不规则四边形。3种耳石形态发育的各阶段与鱼体发育阶段存在一定的对应关系。在生长上,仔稚鱼鱼体、3种耳石的长径和短径与日龄均呈显著的线性关系(P0.001),鱼体全长与3种耳石长径呈现出显著的线性关系(P0.001),仔稚鱼鱼体、3种耳石的特定生长率随日龄增加均呈下降趋势。     

元江水系越鳠年龄鉴定材料的比较

分析比较了元江越鳠(Mystus pluriradiatus)不同年龄鉴定材料的鉴定结果。结果显示:微耳石磨片、星耳石和脊椎骨材料为鉴定元江越鳠年龄的可利用材料,矢耳石细小且脆不适合用于越鳠的年龄鉴定。微耳石磨片和星耳石的年龄判别能力最高,均达到96.0%,脊椎骨的年龄判别能力为93.3%;最大年龄读数出现在微耳石磨片和脊椎骨上,读数为5+龄;微耳石磨片的同一鉴定者两次独立年龄读数的吻合率最高为94.5%、脊椎骨次之为91.7%、星耳石最低为88.1%;两种年龄材料读数之间的吻合率比较中,微耳石磨片与星耳石的吻合率较高为90.9%,两者的年龄读数在低龄组时吻合较好,微耳石磨片与脊椎骨的吻合率为89.3%,3+龄以上时二者的年龄读数吻合率高。结果表明,鉴定低龄个体选择星耳石材料(n+龄≤3+龄),高龄个体用微耳石磨片进行辅助鉴定的年龄鉴定方法对元江越鳠进行年龄鉴定较为准确。     

Otolith microstructure of brown sole <Emphasis Type="Italic">Pseudopleuronectes herzensteini</Emphasis>: validation of daily ring formation and the occurrence of microstructure denoting metamorphosis

Brown sole Pseudopleuronectes herzensteini larvae and juveniles were reared to validate daily otolith ring formation. At 15°C, a check (a distinct ring) formed on the sagittae and lapilli at 6 days after hatching, and clear increments regularly formed outside the check. For both otoliths, the relationship between the number of days after hatching and number of increments was linear, and the slope of the line was approximately 1; therefore, daily formation was validated. At 12°C, the check formed on the lapillus 8 days after hatching. Accessory primordia (AP) began forming on the sagittae of metamorphosing larvae, and the shape of the sagittae became complicated. AP were not formed on the lapillus; concentric rings were formed throughout larval and juvenile stages. Wide and obscure increments formed on the lapilli during metamorphosis (metamorphosing zone, MZ). Based on MZ, concentric rings that have formed on the lapilli of juveniles can be separated into larval and juvenile rings. The morphs of large juveniles’ lapilli were bilaterally asymmetric, and the blind-side lapilli were most suitable for otolith microstructure analysis. This study provides fundamental information for otolith microstructure analysis in wild brown sole.     

Can calcein and alizarin complexone be used for double immersion marking of juvenile qingbo <Emphasis Type="Italic">Spinibarbus sinensis</Emphasis>?

Calcein (CAL) from 50 to 250 mg/l and alizarin complexone (ALC) from 100 to 300 mg/l were used for double immersion marking of juvenile qingbo Spinibarbus sinensis. With the exception of the scales, double immersion for 24 h produced detectable double marks in otoliths (sagittas and asterisci), barbs, fin rays (dorsal, pectoral, ventral, anal, and caudal), and fin spines (dorsal, pectoral, ventral, and anal) after 90 days in a laboratory growth experiment. Green fluorescent rings produced by CAL were considerably closer to the inside of the bony structures (including otoliths, barbs, fin rays, and fin spines) than red fluorescent rings produced by ALC. Sagittas, asterisci, and barbs showed acceptable fluorescent marks at higher concentrations (250 mg/l CAL and ≥ 200 mg/l ALC, ≥ 200 mg/l CAL and 300 mg/l ALC, 150–250 mg/l CAL and 250–300 mg/l ALC, respectively). Fin rays and fin spines treated by 200 mg/l CAL and 250 mg/l ALC and 250 mg/l CAL and 300 mg/l ALC simultaneously had both acceptable CAL and ALC marks. There was no statistically significant difference on the survival or growth of marked fish compared to the controls throughout the experiment (p > 0.05). The results suggest that double immersion with CAL and ALC is suitable for double mass-marking of juvenile S. sinensis, and these double marks are useful in the experimental development of biological research or restocking methodologies.     

