Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed “reproducibility crisis”. In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI₉₅ [34%, 49%]) to 72% (CI₉₅ [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

An experimenter heterogenisation was not sufficient to prevent idiosyncratic results in a multi-laboratory setting. Astonishingly, neither the experimenter nor the laboratory accounted for the main portion of the observed variation, but a high amount of residual variation in fact remained unexplained despite strict standardisation regimes.     

Optimal behaviour can violate the principle of regularity

Understanding decisions is a fundamental aim of behavioural ecology, psychology and economics. The regularity axiom of utility theory holds that a preference between options should be maintained when other options are made available. Empirical studies have shown that animals violate regularity but this has not been understood from a theoretical perspective, such decisions have therefore been labelled as irrational. Here, I use models of state-dependent behaviour to demonstrate that choices can violate regularity even when behavioural strategies are optimal. I also show that the range of conditions over which regularity should be violated can be larger when options do not always persist into the future. Consequently, utility theory—based on axioms, including transitivity, regularity and the independence of irrelevant alternatives—is undermined, because even alternatives that are never chosen by an animal (in its current state) can be relevant to a decision.     

The Genetic Structure of an Invasive Pest,the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Liviidae)

The Asian citrus psyllid Diaphorina citri is currently the major threat to the citrus industry as it is the vector of Candidatus Liberibacter, the causal agent of huanglongbing disease (HLB). D. citri is native to Asia and now colonizes the Americas. Although it has been known in some countries for a long time, invasion routes remain undetermined. There are no efficient control methods for the HLB despite the intensive management tools currently in use. We investigated the genetic variability and structure of populations of D. citri to aid in the decision making processes toward sustainable management of this species/disease. We employed different methods to quantify and compare the genetic diversity and structure of D. citri populations among 36 localities in Brazil, using an almost complete sequence of the cytochrome oxidase I (COI) gene. Our analyses led to the identification of two geographically and genetically structured groups. The indices of molecular diversity pointed to a recent population expansion, and we discuss the role of multiple invasion events in this scenario. We also argue that such genetic diversity and population structure may have implications for the best management strategies to be adopted for controlling this psyllid and/or the disease it vectors in Brazil.     

Sampling phylogenetic tree space with the generalized Gibbs sampler

A recent article published in Cladistics is critical of a number of heuristic methods for phylogenetic inference based on parsimony scores. One of my papers is among those criticized, and I would appreciate the opportunity to make a public response. The specific criticism is that I have re‐invented an algorithm for economizing parsimony calculations on trees that differ by a subtree pruning and regrafting (SPR) rearrangement. This criticism is justified, and I apologize for incorrectly claiming originality for my presentation of this algorithm. However, I would like to clarify the intent of my paper, if I can do so without detracting from the sincerity of my apology. My paper is not about that algorithm, nor even primarily about parsimony. Rather, it is about a novel strategy for Markov chain Monte Carlo (MCMC) sampling in a state space consisting of trees. The sampler involves drawing from conditional distributions over sets of trees: a Gibbs‐like strategy that had not previously been used to sample tree‐space. I would like to see this technique incorporated into MCMC samplers for phylogenetics, as it may have advantages over commonly used Metropolis‐like strategies. I have recently used it to sample phylogenies of a biological invasion, and I am finding many applications for it in agent‐based Bayesian ecological modelling. It is thus my contention that my 2005 paper retains substantial value.     

Dog’s Discrimination of Human Selfish and Generous Attitudes: The Role of Individual Recognition,Experience, and Experimenters’ Gender

Discrimination of and memory for others’ generous and selfish behaviors could be adaptive abilities in social animals. Dogs have seemingly expressed such skills in both direct and indirect interactions with humans. However, recent studies suggest that their capacity may rely on cues other than people’s individual characteristics, such as the place where the person stands. Thus, the conditions under which dogs recognize individual humans when solving cooperative tasks still remains unclear. With the aim of contributing to this problem, we made dogs interact with two human experimenters, one generous (pointed towards the food, gave ostensive cues, and allowed the dog to eat it) and the other selfish (pointed towards the food, but ate it before the dog could have it). Then subjects could choose between them (studies 1-3). In study 1, dogs took several training trials to learn the discrimination between the generous and the selfish experimenters when both were of the same gender. In study 2, the discrimination was learned faster when the experimenters were of different gender as evidenced both by dogs’ latencies to approach the bowl in training trials as well as by their choices in preference tests. Nevertheless, dogs did not get confused by gender when the experimenters were changed in between the training and the choice phase in study 3. We conclude that dogs spontaneously used human gender as a cue to discriminate between more and less cooperative experimenters. They also relied on some other personal feature which let them avoid being confused by gender when demonstrators were changed. We discuss these results in terms of dogs’ ability to recognize individuals and the potential advantage of this skill for their lives in human environments.     

