Caenorhabditis elegans. Here we review general principles of genetic screens in C. elegans, and use a modified binomial strategy to obtain a general expression for the number of mutagenized gametes examined in a genetic screen. We use this expression to calculate optimal screening parameters for a large range of genetic screen types. In addition, we developed a simple online genetic-screen-optimization tool that can be used independently of this paper. Our results demonstrate that choosing the optimal F2-to-F1 screening ratio can significantly improve screen efficiency.In genetic screens, the number of mutagenized gametes examined is an important parameter for evaluating screen progress, the number of genes of a given mutable phenotype, gene size, cost, and labor. Since genetic screens often entail examination of thousands or tens of thousands of animals, strategies for optimizing genetics screens are important for minimizing effort while maximizing the number of mutagenized gametes examined. To date, such strategies have not been described for genetic screens in the nematode  The identification of mutant animals is usually the first step in the genetic analysis of a biological process. In animals amenable to genetic study, such mutants are commonly identified by random mutagenesis, followed by screening for the trait under study. The mutation frequencies conferred by different mutagens in different organisms have been extensively documented Caenorhabditis elegans, typical mutagenesis regimens require isolation and examination of thousands of animals to approach saturation. Depending on the screening approach, examination of such numbers of animals can be quite labor intensive. In these situations, therefore, it is advantageous to identify optimal screening strategies that maximize the number of genomes screened, while minimizing the work involved. Indeed, as we show here, choosing a reasonable, yet suboptimal ratio of F2 to F1 animals, can double or triple the work involved in screening a given number of mutagenized F1 animals, as compared to screening using optimal parameters. Thus, suboptimal screen strategies may unnecessarily prolong screens, and use up excess reagents.For screens in the nematode C. elegans is not available. We therefore set out to develop a general algorithm for optimizing genetic screens in this organism. Here we examine such an optimization approach. We begin by reviewing the variables affecting the number of mutagenized haploid genomes needed to achieve saturation screening. We then derive a general expression, valid for most genetic screening approaches used in C. elegans, for counting the number of mutagenized F1 animals examined in a genetic screen. The expression we derive is, as we show, essentially independent of the total number of animals scored during the course of the screen, and is independent of variations in locus mutability. Although the number of mutagenized F1 animals is often approximated by the Poisson distribution, we demonstrate that for at least one major screen class, the Poisson approximation leads to large errors.Currently, a description of how to optimize genetic screens in F2-to-F1 screening ratio always exists for these screens, and that this ratio is dependent neither on the total number of animals scored, nor on the number of mutagenized gametes examined. Rather, the optimal screening ratio depends only on the type of mutation sought  and on the relative work involved in picking and scoring F1 and F2 animals. We use our results to delineate a simple algorithm for setting up and following the progress of genetic screens in C. elegans.Using the generalized expression described above, we solve optimization equations to maximize the efficiencies of two common genetic screen types. Our solutions reveal that an optimal C. elegans, our results can be simply extended to genetic screens in other organisms.Although valid for C. elegans). are exposed to a mutagen inducing mutations in sperm and oocytes C. elegans gene at an average frequency of one every 2,500 mutagenized P0 gametes The nematode P0 animals are allowed to self-fertilize, to produce F1 progeny. If mutations in either copy of a gene under consideration can be revealed in subsequent analysis, and if n F1 animals are examined, then the number of haploid genomes screened, defined as the number of P0 gametes examined, is given by 2n. More generally, however, the number of haploid genomes examined is given by a times n (an) where a takes on the value of either 1 or 2. For example, if a mutation in a gene of interest is suspected to be lethal or sterile, attempts to induce it on a marked chromosome, opposite a balancer chromosome, are often undertaken a = 1.Next, F1 animal heterozygous for a mutation in a specific gene of interest is influenced by a number of parameters, and can often vary greatly from gene to gene. Thus, for example, smaller genes may be less likely to be hit by mutagen. Furthermore, some mutagens, such as EMS, preferentially alter certain nucleotides F1 animals to be screened is difficult, and screens are generally considered near saturation when multiple alleles of a given gene have been identified.Following mutagenesis, the probability of finding at least one Nonetheless, in many instances, assuming an average mutagenesis frequency can lead to useful estimates regarding screen progress, and deviations from these estimates can often hint at unique features of a gene, such as unusually large or