Novel parasite invasion leads to rapid demographic compensation and recovery in an experimental population of guppies

The global movement of pathogens is altering populations and communities through a variety of direct and indirect ecological pathways.  The direct effect of a pathogen on a host is reduced survival, which can lead to decreased population densities. However, theory also suggests that increased mortality can lead to no change or even increases in the density of the host. This paradoxical result can occur in a regulated population when the pathogen’s negative effect on survival is countered by increased reproduction at the lower density.  Here we analyze data from a long-term capture-mark-recapture experiment of Trinidadian guppies (Poecilia reticulata) that was recently infected with a nematode parasite (Camallanus cotti). By comparing the newly infected population with a control population that was not infected we show that decreases in the density of the infected guppy population were transient. The guppy population compensated for the decreased survival by a density-dependent increase in recruitment of new individuals into the population, without any change in the underlying recruitment function. Increased recruitment was related to an increase in the somatic growth of uninfected fish. Twenty months into the new invasion, the population had fully recovered to pre-invasion densities even though the prevalence of infection of fish in the population remained high (72%). These results show that density-mediated indirect effects of novel parasites can be positive, not negative, which makes it difficult to extrapolate to how pathogens will affect species interactions in communities.  We discuss possible hypotheses for the rapid recovery.

Methods
Field Data (Individual Data and Population Summaries)

We established four experimental populations of guppies in the upper Guanapo drainage in the Northern Mountain Range of Trinidad, West Indies. All four populations were established from a single source population on the lower Guanapo River where guppies live in diverse fish communities and with other species that frequently prey upon guppies. The guppies from these high predation communities were moved to the experimental streams, which previously lacked guppies, but were otherwise similar to natural upstream locations without predators. Upstream barriers prevent the upstream movement of guppies out of the experimental populations. More details of the experimental translocations can be found elsewhere.

During our monthly recapture, each fish greater than 14mm standard length is measured for mass, photographed and, if not previously marked, marked with a unique color combination of subcutaneous elastomer implants (Northwest Marine Technologies). Each fish receives two colors in two of eight locations on the body allowing us to individually identify 4,032 fish of each sex. Fish are lightly anesthetized with MS-222 for processing, housed overnight in medicated water to prevent infections from marking, and returned to the streams the following day.

We estimated the population size, survival, and recruitment of females and male guppies in the population using the POPAN module of program MARK implemented in Program R. For each population, we fitted models that were fully time dependent (apparent survival and probability of capture) and crossed by sex. Because our populations are introduced and we know exactly how many individuals were released into each stream, we constrained the probably of capture parameter for the first time period to be equal to unity. Other than this parameter, the default settings were used for each model.

Lab Data 

Female guppies (n=104) were collected from the extralimital site in the Caigual in January 2019 (month 131).  All guppies were caught using butterfly nets and transported back to the field station in two-liter Nalgene bottles filled with stream water.  A wide range of guppy sizes were chosen to understand the impact of the parasite in all life stages and sizes of the fish.  The initial standard length (SL, distance from the tip of the snout to the hypural plate) of the fish ranged from 7.99 to 33.35 millimeters. 

In the laboratory, the fish were housed in three-gallon tanks that received constant aeration and were under ambient temperature and light conditions.  The fish were not marked but were housed individually for the duration of the experiment, making them individually identifiable by tank number.  The water was changed every third day.

At the start of the experiment and each week thereafter, the standard length of each fish was measured to the nearest 0.01 mm using digital calipers (Mitutoyo) and the fish was weighed to the nearest 0.001 g on an Ohaus Scout scale.  During these weekly measurements, all fish were individually anaesthetized with 0.2% Tricaine S (MS-222) and screened for visible signs of infection using a stereo-microscope (Carl Zeiss Stemi 305).

Fish were fed twice daily with live, freshly hatched brine shrimp napauli (Artemia spp.).  Fish were fed food rations proportional to their size using a glass microliter syringe (Hamilton 50 μl or 250 μl micropipette).  Food amount was calculated each week according to the fish’s most recent weight, using the equation food= mass*e(4-(0.5*mass)) . Tanks were checked twice daily for offspring and dead fish.  Any offspring born during the course of the experiment were counted and housed separately from the mother.  Fish that died during the course of the experiment were immediately preserved in seven percent buffered formalin and saved for later dissection.

            After four weeks, the fish were euthanized with an overdose of MS-222 and preserved in seven percent buffered formalin or pure ethanol. Only females were selected for dissection because we sought to determine the effect of infection on fecundity (number of embryos) and offspring size. The gastrointestinal tract of each fish was examined for worms and, if worms were present, the number and wet weight of the worms was recorded to quantify parasite load.  The number of embryos and regressors (aborted embryos or unfertilized eggs) was recorded.  The somatic tissue, ovary tissue, regressors, and embryos were dried at 50° Celsius for at least 24 hours in a drying oven (Quincy Lab Model 40) and dry weights were recorded.