Representing supraspecific taxa in higher-level phylogenetic analyses: guidelines for palaeontologists

Abstract:  As phylogenetic analyses become larger, one of the greatest methodological difficulties is representing speciose supraspecific clades in higher-level analyses (e.g. trilobites within studies of arthropod phylogeny). Several strategies have been proposed, including using representative single composite terminals or species-level exemplars, and various methods are currently used in the palaeontological literature. However, this is problematic, as simulation studies and empirical arguments in the systematics literature have clearly identified multiple exemplars as the optimal method. The continuing usage of suboptimal strategies in palaeontology may lessen the accuracy of phylogenies and hampers comparison between alternative studies. Here, I outline problems with suboptimal strategies, review arguments in support of multiple exemplars and provide guidelines for palaeontologists undertaking higher-level phylogenetic analyses.     

Ecological theory as a foundation to control pathogenic invasion in aquaculture

Detrimental host–pathogen interactions are a normal phenomenon in aquaculture animal production, and have been counteracted by prophylactic use of antibiotics. Especially, the youngest life stages of cultivated aquatic animals are susceptible to pathogen invasion, resulting in disease and mortality. To establish a more sustainable aquatic food production, there is a need for new microbial management strategies that focus on ‘join them'' and not the traditional ‘beat them'' approaches. We argue that ecological theory could serve as a foundation for developing sustainable microbial management methods that prevent pathogenic disease in larviculture. Management of the water microbiota in aquaculture systems according to ecological selection principles has been shown to decrease opportunistic pathogen pressure and to result in an improved performance of the cultured animals. We hypothesize that manipulation of the biodiversity of the gut microbiota can increase the host''s resistance against pathogenic invasion and infection. However, substantial barriers need to be overcome before active management of the intestinal microbiota can effectively be applied in larviculture.     

基于分子生物学方法的外来入侵物种入侵历史重构

生物入侵是一个世界性的问题。全球每年因生物入侵造成的损失超过1万亿美元。探究入侵物种在入侵地的入侵历史对了解生物入侵的生物生态学机制、制定阻截及防除措施有重要意义。分子标记方法的兴起和大规模应用打开了入侵生物入侵历史研究的新天地。采用分子标记的方法可鉴定入侵物种的种类、追溯其来源地、回溯其扩散路径、分析扩散模式及探究物种入侵过程中对入侵种群本身的变化及其对生态系统所造成的各种影响。分子标记的应用使得多个入侵物种的入侵历史得以重现。由于分子标记方法重构的入侵历史受采样范围、采用的分子标记的种类及数量等因素的影响,该方法呈现入侵历史是否是真实发生的入侵过程还存在争议。     

Eating the competition speeds up invasions

Many introduced species engage in intraguild predation (IGP), the consumption of species with which they compete for shared resources. While the factors influencing local persistence of IG predator and prey species are well-understood, using these factors to predict the invasion speed of an introduced IG predator has received less attention. Existing theory predicts that native competitors slow invasions via depletion of shared resources, but this fails to account for additional resources acquired when an invader consumes competitors. Here, I outline a general framework for understanding the effect of IGP on invasion speeds. I find that invaders that consume native competitors may be able to spread where invasion by pure competitors would fail, and that invasion speed increases with increasing levels of IGP. Notably, if the benefit from consuming competitors outweighs the loss of shared resources to competitors, invasion proceeds faster than invasion in the absence of competitors. This may explain empirical observations of rapid spread rates of invaders that feed at multiple trophic levels.     

Evolution of the silver and gold stains in neurohistology

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its “golden years,” 1870-1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Reproduction and the central project of evolutionary theory

In much of the discourse of evolutionary theory, reproduction is treated as an autonomous function of the individual organism — even in discussions of sexually reproducing organisms. In this paper, I examine some of the functions and consequences of such manifestly peculiar language. In particular, I suggest that it provides crucial support for the central project of evolutionary theory — namely that of locating causal efficacy in intrinsic properties of the individual organism. Furthermore, I argue that the language of individual reproduction is maintained by certain methodological conventions that both obscure many of the problems it generates and serve to actively impede attempts to redress those difficulties that can be identified. Finally, I suggest that inclusion of the complexities introduced by sexual reproduction — in both language and methodology — may radically undermine the individualist focus of evolutionary theory.I am indepted to the Rockefeller Foundation for a Humanities Fellowship that supported this research during the spring of 1986. I am also grateful to Richard Lewontin, Diane Paul, and Lisa Lloyd for many extremely helpful conversations.     

The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.     

Estimation of age from the pubic symphysis by means of multiple regression analysis.

This study has been carried out to assess the age from the pubic symphysial surface employing a multiple regression analysis and a quantification theory model I analysis. Using partial regression coefficients and/or normalized scores obtained from the analyses, ages of skeletal remains can be quantitatively estimated with a fairly high reliability. The use of this method is, however, limited to the samples between 18 and 38 years of age, because age changes in the symphysial surface show large variations after about 40 years. The reliability of this method was also examined.     

