嗜水气单胞菌UDP-乙酰葡萄糖胺-4-差向异构酶基因的克隆表达及酶的性质

通过水生动物源性嗜水气单胞菌（Aeromonas hydrophila，Ah）强毒株J-1株与弱毒株MR-1株抑制差减杂交得到gneJ差减片段，该片段与创伤弧菌（Vibrio vulnificus）和人源嗜水气单胞菌的UDP-乙酰葡萄糖胺-4差向异构酶基因有较高的同源性。扩增完整的gneJ基因，进行分布检测并将其克隆至质粒pET32a（＋），在大肠杆菌（Escherichia coli）BL21中进行异源表达，SDS-PAGE结果显示，重组表达蛋白的分子量约59．7kD，与理论推测值基本相同。酶活性分析表明，该重组蛋白可将UDP-乙酰葡萄糖胺转化为UDP-乙酰半乳糖胺，确证gneJ基因编码蛋白为UDP-乙酰葡萄糖胺-4-差向异构酶。Western Blot分析显示，嗜水气单胞菌J-1株胞外产物的兔抗血清可识别该重组蛋白，表明该蛋白与天然蛋白有相同的抗原性。以重组蛋白为免疫原，对小鼠进行动物保护实验，结果显示，该重组蛋白对小鼠有60％的保护率，证明UDP-乙酰葡萄糖胺-4差向异构酶可作为亚单位疫苗的侯选成分。     

嗜水气单胞菌细胞外膜蛋白及S层蛋白分析

用十二烷酰肌氨酸盐抽提结合超速离心纯化，制备了19株典型嗜水气单胞菌（Aeromonas hydrophila)和1株温和气单胞菌（A.sobria)的主要外膜蛋白（MOMP）。根据SDS-PAGE图谱，将其中的14株菌分为3个MOMP组，I组的主要蛋白带为40kD和43kD，Ⅱ组的主要蛋白带为40kD和50kD,Ⅲ组的主要蛋白为40kD、46kD和50kD.MOMP组和血清型间没有严格的关系。用兔抗嗜水气单胞菌体抗原为第一抗体进行免疫印迹实验。结果显示，所有被试菌株都能产生强阳性反应，其中40kD的蛋白带为大多数菌株所共有，且具有相似的免疫原性，是构成菌体抗原的重要免疫原。用0.2mol/L甘氨酸缓冲液（pH4.0)处理上述菌株培养物，结果仅有嗜水气单胞菌ZY01-g3能够分离到丰富的S层样蛋白，其分子量约为52kD，而分子量约为52kD，而其他菌株未能分离到典型的S层样蛋白，表明S层蛋白的分布有限。     

筛选用转座子Tn916诱变的具有_省略_疫原性的嗜水气单胞菌蛋白酶缺失株

以携带质粒pAM120(Apr、Tcr/Tn916)的大肠杆菌CG120株为供体菌，与受体菌嗜水气单胞菌J-1株，采用滤膜接合法进行接合转移，在选择平板(含Tc、cfz和脱脂奶)上进行筛选，筛选出6株蛋白酶缺失株(ECPase-)，ECPase-菌株其生长速度，对正常鲫血清的抗性降低，用脱脂奶平板法及底物法检测，蛋白酶产量明显降低。与亲本J-1株相比，突变株致病力降低，对鲫的半数致死量大于108(J-1株对鲫的半数致死量为5×103)。用ECPase-突变株MJ-1株的18h培养物(5×106CFU)接种健康鲫，不产生亲本J-1株引起的病变及死亡。用MJ-I活菌免疫鲫，在第5周产生较高的凝集抗体，且对强毒株J-1的攻击有保护作用。表明蛋白酶是嗜水气单胞菌的一个重要的毒力因子，ECPase-突变株仍具有免疫原性。     

嗜水气单胞菌外膜蛋白基因ompTS的克隆与序列分析

根据已发表的外膜蛋白基因ompⅡ的核苷酸序列设计引物，从分离 自患红底板病的中华鳖的嗜水气单胞菌中扩增得到了ompTS基因，对ompTS 基因进行序列分析，发现其与ompⅡ基因的核苷酸序列有83.5%的同源性。ompTS基因最长的开放阅读框（ORF）为1068nt，编码由355个氨基酸组成，分子量为38.9kDa的蛋白质OmpTS，其氨基酸序列的前20个氨基酸残基可能组成信号肽。由ompTS基因的编码氨基酸序列与其它细菌外膜蛋白的氨基酸序列的比较结果，进一步证实细菌外膜蛋白氨基酸序列的N端存在高度保守区。根据序列分析结果推测，ompTS基因很可能是一个新的基因，编码38.9kDa的嗜水气单胞菌外膜蛋白OmpTS，该蛋白质在膜中极有可能形成孔道，具备与孔蛋白相似的性质。     