病原体的全球扩散正通过多种直接与间接的生态途径改变种群与群落结构。病原体对宿主的直接作用是降低其存活率，进而可能导致种群密度下降。但相关理论亦指出，死亡率上升可能不会使宿主种群密度产生变化，甚至反而使其升高。当病原体对存活率的负面影响被低密度下宿主繁殖率的提升所抵消时，这种矛盾的结果便会在受调控的种群中出现。
本研究基于近期感染驼形线虫（Camallanus cotti）的特立尼达孔雀鱼（Poecilia reticulata）长期标记重捕（capture-mark-recapture）实验数据展开分析。通过将新感染种群与未感染的对照组种群进行对比，我们发现受感染孔雀鱼种群的密度下降仅为暂时性现象。孔雀鱼种群通过密度依赖的新个体补充量提升，抵消了存活率下降带来的影响，且其基础补充函数未发生任何改变。补充量的提升与未感染个体的躯体生长加快相关。在寄生虫完成入侵的20个月后，尽管种群内鱼类的感染率仍高达72%，该种群已完全恢复至入侵前的密度水平。上述结果表明，新型寄生虫由密度介导的间接效应可能为正向而非负向，这使得我们难以推断病原体将如何影响群落内的物种互作。本研究还针对种群的快速恢复现象探讨了若干潜在假说。

研究方法
野外数据（个体数据与种群汇总数据）
我们在西印度群岛特立尼达岛北部山脉的瓜纳波河上游流域建立了4个孔雀鱼实验种群。这4个实验种群均源自瓜纳波河下游的单一源种群，该区域的孔雀鱼栖息于物种丰富的鱼类群落中，且面临多种捕食者的威胁。我们将这些来自高捕食压力群落的孔雀鱼转移至此前无孔雀鱼分布的实验溪流中，这些溪流的环境与上游无捕食者的自然栖息地高度相似。上游的拦阻屏障可防止孔雀鱼从实验种群中向上游逃逸。关于实验移殖的更多细节可参考其他相关文献。
在每月一次的重捕过程中，我们会对体长超过14mm的个体测量体质量、拍摄照片，若个体未被标记，则采用皮下弹性体植入（Northwest Marine Technologies）的独特颜色组合进行标记。每个个体可在身体8个部位中的2个位置植入两种颜色的标记，这使得我们能够分别对雌雄各4032尾个体进行个体识别。实验过程中，我们使用MS-222对鱼类进行轻度麻醉，将其置于含药物的水中过夜以防止标记操作引发感染，次日将其放回溪流中。
我们借助R语言中运行的Program MARK软件的POPAN模块，估算了种群中雌雄孔雀鱼的种群规模、存活率与补充量。针对每个种群，我们构建了完全随时间变化（包括表观存活率与捕获概率）且考虑性别差异的模型。由于实验种群为人工引入且已知每个溪流的初始放归个体数，我们将第一个采样周期的捕获概率参数约束为1。除该参数外，所有模型均采用默认设置。

实验室数据
2019年1月（采样第131期），我们从凯格瓦尔的域外位点采集了104尾雌性孔雀鱼。所有个体均通过蝶形网捕获，并使用装有溪流流水的2升Nalgene瓶运至野外工作站。我们选取了不同体长范围的孔雀鱼，以探究寄生虫对鱼类各生命阶段与体型的影响。这些个体的初始标准体长（SL，即吻端至尾下骨板的距离）范围为7.99~33.35mm。
在实验室中，我们将鱼类饲养于容积3加仑的水族箱中，水族箱配备持续充气装置，环境温度与光照条件均为自然状态。实验期间，鱼类未被标记，但均单独饲养，因此可通过水族箱编号进行个体识别。每3天更换一次饲养用水。
实验开始时及之后每周，我们使用三丰（Mitutoyo）数显游标卡尺测量每尾鱼的标准体长，精度可达0.01mm；使用奥豪斯探险家（Ohaus Scout）电子天平称量体质量，精度可达0.001g。在每周的测量过程中，我们使用0.2%的MS-222对每尾鱼进行个体麻醉，并通过蔡司Stemi 305体视显微镜筛查可见的感染迹象。
我们每日两次投喂鲜活的刚孵化的卤虫无节幼体（Artemia spp.）。使用玻璃微量注射器（汉密尔顿50μl或250μl微量移液管），按照与鱼体大小成正比的比例投喂饵料。饵料投喂量每周根据鱼体最新体质量进行计算，计算公式为：饵料量=体质量×e^(4-0.5×体质量)。每日两次检查水族箱，记录幼鱼与死亡个体。实验期间产出的所有幼鱼均会被计数，并与母鱼分开饲养。实验期间死亡的个体将立即置于7%的缓冲福尔马林中保存，用于后续解剖分析。
实验进行4周后，我们使用过量MS-222对鱼类实施安乐死，并将其保存于7%的缓冲福尔马林或纯乙醇中。仅选取雌性个体进行解剖，因为我们旨在探究感染对繁殖力（胚胎数量）与幼体体型的影响。我们对每尾鱼的胃肠道进行检查以寻找线虫，若发现线虫，则记录其数量与湿重，以量化寄生虫负荷。同时记录胚胎与退化卵（流产胚胎或未受精卵）的数量。将躯体组织、卵巢组织、退化卵与胚胎置于Quincy Lab 40型干燥箱中，在50℃下干燥至少24小时后记录其干重。