Rethinking the role of theory in exploratory experimentation

To explain their role in discovery and contrast them with theory-driven research, philosophers of science have characterized exploratory experiments in terms of what they lack: namely, that they lack direction from what have been called “local theories” of the target system or object under investigation. I argue that this is incorrect: it’s not whether or not there is direction from a local theory that matters, but instead how such a theory is used to direct an experiment that matters. Appealing to contemporary exploratory experiments that involve the use of experimental techniques—specifically, examples where scientists explore the interaction of neural activity and human behavior by magnetically stimulating brains—I argue that local theories of a target system can inform auxiliary hypotheses in exploratory experiments, which direct these experiments. These examples illustrate how local theories can direct the exploration of target systems where researchers do not aim to evaluate these theories.     

To change or not to change experimenters: caveats for repeated behavioural and physiological measures in Montagu's harrier

Animals facing anthropogenic disturbances may exhibit different anti‐predator responses depending on their perception of the risk. Experimental measures of behavioural and physiological traits may be impacted by the disturbance due to experimenter handlings and activities. In this study, we assessed the behavioural and physiological responses of Montagu's harrier chicks Circus pygargus from 14 nests, visited four times from hatching to fledgling by either the same (group I) or different (group NI) experimenters. Escape, aggressiveness and stress‐induced corticosterone concentration increased for both groups over time but chicks from group I showed a higher increase in their behavioural and physiological responses than chicks from group NI. This increase occurred after the second visit when experimenters took the first blood sampling on nestlings. Handling by the same experimenters throughout the study did not result in habituation but in sensitization. Our results suggest that Montagu's harrier nestlings may discriminate between the people who visit their nest and respond to experimenters’ identity. Therefore, we urge researchers working on behaviour and/or the physiology of stress including repeated measurements (such as animal personality studies for instance) to consider whether the experiments should or should not be conducted by the same experimenters (as usually done to minimize bias) as it can influence the outcomes of the experiments.     

Reputation,a universal currency for human social interactions

Decision rules of reciprocity include ‘I help those who helped me’ (direct reciprocity) and ‘I help those who have helped others’ (indirect reciprocity), i.e. I help those who have a reputation to care for others. A person''s reputation is a score that members of a social group update whenever they see the person interacting or hear at best multiple gossip about the person''s social interactions. Reputation is the current standing the person has gained from previous investments or refusal of investments in helping others. Is he a good guy, can I trust him or should I better avoid him as a social partner? A good reputation pays off by attracting help from others, even from strangers or members from another group, if the recipient''s reputation is known. Any costly investment in others, i.e. direct help, donations to charity, investment in averting climate change, etc. increases a person''s reputation. I shall argue and illustrate with examples that a person''s known reputation functions like money that can be used whenever the person needs help. Whenever possible I will present tests of predictions of evolutionary theory, i.e. fitness maximizing strategies, mostly by economic experiments with humans.     

Microbiology of the phyllosphere: a playground for testing ecological concepts

Many concepts and theories in ecology are highly debated, because it is often difficult to design decisive tests with sufficient replicates. Examples include biodiversity theories, succession concepts, invasion theories, coexistence theories, and concepts of life history strategies. Microbiological tests of ecological concepts are rapidly accumulating, but have yet to tap into their full potential to complement traditional macroecological theories. Taking the example of microbial communities on leaf surfaces (i.e. the phyllosphere), we show that most explorations of ecological concepts in this field of microbiology focus on autecology and population ecology, while community ecology remains understudied. Notable exceptions are first tests of the island biogeography theory and of biodiversity theories. Here, the phyllosphere provides the unique opportunity to set up replicated experiments, potentially moving fields such as biogeography, macroecology, and landscape ecology beyond theoretical and observational evidence. Future approaches should take advantage of the great range of spatial scales offered by the leaf surface by iteratively linking laboratory experiments with spatial simulation models.     

Evolution of the silver and gold stains in neurohistology

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its “golden years,” 1870–1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Evolution of the silver and gold stains in neurohistology.

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its "golden years," 1870-1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

A Defence of the Counterfactual Account of Harm

In order to determine whether a particular course of conduct is ethically permissible it is important to have a concept of what it means to be harmed. The dominant theory of harm is the counterfactual account, most famously proposed by Joel Feinberg. This determines whether harm is caused by comparing what actually happened in a given situation with the ‘counterfacts’ i.e. what would have occurred had the putatively harmful conduct not taken place. If a person's interests are worse off than they otherwise would have been, then a person will be harmed. This definition has recently faced challenges from bioethicists such as John Harris, Guy Kahane and Julian Savulescu who, believing it to be severely flawed, have proposed their own alternative theories of the concept. In this article I will demonstrate that the shortcomings Harris, Kahane and Savulescu believe are present in Feinberg's theory are illusory and that it is their own accounts of harm that are fraught with logical errors. I maintain that the arguments presented to refute Feinberg's theory not only fail to achieve this goal and can be accommodated within the counterfactual account but that they actually undermine the theories presented by their respective authors. The final conclusion will be that these challenges are misconceived and fail to displace the counterfactual theory.