small size F1 animal heterozygous for a mutation in a gene of interest among n F1 progeny of mutagenized P0 animals, assuming a = 2, is 1−−. For common mutagenesis frequencies, the last term is exceedingly small and can be ignored without significant loss of accuracy. Thus, the probability of finding a heterozygous F1 animal, π(n), is given byF1 animals expected, on average, to carry a mutation in a gene of interest. As described above, r = 1,250 for loss-of-function mutations obtained by EMS mutagenesis.The probability of finding at least one F1 animals should be examined, on average, to obtain at least one animal carrying a loss-of-function mutation in a gene of interest. For example, to achieve a 95% probability (π(n) = 0.95) of obtaining an F1 carrier, we subtract 1 from both sides of equation (1), divide both sides by −1, take the logarithm of both sides of the equation, and rearrange to obtain n = ln0.05/ln(1−1/r). For large values of r, as is the case for most mutagenesis regimes, ln(1−1/r)≈−1/r. We thus obtain the expression n/r = −ln0.05, or n/r≈3, a commonly used result , we can calculate how many a = 1 (see example above) is more complex if the F1 animals, in which the mutation of interest is induced on the balancer chromosome, cannot be readily distinguished from those in which the mutation is induced on the marked chromosome, and we proceed as follows. Of a collection of n F1 animals, assume that q are heterozygous for mutations in a given gene. The odds that this is the case are given by the probability of obtaining q heterozygotes, (1/r)q, multiplied by the probability of the remaining n−q F1 animals being non-carriers, (1−1/r)n−q, multiplied by the number of ways such an arrangement can occur, given by the binomial coefficient q animals carries the mutation on the marked chromosome is given by 1−(1/2)q, where (1/2)q is the probability that all q heterozygotes carry the mutation on the balancer chromosome. Thus, the probability that of n F1 animals, q are heterozygous for mutations in a given gene, and at least one of these q animals carries the mutation on the marked chromosome is F1 animal is present among the n F1 animals picked, we sum the individual probabilities for each value of q to obtainA similar calculation for n and r, equation (2) can be accurately approximated using the Poisson distribution > πa = 1(n), a result illustrated in n) are plotted for values of n between 1 and 20,000, assuming r = 1,250.For a given F1 animals must be sifted through to identify a mutant of choice. However, the question arises as to whether an optimal screening strategy, independent of locus mutability, might exist, and if so, what are its properties.The discussion of the previous section suggests that gene-to-gene variations in mutability make it difficult to predict precisely how many mutagenized F1 animals have been examined during a genetic screen. In this section and the following sections we make a distinction between the number of F1 animals picked for analysis, n, and the number of F1 animals whose mutation content has actually been examined, actn. If the mutation being sought is predicted to behave in a simple dominant fashion, then counting how many F1 animals were examined is trivial, and is precisely equal to the number of F1 animals picked, that is, actn = n , (2), and (3) to obtain π(n).To begin to address this issue, we must first determine how many mutagenized = n e.g.. Howeveractn, we begin by considering, as an example, a genetic screen in which all n F1 animals are picked to a single plate, from which m F2 animals are then scored for the mutant phenotype. We assume that all F1 animals produce approximately the same number of F2 animals. Indeed, for standard EMS screens in C. elegans, only about 5% of F1 animals have greatly reduced fertility, and fewer than 1% of F1 animals have reduced fertility using one common protocol for trimethylpsoralen and ultraviolet light mutagenesis . Furthermore, for many mutagenesis schemes, only healthy F1 animals are picked for subsequent screening, further reducing the number of animals producing low brood counts.To obtain a general expression for actn can be approximated fairly accurately in two limiting cases. First, we consider a screen in which m≫n. In this case, many F2 progeny have been scored for each F1 animal, making it very likely that the mutation content of all F1 animals has been established. Therefore, actn≈n.For such a screen design, n≫m. Here, the likelihood that two or more of the F2 animals scored derive from the same F1 animal is small. Thus, actn≈m(1−p), where p, is the probability that a scored F2 has failed to reveal whether its F1 parent had the mutation of interest. In the case of simple screening schemes involving recessive mutations, p = 3/4.Second, we consider the case for which F1 animals, F2 animals, or both, may fail to satisfy the limiting conditions considered above. To calculate actn for the general case of this example, we proceed as follows.Although these limiting cases have important uses, genetic screens that involve manually picking m scored F2 animals are represented q progeny of a particular F1 animal is given by the binomial termn)q is the probability of scoring q progeny of a particular F1 animal, (1−1/n)m−q is the probability of the remaining scored F2 animals not being progeny of the particular F1 animal under consideration, and The probability that among the q scored F2 animals is informative about the presence of a