不同来源嗜水气单胞菌外膜蛋白基因检测及序列分析

采用PCR方法,对分离于发病宽体金线蛭(Whitmania pigra)、中国林蛙(Rana temporaria chensinensis)、牙鲆(Paralichthys olivaceus)、草鱼(Ctenopharyngodon idellua)、青鱼(Mylopharyngodon piceus)、鲤(Cyprinus carpio)的42株致病性嗜水气单胞菌(Aeromonas hydrophila)进行了外膜蛋白(OMP)基因的检测;将代表菌株的外膜蛋白基因通过DNAStar软件与GenBank中报道的4株嗜水气单胞菌(FJ437030.1、AF183931.1、AF276639.2、CP000462.1)和1株温和气单胞菌(A.sobria,FJ437029.1)参考菌株进行了核苷酸同源性比较分析。结果表明,所检不同来源嗜水气单胞菌的OMP基因携带率为100%;所测代表菌株的OMP基因与参考菌株的同源性在86.0%～99.2%之间。所测得的不同来源嗜水气单胞菌OMP基因的同源性98.3%～100%,其中分离于宽体金线蛭的HTQ010505-1与青鱼的HL060811-4两菌株间同源性高达100%。     

中华鳖气单胞菌菌细胞苗和菌化学成份疫苗研制

利用分离自中华鳖的嗜水气单胞菌Ａｈ９６１００４、Ａｈ９６０９１６和Ａｈ９７０５１６与温和气单胞菌Ａｓ９７０５１０菌体及其上清制备灭活苗细胞苗和化学成份疫苗。肌肉注射中华鳖进行免疫,加强免疫１５ｄ后,用菌株Ａｈ９６１００４腹腔注射进行攻击,观察２０ｄ。结果表明,温和气单胞菌Ａｓ９７０５１０株的菌苗及其胞外分泌物（ＥＣＰ）苗组和嗜水气单胞菌Ａｈ９６１００４株的外膜成份组对嗜水气单胞菌的攻击的保护率最高     

嗜水气单胞菌J-1株丝氨酸蛋白酶基因克隆与序列分析

根据已发表的气单胞菌胞外蛋白酶基因核苷酸序列，设计和合成了一对引物，以嗜水气单胞菌AhJ—1的基因组DNA为模板，通过PCR技术，扩增到约900bp的丝氨酸蛋白酶基因片段，并克隆到质粒载体pGEM—T中进行测序和分析，结果表明扩增的丝氨酸蛋白酶基因片段与已发表的嗜水气单胞菌丝氨酸蛋白酶Ahe2的同源性有87％，扩增片段编码343个氨基酸，推测的分子量为35700，计算机软件分析表明编码的氨基酸有较高的抗原性，可作为核酸疫苗的侯选基因片段。     

嗜水气单胞菌感染后牛蛙血清中抗菌物质的初步研究

用1×107 mL-1的嗜水气单胞菌腹腔注射感染牛蛙，注射后7 d内试验组牛蛙血清抗菌活性都高于对照组，其中在3 d、7 d时存在显著性差异，峰值出现在感染后第3天。收集第2批人工感染后3 d时牛蛙的血清。该血清经Sephadex G25凝胶柱分离后,并在波长280 nm处测定紫外吸收值，得到两个吸收峰。以嗜水气单胞菌检测分离物的抗菌活性,结果表明经诱导的血清抗菌物质主要集中在第1个吸收峰。通过对金黄色葡萄球菌等10株指示菌抗菌谱的测定，其对大肠杆菌、枯草芽孢杆菌、铜绿假单胞菌等具有明显的抗菌作用；SDSPAGE分析显示抗菌蛋白分子量较大。结果说明：牛蛙被嗜水气单胞菌感染后能很快产生多种抗菌物质，主要的抗菌物质是分子量比较大的物质，这些抗菌物质对部分革兰氏阴性菌和革兰氏阳性菌有抗菌作用。图3表1参24 关键词：牛蛙；嗜水气单胞菌；抗菌蛋白；抗菌活性     

