Timing is everything: survival of Atlantic salmon (Salmo salar) postsmolts during events of high salmon lice densities

Atlantic salmon in aquaculture act as reservoir hosts and vectors of parasites like salmon lice and this parasite is shown to harm wild salmonid populations.
In the present study, n=29817 tagged Atlantic salmon were studied in four release trials. Half of the released fish were given prophylactic treatment against lice, the other half represented sham control fish. We used a nested design comparing years with low and high lice density and seasonal dynamics in infestation pressure. The released Atlantic salmon thus experienced highly variable lice infestation pressures, which we linked to survival and growth in returning fish. The fish were released in a protected “National Salmon Fjord“ and n=559 Atlantic salmon were recaptured after spending 1-4 years at sea.
In most experimental groups 1 – 2.5 % of the fish were recaptured at return. However, survival of unprotected fish was extremely low for the trial released at the highest density of lice: only 0.03 % of these Atlantic salmon returned to the river, compared to 1.86 % in the protected group.

Synthesis and applications . We document that a high lice density can cause a more than 50 times higher mortality risk in Atlantic salmon on their sea migration, even in a fjord with protected status. Fine-tuned and hard-to-predict year-to-year differences in timing, both for the wild smolt migration and the population build-up of lice released from aquaculture, means life or death to wild salmon. Management actions such as spatial segregation of farmed fish and lice (e.g. closed farm pens), and/or moving farms away from vulnerable habitats for wild salmonids (fjords and coastal areas), may be needed to ensure sustainable co-existence of wild and farmed Atlantic salmon.

Methods
Materials and Methods

Study area

The study was carried out in River Etne, draining in the outer parts of Hardangerfjord, in Hordaland county, western Norway (Fig. 1). The Hardangerfjord is among the most intensively used areas on the Norwegian coast for salmon production, with a standing stock of farmed Atlantic salmon of about 80 000 and 95 000 metric tonnes in 2013 and 2014, respectively (Fiskeridirektoratet 2019). For further details on the study area see (Halttunen et al. 2018).

Experimental design

The experiment started in 2013 and was replicated in 2014; two groups of Atlantic salmon were released in May and June, each year (Table 1). All fish were released close to the mouth of River Etne. Returning adult individuals were caught in the trap in River Etne after 1-4 years at sea. 

Table1. Summary of released salmon smolts and sample sizes for treatment (prophylaxis) and control groups in the four trials. Fish weights in gram ± SD.

Year

Release date

Prophylaxis

Control

Weight (g)

2013

May 18th

3791

3972

72 ± 21

2013

June 9th

3801

3868

74 ± 16

2014

May 18th

3819

3818

47 ± 11

2014

June 9th

3770

2978

42 ± 10

Fish used in this study were 1. generation one-year old hatchery-reared Atlantic salmon post smolts produced from eggs and sperm stripped from broodstock caught in River Etne. Fish were reared at Matre Research Station (IMR) and made ready for release in saltwater. Prior to release, all salmon smolts were tagged using coded wire tags (CWTs) inserted in their snout, which enable fish identification to i) treatment/control, and ii) timing of release. In addition, all fish had their adipose fin removed to enable us to distinguish experimental fish from wild fish in the trap on return to the river.

For the prophylactic antiparasitic treatment, we used a 30-minute bath of Substance EX (Pharmaq), hereafter termed SubEX, at a concentration of 2 ppm in oxygenated water. This treatment was applied to 50 % of the fish, randomly selected, securing a balanced design. SubEX protects the fish by preventing attached copepodids to develop into the next life stage for up to 16 weeks after treatment (Skilbrei et al. 2015). Identical (sham) treatment was performed on the control fish. This process was performed three days before each of the four releases to allow recovery of the treated fish.

After tagging and treatment, fish were transported in closed oxygenated tanks to Etne by car to a 5 m3 cage in the sea, close to the outlet of River Etne. The fish were kept in the cage for approximately 48 hours before they were released by lowering the net in the cage. The release was done by night to prevent immediate predation form birds. Prior to release a sample of 30 fish (randomly picked from the net) were killed to measure length and weight.

From 2014-2017, i.e.  1-4 years after release, all experimental fish returning to River Etne were caught in the fish trap and killed (wild Atlantic salmon not belonging to the experiment were released above the trap). Data on body length, weight and sex were registered at the return date.