mutation of interest in the particular F1 animal under consideration, is given by 1−qp, where p is the probability of the F2 animal not being informative. Therefore, by analogy to the calculation in the previous section, the probability that at least one informative F2 progeny of a particular F1 animal is found among the m F2 animals scored is given byThe probability that at least one of the F2 to F1 animals, y = m/n. It will become useful, therefore, to represent the functions B and P as functions of y and N = n+m, the total number of animals picked in the screen. By making the appropriate substitutions we can rewrite these functions asIn this paper we aim to describe optimal genetic screening strategies, and are, in general, interested in the optimal screening ratio of F1 animals examined for possession of a mutation in a specific gene, then, is given byandactn/N as a function of log10y for a simple recessive mutation with p = 0.75 and N = 1000. As expected, for n≫m, actn/N is asymptotic to the curve m/(4N) = y/(4(1+y)) (red line). For m≫n, actn/N is asymptotic to the curve n/N = 1/(1+y) (blue line). As is evident from the figure, for p = 0.75, the asymptotic curves overestimate actn by only 5% or less for n and m such that y = m/n≤0.4 or m/n≥12.2. However, in between these values, a very large error, that may exceed 100% of the true value of actn, can occur (not shown), justifying the more detailed analysis presented in this section. For large m and n, equations (7) and (8) are very closely approximated using the Poisson distribution  shows that actn/N is dependent on the value of N, m and n, actn/N becomes entirely independent of N, making actn/N useful for the analysis of most screens.p = 0.75 are the most commonly sought mutations p≠0.75. For example, if the F1 animals are heterozygous for a balancer chromosome and an unmarked homologous chromosome, such that animals homozygous for the balancer are dead, or easily identifiable, then p becomes 2/3 = 0.67 for a fully penetrant recessive mutation. As another example, dominant mutations that are rescued by a wild-type maternal genotype cannot be isolated in the F1 generation, but can be sought among F2 animals. For strictly dominant mutations of this type, p = 0.25.Although fully penetrant recessive mutations for which p = 0 occurs when F1 animals are heterozygous for a balancer and a marked chromosome, with the mutation of interest induced on the latter. In this case, only marked F2 animals are scored, and these should all be homozygous for the mutation of interest. The condition p = 0 also holds for rare screens where dominant maternal-effect mutants are sought. In such screens, heterozygous F1 animals do not have the phenotype of interest, but all of their progeny do.The case p = 0 arises frequently in genetic screens of haploid organisms. In this case, actn/N is a measure of the representation of an initial pool of mutagenized organisms in the progeny that have been examined.The condition actn/N vs. log10y for a number of possible values of p. The curves agree with the expectation that the more informative an F2 animal is about the mutation state of its F1 parent , the fewer F2 animals must be examined to achieve a specific value of actn/N.In F1 animals are placed on a single plate, from which F2 progeny are sampled. However, usually, it is necessary for F1 animals to be placed individually, or in groups, on multiple plates. F2 animals are then drawn from each plate. Individual plating of F1 animals is a particularly common scheme that is utilized if the expected mutation is lethal, and heterozygous F2 siblings need to be recovered, or in a situation where males are present in the population and mating between F1 animals must be avoided. Should F1 plating strategy affect actn? Consider the case where single F1 animals are placed on individual plates, and equal numbers of F2 progeny are drawn from each plate. Because we know for certain that in such a scheme F2 animals scored are derived from every F1 animal picked, it is predicted that for a given y, actn should be greater than in a scheme in which all F1 animals were plated on a single plate, where some F1 animals may not be sampled in the F2 generation. Thus, for the same number of F1 and F2 animals, actn will indeed be dependent on plating strategy.The analysis described in the preceding two sections holds for the specific case in which all actn explicitly, we let ν equal the number of F1 animals picked to a plate, and μ equal the number of F2 animals scored per plate. In general, ν and μ can be different for every plate. F1 animals examined on the ith plate, is given, as in equation (8), byactn/N, is, therefore, obtained by summing the actual number of F1 animals examined over all plates, orTo calculate the general expression for F1 and F2 animals are plated and scored per plate. In this case, νi, μi, and β are identical for each plate, and equation (13) can be written ask is the number of plates examined.Equation (13) is valid for essentially every type of genetic screen, involving any plating strategy. The equation can be significantly simplified if we assume that the same number of y as follows. To express μ as a function of y we note that y = m/n = kμ/(kν) = μ/ν. Rearranging terms yields μ = νy. Also, kν/N = n/(n+m) = 1/(1+m/n) = 1/(1+y), yieldingEquation (14) can be expressed in terms of F1 animal/plate, equation (15) can be reduced toF1 animals on the same plate, ν becomes n, and β becomes B, as in equation (8).Four points regarding equation (15) are of note. First, for the common plating strategy of one N, is not well-approximated by the Poisson distribution if a small number of F1 animals is plated per plate, which is generally the case for clonal screens. In actn/N by using the Poisson approximation for different values of p for the case of one F1 animal per plate. Note that for p = 0.25, the error can be nearly 30% off the exact value. Indeed, it can be shown that the fractional error, [(actn/N)exact−(actn/N)Poisson]/(actn/N)exact, plotted in p−ep−1)(/(p−1) as y→1  and (8), equation (15), even for large  y→1 see .actn/N is independent of N, although for a given N only values of μ+ν that are divisors of N are possible.Third, equation (15) reveals that N, the larger the number of F1 animals per plate (kØ1), the better is equation (15) approximated by equation (10). Simulations for different ratios of n to k reveal that for n/k≥30 equation 10 gives an excellent estimate of actn .Fourth, for a given C. elegans. In general, a measure of screen efficiency should take into account the amount of work performed in a screen as well as the total number of mutagenized F1 animals examined, actn. Work can be defined in a number of ways. Here, we will generally define work as the amount of time spent picking and scoring animals. Alternatively, work can be a measure of the total amount of reagents needed for the screen, etc., and much of the analysis that follows would still be valid using this definition. Regardless of the precise definition of work used, the work expended in a screen must be of the form γn+αm, where γ and α represent work per animal and have values between 0 and ∞. If we measure work in such a way that γ = α = 1 defines a unit of work, we can write the total work expended as W = γn+αm. Given this definition, we now propose to define the efficiency of a genetic screen asF1 animals examined per unit of work. As we will demonstrate below, this definition allows us to make quantitative estimates of the optimal screening ratio, y, for use in a broad range of genetic screen types.The results described in the previous sections provide us with the appropriate tools to consider how to optimize genetic screens in F1 and pick and score F2 animals, we define γ = α = 1; then, W = N, and ε = actn/N, which is the parameter we have used throughout this paper  that a maximum indeed exists for every value of γ and α>0.We can now pose the optimization problem for genetic screens as follows: what is the actn as a function of log10y for three different values of W where γ = α = 1. The minimal amount of work, minW, required to screen 5,000 mutagenized F1 animals, reqn = 5,000, occurs at W = 37,642. Values of W smaller than this will never achieve reqn, while values of W greater than 37,642 do not minimize W, by definition. We can therefore formulate the following general criteria: minW is the value of W satisfying the two conditions, actn = reqn. Although these equations can be numerically solved for the general case represented by equation (13), we restrict our analysis below to the two most common screen cases for which either equations (10) or (16) are valid.Consider F1 animals on a single plate or on a small number of plates relative to the total number of F1 animals examined, we can use the Poisson approximation to write the efficiency of a screen as:For genetic screens involving plating all y and set the result equal to 0 to obtain the following transcendental equation for maxy, the optimal F2-to-F1 screening ratio:We differentiate this equation with respect to maxy is only dependent on the relative values of γ and α. Thus, the optimal screening ratio, maxy, is independent of the absolute work expended to pick and pick and score F1 and F2 animals, respectively.Two features of this equation are of interest. First, the equation cannot be solved analytically, and must be evaluated numerically. Second, and more importantly, the value of maxy always exist? Inspection of equation (19) reveals that it is of the general form x = ln(a+x), where x = y(1−p), and a is always greater than 1. Consider the range of possible values of x, from 0 to ∞. At x = 0, the left hand side of the preceding equation is always smaller than the right hand side (lna). As x→∞, ln(a+x)≈ln(x), and thus the right hand side of the equation is always smaller than the left hand side. Since both x and ln(a+x) are continuous functions, these observations mean that there always exists an intersection point of the functions x and ln(a+x), defining maxy. Therefore, maxy exists for all values of α and γ. Differentiation of ε twice with respect to y shows that maxy, guaranteeing that maxy indeed represent a global maximum of ε.Does maxy into equation (18) yields εmax for given values of α and γ. max for a range of values of α and γ and for different values of p. As expected, the smaller the values of α and γ, the larger is ε, and the more efficient the screen.F1 animal per plate, the Poisson approximation cannot be used (see above), and instead we use equation (16) to write the efficiency of a screen as:For genetic screens involving plating one maxy:As above, we differentiate equation (20) to obtain the following transcendental equation for y. However, the value of y obtained here cannot be used directly to compute maxy or to calculate εmax, for two reasons. First, unlike the previous section, y cannot be treated as a continuous variable here, and the maximum calculated