Transgenerational marking of brown trout Salmo trutta f.f., using an 84Sr spike

The maternal transmission of a naturally occurring and non‐toxic minor isotope of strontium (⁸⁴Sr) to the central otolith region of the progeny of a typical European freshwater fish species, brown trout, Salmo trutta f.f. L. was accomplished. The focus was to apply minimum doses of a non‐toxic solution at physiological salt concentration to minimise potential adverse effects on the fish. Female spawners were intraperitoneally injected with doses of 12.5 and 30.2 μg ⁸⁴Sr kg^?1 fish. Eggs were stripped and the resulting progeny were reared in a hatchery for about 1 year before sampling the otoliths. Strontium isotope ratios in the otoliths of the offspring were measured by cross‐sectional line scans using laser ablation‐multiple collector‐inductively coupled plasma mass spectrometry (LA‐MC‐ICPMS). Otolith cores of marked juveniles showed a significantly elevated ⁸⁴Sr/⁸⁶Sr ratio compared with control fish and to otolith regions created after hatching. Mass marking of cohorts of progeny from individually spiked brown trout with Sr isotopes is therefore possible for dispersal and migration studies without the necessity of handling eggs and applying other larval marking methods like immersion.     

Daily and sub-daily otolith increments of larval and juvenile walleye pollock, Theragra chalcogramma (Pallas), as validated by alizarin complexone experiments

Walleye pollock (Theragra chalcogramma) were reared from eggs to the juvenile life stage to study daily increment formation in the sagittae otoliths, which are routinely used for age and growth analyses. The apparent deposition of sub-daily growth increments becomes problematic for determining fish age from the late larval stage throughout the juvenile (young-of-the-year) development stage. Otolith marking experiments were conducted to determine interpretation criteria to differentiate between daily and sub-daily increments. Immersion of larval and transforming walleye pollock in 25 mg/l of alizarin complexone (ALC) for 6 h once a week produced a fluorescent mark on the day of staining. Evidence of six well defined and equally spaced increments counted between the weekly ALC marks validated the deposition of daily increments. The daily increments gradually increased in width as the fish/otolith grew. The criteria for determining the presence of sub-daily increments between the daily increments were (1) weak optical definition and (2) a sudden change in incremental distance that lasted for one or two increments and were approximately <0.5 μm in width. Growth problems that occurred during the experiments were identified on otoliths as reductions in daily incremental widths and optical definition, which continued for several days. Otoliths from field-collected fish have also shown similar changes in daily increment properties during the juvenile stage, which may be an indicator of an environmental influence. The criteria for defining different increment types help to resolve our current age determination issues for late larval and early juvenile stage walleye pollock from the Gulf of Alaska.     

大黄鱼耳石锶标志技术

利用六水合氯化锶进行大黄鱼(Larimichthys crocea)幼鱼耳石的标记,探讨了锶元素对大黄鱼幼鱼耳石的元素指纹标记效果,分析了锶元素标记对大黄鱼幼鱼生长和存活的影响。结果表明:(1)阶段性倍增饲养水体中锶离子浓度可使标记组个体在特定耳石区段的Sr/Ca比值显著提升,形成与对照组个体和野生个体在该耳石区段Sr/Ca比值的显著差异(P0.01),标记组、对照组和野生个体在该耳石区段的Sr/Ca比值分别为(3.58±1.09)mmol/mol、(1.73±0.08)mmol/mol和(1.09±0.35)mmol/mol。此区段形成的Sr/Ca比值峰值可视作标记组个体的耳石锶元素人工标记。(2)标志组和对照组大黄鱼幼鱼的生长速率和存活率均无显著性差异(P0.05),表明此项标记技术对受标个体的生长和存活状况不会产生显著负面影响,可用作大黄鱼增殖放流鱼苗的规模化标记手段。