Estimation of lice infestation pressure

Salmon lice densities were estimated based on sentinel cages (Bjørn et al. 2011) stocked with 30 farmed Atlantic salmon post smolts and positioned in the area the fish would migrate through (Fig. 1). We extracted lice counts from periods that approximately matched the times of release for the fish, i.e. in a 14-day period after May 18th and June 9th in 2013 and 2014. We included all life-stages of lice (from copepodites to adult stages) and calculated the total added number of lice per fish for a standardized period of 14 days (using modeled means of each cage mean, c.f. Fig. 2). These numbers were used to represent the environmental infestation pressure of lice in this study, hereafter termed Lice Infestation Pressure, for each of the four experimental releases. The positioning of the cages was the same between years.

To visualize the spatial distribution of lice infestation pressure in the whole area of interest (Fig. 3) we used the Relative Operating Characteristic (ROC) method to identify where the lice densities from the hydrodynamic lice dispersion model (see www.lakselus.no) were low (< 1 lice per fish), medium (1-10 lice per fish) or high ( > 10 lice per fish) (Sandvik et al. 2016). The hydrodynamic lice model is described in detail in earlier studies (Johnsen et al. 2014, Myksvoll et al. 2018).

Risk Ratio (RR)

The Risk Ratio or relative risk quantifies how much more likely the treated group is to return to the home river, compared to the control group. We analyzed differences in return rates between treated and non-treated fish, for each of the 4 experimental releases, with the following formulae:

 RR=ET/(ET+NT)EC/(EC+NC)=ET(EC+NC)EC(ET+NT)                                                                                                                                 (1)

where ET is the number of return events (E) in the treatment (T) group; NT is the number of non-return events (N) in the treatment (T) group; EC is the number of return events (E) in the control (C) group; and NC is the number of non-return events (N) in the control (C) group.

RR-values higher than 1 show higher adult salmon returns of treated fish as compared to control fish, RR-values lower than 1 show higher returns of the controls. We calculated confidence intervals for the RR with the formulae:

                                                                                               (2)

Where n1 and n2 = sample size of treated and non-treated fish released, respectively; x1 and x2 are sample size of returned fish in treated and control group, respectively. For 95 % confidence intervals we used z = 1.96.

Survival probability

The survival probability (probability of return) was modeled by logistic regression:

glm (Returned fish ~ Lice Infestation Pressure * Treatment, family = ’binomial’),                                 (3)

where Returned fish represents the probability for surviving 1-4 years in the sea and returning to the river (1 for returning fish, 0 for non-returning fish), Lice Infestation Pressure is the estimated environmental infestation pressure (standardized with mean = 0 and SD = 2) of lice and Treatment is prophylaxis against lice versus control. We also tested whether Releaseweight (average fish weight for the group at release) was a significant covariate in the model. As Releaseweight was a non-significant covariate (Estimate = -0.0054, Z = -1.471, p = 0.14), and did not improve the model (AIC), we used the simpler model without this factor. For model validation, we inspected residuals and re-run the model excluding one outlier fish. However, as the results were practically the same, we decided to include all data points.

Growth at sea

The growth of the fish during its sea migration was evaluated with a linear regression model:

lm (Weight ~ Lice Infestation Pressure + Treatment + Seawinter + Sex)                                                  (4)

where Weight is individual fish body mass at return, Lice Infestation Pressure is the environmental lice infestation pressure (standardized with mean = 0 and SD = 2), Treatment is prophylaxis or control, Seawinter is the number of years at sea before returning to the river (standardized for 2SW fish by subtracting 2 from the number of seawinters) , and Sex differentiate males from females. Fish that spent 4 winters at sea was excluded from the analysis since these were only observed in one of the trials. We standardized Lice Infestation Pressure and Seawinter in order to have comparable effect sizes between factors and covariates in the model (Schielzeth 2010). For model validation, residuals were inspected visually (versus fitted values and leverage, qq-plot, scale-location). We also re-run the model without two potential outliers, but decided to include all fish in the data set.

Statistical analyses were carried out in R statistical package version 3.5.1 (R-Developmental-Core-Team 2019).