in equation (21) makes this assumption. Second, y can only take on integer values ≥1. Thus, to identify maxy, we numerically calculate the solution to equation (21). We then check whether the obtained value of y is smaller than 1. If so, then maxy = 1. If not, we calculate the efficiency of screening, using equation (20) for the two nearest integer values of y, and choose maxy as the value giving the highest value of ε. The results of these calculations for εmax and maxy are presented in Similar reasoning to that of the previous section guarantees the existence of a solution for W, to the minimal work, minW, calculated using equations (17), (18), and (19), as a function of m/n, for a screen in which scoring and picking F2 animals requires ten times more work than picking F1 animals (α/γ = 10). Such parameters are often encountered, and may serve as a model for screens in which F2 animals need to be examined individually on a compound microscope, for example. As the figure demonstrates, relatively small deviations in the screening ratio can have a large impact on the amount of work carried out to screen the same number of mutagenized F1 animals. For example, screening five F2 animals for every F1 parent for recessive mutations nearly doubles the work required to screen the same number of mutagneized F1 animals compared to the optimal screening ratio of 0.86. The differences are even more dramatic when dominant mutations are sought.In C. elegans.These results clearly show that using optimized screen parameters can have a significant impact on the progress and output of genetic screens in C. elegans. We demonstrate two key points. First, an optimal screening strategy always exists for every genetic screen of the types considered here. Second, calculation of this optimal strategy is possible. F2-to-F1 screening ratios for a large range of screen parameters. As shown in F1 animals and picking and scoring F2 animals; and when the mutations sought manifest themselves in the F2 population in increased proportion ), that is valid for a large number of genetic screens. Fourth, we demonstrate that two limiting cases of this equation, in which a large number of F1 animals are placed on a small number of plates, or in which F1 animals are plated individually, yield simplified equations (equation (10), based on the Poisson approximation, and equation (16)), that are well known and of considerable practical use. Finally, analysis of these limiting cases also reveals that the number of mutagenized F1 animals examined is dependent on the mode in which F1 animals are plated. In general, we show that plating F1 animals individually, followed by scoring their F2 progeny, allows more mutagenized F1 animals to be examined than plating the same number of F1 animals on a single or small number of plates. However, it should be noted that because plating F1 animals individually can be more time consuming and may require more reagents, the overall screen efficiency may or may not be higher using this strategy (see below).In addition to the key results discussed above, we have also derived a number of other useful results. First, we have shown that the optimal screening strategy does not depend on the total amount of effort expended in a screen, but only depends on the ratio of the work involved in picking C. elegans can be divided into three general categories, based on the difficulties involved in picking F1 animals  and (16), can be used to determine actn and follow screen progress.Picking F1 animals is much harder than picking F2 animals (α/γ→0). As shown in F2 animals should be scored for each F1. As we showed in the beginning section of the paper, under such conditions actn≈n, and screen progress is limited by the number of F1 animals that can be examined.Picking F2 animals is much more difficult than picking F1 animals (α/γ→∞). F1 animals picked should be much greater than the number of F2 animals scored. Under these conditions, actn≈m(1−p), and progress is determined by the type of mutation being sought , and by the number of F2 animals screened.Picking/scoring actn) is very simple and does not depend on plating strategy. Screens of type I, however, require a more detailed study of the screen and plating parameters.The merits of the classification system described above are that it allows a quick determination of whether an in-depth analysis of screen parameters is required to follow screen progress. Specifically, for screens of types II and III, calculating screen progress . The table illustrates a number of points. First, it provides estimates of the amount of work expended for each screen. Although the table provides exact numbers, it is important to note that these numbers only approximate the actual work involved because some of the parameters used in calculating the work, such as α and γ, may not be exact. Second, it provides specific applications of the different screen strategies to common screens undertaken in C. elegans. Third, the table demonstrates that strategies that minimize both α and γ are, as expected, most efficient , of screens of type II approaches 1/γ, whereas the efficiency of screens of type III approaches (1−p)/α. Thus, if one is debating between screening strategies of type II and III where γ(type II)≈α(type III), screens of type II will always be more efficient.Interestingly, examination of F2-to-F1 screening ratio. Indeed, as equations (19) and (21) show, only the ratio of α to γ is relevant. It turns