鳜嗜水气单胞菌GYK1株胞外产物提取、纯化及生物学活性分析

以鳜分离的嗜水气单胞菌GYK1株为代表,采用玻璃纸覆盖技术提取了8株嗜水气单胞菌和1株温和气单胞菌的胞外产物,SDS-PAGE电泳分析显示,8株嗜水气单胞菌的胞外产物均有35kDa的共同蛋白带。酶活性和溶血性分析表明,GYK1株胞外产物具有酪蛋白酶、淀粉酶、脂肪酶、明胶酶活性,但不具备脲酶活性;溶血性较强,蛋白浓度为365μg·mL-1的胞外产物对鳜血细胞的溶血价为213,对加州鲈、银鲫、小白鼠、兔、绵羊、人O型血的红细胞的溶血价在211~214之间。应用聚丙烯酰胺葡聚糖凝胶柱层析,对GYK1株的胞外产物进行了初步纯化,得到分子量为35kDa的蛋白带,该纯化产物具溶血性,但溶血性比粗胞外产物降低,对鳜和小鼠血细胞的溶血价均为25。     

鳗源嗜水气单胞菌β—溶血素基因的克隆及表达

应用PCR技术,从1株鳗源嗜水气单胞菌ML316中扩增得到β 溶血素基因AHL316HEM,将AHL316HEM克隆到pGEM TEasyVector,经分析验证后重组到pcDNA3.0中,构建了重组质粒PDLH。转化重组质粒PDLH的大肠杆菌(Escherichiacoli)DH5α能在血琼脂培养皿中形成明显的β 溶血斑,重组菌纯化的胞外产物溶血价为1.28×104HU·mg-1,同时重组菌的胞外产物能被原始菌株的高免血清特异性地识别。结果证实,克隆到鳗源嗜水气单胞菌β 溶血素基因,重组质粒PDLH能够表达具有天然生物学活性的β 溶血素,为构建嗜水气单胞菌核酸疫苗奠定了基础。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

An overview of stress physiology of fish transport: changes in water quality as a function of transport duration

This study brings an integrated analysis about the relationship between water deterioration and its physiological consequences in live fish transport. The analysis was focused on the transport water and its deterioration, and physiological challenges imposed on the fish. Usual commercial handling procedures employed to mitigate fish stress during transport were discussed. Future topics of research for the establishment of safer fish transport protocols were proposed. Transport was classified into short (≤8 h) or long transport (>8 h). The main issue in short transports should be the prevention of water pH reduction, while in long transports it is the increase in ammonia. Plasma cortisol is the most employed marker for stress and is acutely elevated upon short episodes of transport, but remains elevated even in long‐transport events. Plasma glucose is perhaps a better marker for handling stress. Plasma lactate, pH, osmolality CO₂ and ions should be more often evaluated. Plasma Na⁺ and Cl^‐ are very useful markers of acidosis, due to their respective exchange for H⁺ and , for acid–base regulation. The establishment of species‐specific transport protocols should be preceded by such combined analyses of water and physiological parameters.     

Are hatchery‐reared abalone naïve of predators? Comparing the behaviours of wild and hatchery‐reared northern abalone,Haliotis kamtschatkana (Jonas, 1845)

Abalone populations have declined worldwide, generating interest in enhancement using hatchery‐reared individuals. In many cases, such restoration efforts have met with limited success due to high predator‐induced mortality rates. Furthermore, the mortality rates of outplanted hatchery abalone are often considerably higher than for wild individuals. This study uses northern abalone (Haliotis kamtschatkana) as a case study to determine whether hatchery‐reared abalone behave differently than their wild counterparts. In the field, outplanted hatchery‐reared abalone were significantly less responsive than wild abalone, in terms of number of abalone responding and intensity of response, to nearby movement and to physical contact with an inert probe. Also, when encountering a cue to which all abalone responded (a seastar predator), hatchery‐reared individuals remained subdued. Anti‐predator behavioural deficits in hatchery‐reared abalone were more pronounced in 4‐year‐old individuals than in 1‐year‐old individuals, suggesting an influence of either age or amount of time spent in the hatchery environment. These behavioural differences are expected to increase the vulnerability of hatchery‐reared abalone to predators, and are likely a major cause of their elevated predator‐induced mortality when outplanted.