养殖大西洋鲑可作为鲑虱等寄生虫的储存宿主与传播媒介，该寄生虫已被证实会对野生鲑科鱼类种群造成危害。
本研究针对4次放流试验中的29817尾标记大西洋鲑展开分析。其中半数放流个体接受了鲑虱预防性驱虫处理，其余个体作为假处理对照组。本研究采用嵌套设计，对比了鲑虱密度高低年份以及侵染压力的季节动态差异。放流的大西洋鲑因此经历了差异显著的鲑虱侵染压力，我们将这一压力与洄游个体的存活率及生长情况相关联。研究放流区域为受保护的“国家鲑鱼峡湾”，最终共有559尾大西洋鲑在海洋中生活1-4年后被重捕。
多数试验组的重捕率为1%~2.5%。但在鲑虱密度最高的试验组中，未接受保护的个体存活率极低：仅0.03%的该组大西洋鲑返回河流，而同期保护组的重捕率为1.86%。

综合与应用
本研究证实，即使在受保护的峡湾中，高鲑虱密度也会使大西洋鲑海洋洄游阶段的死亡风险提升50倍以上。野生鲑鱼幼体洄游时间与养殖源鲑虱种群增殖时间之间细微且难以预测的年际差异，会对野生鲑鱼的生存产生决定性影响。为实现野生与养殖大西洋鲑的可持续共存，需采取空间隔离养殖设施与鲑虱（如封闭养殖网箱）、或将养殖场迁出野生鲑科鱼类的脆弱栖息地（峡湾与近岸区域）等管理措施。

材料与方法
研究区域
本研究于挪威西部霍达兰郡哈当厄尔峡湾外围的埃特内河（River Etne）开展（图1）。哈当厄尔峡湾是挪威海岸鲑鱼养殖利用强度最高的区域之一，2013年与2014年的养殖大西洋鲑存栏量分别约为80000与95000公吨（渔业管理局Fiskeridirektoratet 2019）。关于研究区域的详细信息可参考Halttunen等（2018）的研究。

试验设计
本试验于2013年启动，并于2014年重复开展。每年5月与6月各放流两组大西洋鲑（表1）。所有试验鱼均放流于埃特内河河口附近。在海洋中生活1-4年后，洄游的成熟个体将被埃特内河内的诱捕笼捕获。

表1 4次试验中放流的鲑鱼幼体（洄游幼鲑）概况及驱虫处理组、对照组的样本量。鱼体重单位为克，数据以均值±标准差表示。
| 年份 | 放流日期 | 驱虫处理组 | 对照组 | 体重（g） |
|------|----------|------------|--------|----------|
| 2013 | 5月18日 | 3791 | 3972 | 72±21 |
| 2013 | 6月9日 | 3801 | 3868 | 74±16 |
| 2014 | 5月18日 | 3819 | 3818 | 47±11 |
| 2014 | 6月9日 | 3770 | 2978 | 42±10 |

本研究使用的试验鱼为捕获自埃特内河的亲本亲鱼繁育的一代龄孵化场养殖大西洋鲑海化幼鲑（post smolts），由亲鱼获取的卵与精子培育而成。试验鱼在马特研究站（IMR，挪威海洋研究所）培育至适合海水放流的阶段。放流前，所有鲑鱼幼鲑均通过在鼻部植入编码线标（coded wire tags, CWTs）完成标记，该标记可用于识别试验鱼的分组（处理组/对照组）与放流时间。此外，所有试验鱼均被切除脂鳍（adipose fin），以便在重捕时将试验个体与野生个体区分开。

针对寄生虫的预防性驱虫处理采用浓度为2ppm的Substance EX（Pharmaq公司，以下简称SubEX）进行30分钟药浴。该处理随机应用于50%的试验鱼，以保证试验设计的均衡性。SubEX可通过阻止附着的桡足幼体发育至下一生活史阶段，为试验鱼提供最长达16周的保护（Skilbrei等2015）。对照组鱼接受完全相同的假处理操作。上述操作于每次放流前3天完成，以使处理鱼得以恢复。

完成标记与处理后，试验鱼通过封闭充氧运输箱经陆路运至埃特内河附近的5立方米海上网箱。试验鱼在网箱中暂养约48小时后，通过下放网箱网衣完成放流。放流操作选择在夜间进行，以避免鸟类即时捕食。放流前，随机选取30尾试验鱼进行处死，以测量其体长与体重。

2014-2017年，即放流后1-4年，所有返回埃特内河的试验鱼均被诱捕笼捕获并处死（非本试验的野生大西洋鲑会被释放至诱捕笼上游区域）。记录重捕个体的体长、体重与性别信息。