out, however, that even an exact measurement of this ratio is not needed in practice. The reason for this is shown in F2-to-F1 screening ratio by two orders of magnitude . Thus, the screening ratio is not very sensitive to variations in α and γ. This observation is of clear practical importance, since it is not always easy to precisely compare the work involved in picking F1 animals and picking and scoring F2 animals. These results suggest that order-of-magnitude estimates of the relative work involved will give good estimates of the appropriate screening ratio.As we describe in the Results section, precise values of α and γ are not important for calculating the optimal F1 animals and pick and score a fixed number of F2 animals. Each work segment is then divided, respectively, by either the number of F1 or F2 animals to obtain γ and α, respectively. Values for these parameters can also be adjusted as a screen proceeds, based on estimates derived from earlier stages of screening.In C. elegans can follow a simple set of rules. In F1 animals will be plated onto individual plates  or onto a small number of plates (bulk). This choice is usually not driven by efficiency, but by constraints of the screen. For example, screens that require a clonal strategy, because the identity of the F1 parent is important , demand plating F1 animals individually.Our results suggest that the design of many types of genetic screens in F1 parents, and for which F2 animals must be picked to individual plates for scoring , and these are used to identify the optimal F2-to-F1 screening ratio ( = m/n)  . From thhttp://b5.rockefeller.edu/cgi-bin/labheads/shaham/genetic_screens/screenfrontpage.cgi”.To aid with the calculations described here, in C. elegans, and calculated optimal F2-to-F1 screening ratios for a large range of screen parameters. Calculation of these parameters was aided by obtaining a general expression for counting the number of mutagenized gametes examined in the course of a genetic screen. The strategies described here can, in principle, be applied, with relevant modifications, to the evaluation of equivalent parameters in genetic screens in other organisms.We have presented a systematic approach for optimizing genetic screens in m and n,m and n, we simplify the expression above toIn this paper, we employ expressions of the following formf = [(actn/N)exact−(actn/N)Poisson]/(actn/N)exact. Using equations 10 and 16, the fractional error is written asy approaches 1, this expression, therefore becomesThe fractional error in using the Poisson approximation is defined as


The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning , object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests. Varicellovirus of the subfamily AlphaherpesvirinaePseudorabies virus is classified into the genus Pseudorabies virus expresses a single immediate-early protein, IE180, consisting of 1460 amino acid residues Based on these findings, we had hypothesized that expression of pseudorabies IE180 would cause the developmental neurological abnormalities in host animals without viral infection and replication. In fact, we previously found that transgenic expression of IE180 in two months old mice caused severe ataxia and cerebellar defects, such as size reduction and disorganized lamination, without any abnormality in other parts of the brain such as hippocampus and cerebral cortex Accordingly, we used TgIE96 mice here as an experimental model to study the involvement of cerebellar circuits in different learning tasks. Classical conditioning of eyelid responses was carried out in wild-type and TgIE96 mice, using both trace and delay paradigms ad libitum access to commercial mice chow and water. All the experiments were carried out during the light cycle and according to EU (2003/65/CE) and Spanish  guidelines for the use of laboratory animals for chronic behavioral experiments. All experimental protocols were also approved by the Ethics Committee of the Pablo de Olavide University (07/4-20/12/2008).Experiments were carried out on male TgIE96 mice and on wild-type littermates having a C57BL/6 genetic background, obtained from the Laboratory of Biomedicine Center of Biomedical Research, Kyushu University . Animals were three months old upon their arrival at the Animal House of the Pablo de Olavide University , and were kept on a 12-h light/dark cycle with constant temperature (21±1°C) and humidity (50±7%). Animals were allowed Under deep anesthesia , animals were implanted with four electrodes in the upper eyelid of the left eye. Electrodes were made of Teflon-insulated, annealed stainless steel wire . One pair of electrodes was aimed at the supraorbitary branch of the trigeminal nerve, and served for the presentation of electrical stimuli. The second pair of electrodes was implanted in the ipsilateral orbicularis oculi muscle, and served for recording its electromyographic (EMG) activity. The four electrodes were connected to a 4-pin socket  which was fixed with dental cement to the cranial bone. For a week after surgery, animals were kept in independent cages, with free access to food and water, for a proper recovery before the beginning of the experiment. They were maintained in individual cages for the rest of the experiment.For classical conditioning of eyelid responses, animals were placed individually in a (5 cm ×15 cm ×15 cm) plastic chamber, inside a larger Faraday box (30 cm ×30 cm ×20 cm) to eliminate electrical interferences. Both trace and delay