鲑虱侵染压力估算
鲑虱密度通过哨兵笼法估算（Bjørn等2011）：每个哨兵笼放养30尾养殖大西洋鲑海化幼鲑，放置于试验鱼洄游途经区域（图1）。我们提取与各批次放流时间匹配的时段内的鲑虱计数数据，即2013年与2014年5月18日、6月9日后的14天窗口期。统计范围涵盖鲑虱所有生活史阶段（从桡足幼体至成虫阶段），并基于标准化14天窗口期计算每尾鱼的累计鲑虱数量（采用各哨兵笼的模型均值，详见图2）。该数值用于代表本研究中的环境鲑虱侵染压力，以下简称“鲑虱侵染压力”，对应4次试验放流批次。哨兵笼的布设位置在各年份间保持一致。

为可视化整个研究区域内鲑虱侵染压力的空间分布（图3），我们采用受试者工作特征（Relative Operating Characteristic, ROC）法，将来自水动力鲑虱扩散模型（详见www.lakselus.no）的鲑虱密度划分为低（<1尾/鱼）、中（1~10尾/鱼）与高（>10尾/鱼）三个等级（Sandvik等2016）。该水动力鲑虱模型的详细信息可参考既往研究（Johnsen等2014，Myksvoll等2018）。

风险比（Risk Ratio, RR）
风险比（相对风险）用于量化处理组相较于对照组返回原河的概率倍数。我们针对4次试验批次分别分析处理组与非处理组的重捕率差异，计算公式如下：
RR = [E_T/(E_T + N_T)] / [E_C/(E_C + N_C)] = E_T(E_C + N_C) / [E_C(E_T + N_T)] (1)
其中，E_T为处理组（T）的重捕事件数，N_T为处理组的未重捕事件数；E_C为对照组（C）的重捕事件数，N_C为对照组的未重捕事件数。
RR值大于1表示处理组的成鲑返回率高于对照组，RR值小于1则表示对照组返回率更高。我们采用如下公式计算风险比的95%置信区间：
其中n1与n2分别为处理组与非处理组的放流样本量；x1与x2分别为处理组与对照组的重捕样本量。95%置信区间对应的z值为1.96。

存活率估算
存活率（返回原河的概率）通过逻辑回归（logistic regression）建模：
glm(返回个体 ~ 鲑虱侵染压力 * 处理方式, family = 'binomial') (3)
其中，“返回个体”代表试验鱼在海洋中生活1-4年后返回河流的概率（重捕个体记为1，未重捕个体记为0）；“鲑虱侵染压力”为估算得到的环境鲑虱侵染压力（经标准化处理，均值=0，标准差=2）；“处理方式”为鲑虱预防性驱虫处理与对照组。我们同时检验了放流体重（试验组放流时的平均体重）作为协变量的显著性。由于放流体重的协变量效应不显著（估计值=-0.0054，Z=-1.471，p=0.14），且未提升模型拟合度（AIC值无改善），因此我们采用不含该协变量的简化模型。模型验证环节，我们对残差进行了检验，并在剔除1个异常值后重新运行模型，但由于结果无实质性差异，最终保留所有数据点。

海洋生长情况
采用线性回归（linear regression）模型评估试验鱼海洋洄游阶段的生长情况：
lm(体重 ~ 鲑虱侵染压力 + 处理方式 + 海历冬季数 + 性别) (4)
其中，“体重”为试验鱼重捕时的个体体质量；“鲑虱侵染压力”为环境鲑虱侵染压力（经标准化处理，均值=0，标准差=2）；“处理方式”为预防性驱虫处理或对照组；“海历冬季数”为试验鱼返回河流前的海洋越冬年数（针对2次越冬个体，通过将越冬年数减2完成标准化）；“性别”用于区分雌雄个体。由于仅在1个试验批次中观察到在海洋中越冬4年的个体，因此将该类个体从分析中剔除。我们对鲑虱侵染压力与海历冬季数进行标准化处理，以保证模型中各因子与协变量的效应量具有可比性（Schielzeth 2010）。模型验证环节，我们通过可视化方式检验了残差（与拟合值、杠杆值、QQ图、尺度-位置图）。我们同时在剔除2个潜在异常值后重新运行模型，但最终决定保留所有数据点。

本研究的统计分析均通过R统计软件包3.5.1版本完成（R开发核心团队2019）。