conditioning paradigms were carried out . For this, animals were presented with a tone  as a conditioned stimulus (CS), followed 250 ms from CS onset by an electrical stimulation  as an unconditioned stimulus (US). Intervals between paired CS-US presentations were separated at random by 30±5 s. For habituation and extinction sessions, the CS was presented alone, also at intervals of 30±5 s. A total of two habituation, five conditioning, and four extinction sessions  were presented to each animal across eleven successive days. Only the first fifty trials of each session were analyzed. An additional group of wild-type animals (n = 8) were pseudoconditioned to test the reliability of the task. For pseudoconditioning, unpaired CS and US presentations were carried out for 5 sessions . Pseudoconditioned animals also received two habituation and five extinction sessions as indicated above The EMG activity of the orbicularis oculi muscle was recorded using differential amplifiers with a bandwidth of 1 Hz to 10 kHz . Data were stored directly on a computer through an analog/digital converter , at a sampling frequency of 11–22 kHz and an amplitude resolution of 12 bits. Data were analyzed off-line for quantification of conditioned responses (CRs) with the help of the Signal Average Program . We considered a response to be conditioned when the rectified EMG activity, during the CS-US period, presented the following conditions: i) the EMG activity lasted >10 ms; ii) the EMG was not preceded by any spontaneous activity in the 200 ms preceding CS presentation; iii) the EMG activity was initiated >50 ms after CS onset; and iv) the integrated EMG activity was at least 2.5 times greater than the activity recorded 200 ms before CS presentation For the object recognition task, all the animals (n = 10 per group) were individually habituated to an open field (40 cm ×25 cm ×15 cm), under low illumination conditions, and with no objects, for 5 min. During the training session, two unknown but identical objects (O1 and O2) were placed into the open field, and the animals were allowed to explore them freely for 10 min. The time spent exploring each object and the total approach time were quantified. After each trial, the apparatus and the objects were thoroughly cleaned with 70% ethanol to avoid odor recognition. One hour after the first training, mice were allowed to explore the open field for another 10 min, when one of the two familiar objects (O1 or O2) was replaced by an identical object (O3), and the other (O1 or O2) by a novel object (B1). The time spent exploring each object and the total approach time were quantified again. Within each experimental group, the object O1 was replaced by the new object for half of the animals, whereas object O2 was changed for the other half. The aim was to avoid any issue related to spatial preference associated, or not, with the location of the two objects. Twenty-four hours after the initial training, mice were tested again, with a new object (C1) and an object identical to the old one (B2). The same procedure was carried out 72 h after the initial training  unless the animal reached the platform in a shorter time. The time taken by the animal to reach the hidden platform was quantified. If an animal failed to find the platform within 60 s, the test was ended and the animal was helped to reach the platform by hand. In either case (whether it reached the platform by itself or not), the mouse was maintained on the platform for 30 additional seconds. The time spent on reaching the platform was recorded on-line, and its percentage with respect to the time limit (60 s) was computed.The different parameters of the startle reflex and the prepulse inhibition tests were assessed in both wild-type and TgIE96 mice (n = 10 wild-type and 9 TgIE96). Animals were placed individually inside a startle chamber . The startle response was measured using a piezoelectric accelerometer controlled by a computer, using the protocol described elsewhere Experiments were carried out in a passive avoidance device . In accordance with procedures described elsewhere Data collected from classical conditioning experiments were quantified, through a purpose-designed Excel worksheet, as the percentage of CRs per session – i.e., the proportion of paired CS-US stimulations within a session of 50 presentations that generated an EMG activity satisfying the above-mentioned criteria post hoc test was performed when necessary For data collected from object recognition, water maze, prepulse inhibition, and passive avoidance tasks, we compared the statistical differences between groups, using the two-way repeated measures ANOVA test, performed with the same SPSS package. In addition, the Bonferroni P ≤ 0.814, one-way ANOVA) were observed between reflexively evoked blink responses in the two groups of animals, it was possible to use the classical conditioning of eyeblink responses to test their learning capabilities. Although systematic differences in reflexively evoked eyelid responses would not preclude the possibility of examining whether blink responses may be acquired in an associative task, putative differences in the acquisition process between both groups of animals could be ascribed to the learning process and not to any performance deficit.In a preliminary series of experiments, we checked whether the neural premotor circuits involved in the generation of eyelid responses functioned normally in both wild-type and TgIE96 mice. The blink reflex can be characterized by measuring the latency of its early (R1) and late (R2) components  = 0.60; P = 0.44]. In contrast, the two-way ANOVA applied to the conditioning data showed a significant difference in the evolution of the percentage of CRs for the two experimental groups with respect to values collected during the habitation sessions . As shown by the Bonferroni post hoc test, the wild-type group presented values significantly greater (P ≤ 0.01) than those collected during the habituation sessions from the 3rd to the 5th conditioning sessions, whilst the TgIE96 group presented percentages of CRs significantly (P ≤ 0.01) different from habituation values from the 2nd to the 5th conditioning sessions. During the extinction process, animals presented a decrease in the percentage of CRs, reaching 20.6±5.8% for the wild-type group, and 18.2±4% for the TgIE96 group by the 4th extinction session. No significant statistical difference could be detected between the two groups during the extinction sessions , suggesting a similar evolution of the percentage of CRs during the extinction process. Moreover, for both experimental groups no statistical difference was found between CR values collected during the 4th extinction session and those corresponding to the habituation session .During conditioning sessions using a trace paradigm A, C, co = 1.62; P = 0.22]. The two-way ANOVA applied to the conditioning data showed a significant difference in the percentage of CRs for the two experimental groups with respect to values collected during the habituation sessions . As shown by the Bonferroni post hoc test, the wild-type group presented values significantly higher (P ≤ 0.01) than those collected during the habituation sessions from the 2nd to the 5th conditioning sessions, whilst the TgIE96 group presented percentages of CRs significantly (P ≤ 0.01) different from habituation values from the 1st to the 5th conditioning sessions. During the extinction process, animals presented a decrease in the percentage of CRs, reaching 53.4±8.8% for the wild-type group and 47.6±8.4% for the TgIE96 group by the 4th extinction session. No statistical difference could be detected between the two groups during extinction sessions , suggesting a similar decay in the percentage of CRs during the extinction process. In addition, no statistical difference was found between CR values collected during the 4th extinction session and those of the habituation session for the two experimental groups .For the delay paradigm B, D, wi = 1.12; P = 0.35; data not illustrated]. This result confirms the reliability of the conditioning protocols used in the present study.Finally, the percentage of CRs for the pseudoconditioned animals (n = 8) did not present differences between habituation, conditioning, or extinction sessions  and TgIE96 animals .In another series of experiments, we compared the learning capabilities of wild-type and TgIE96 animals for object recognition. As illustrated in  = 15.5; P<0.001] and TgIE96  animals presented a significant increase in the percentage of attention devoted to the novel object  = 15.8; P<0.001] and TgIE96 animals .During the first choice trial, 1 h after the initial training session, mice were allowed to explore a novel object (B1) and a familiar one (O3). In this case, both wild-type .During the last choice trial, 72 h after the training session , mice weIn summary, and as already described for classical eyeblink conditioning, TgIE96 mice performed the object recognition task with the same efficiency as the control group (or ― during the 72 h session ― even better). = 96.5; P<0.001] shorter times than the TgIE96 group. For example, during the first training day, the control group reached the hidden platform at an escape latency (62.3±7.1%) significantly (P<0.001) shorter than that for the TgIE96 group (95.6±2.9%). Results collected during the following two tests showed that while wild-type animals improved their performance across training (P<0.001 when comparing the 1st vs. the 3rd session), the TgIE96 group did not improve their performance (P = 0.23). Taken together, these results indicate that TgIE96 animals presented evident spatial learning and memory deficits as compared with their littermate controls.To determine spatial learning and memory abilities of the two groups of animals included in this study we used a modified version of the Morris water maze test. Animals had to find a hidden platform with the help of the visual cues provided. The task consisted of three sessions (three days) each of four trials, separated by 20 min, with each trial lasting a maximum of 60 s. The score obtained in each session was the average of the four scores for each animal. Data were expressed as the percentage (%) of the time limit (60 s). As shown in In accordance with previous descriptions  = 1.43; P = 0.24] and peak  latencies between wild-type and TgIE96 groups. Nevertheless, the index for maximum peak response was significantly  greater for wild-type (performance index = 27.5±5) than for TgIE96 (index = 9.3±2) animals. Moreover, there were significant  differences between the two groups with regard to the index for total response area (indexes = 32.4±6.3 for wild-type and 14.7±4.6 for TgIE96 mice). Thus, TgIE96 mice presented some functional deficit in the elaboration of prepulse inhibition when compared with the performance of wild-type animals.For the sake of homogeneity, and as explained in the  = 8.5; P<0.05]. As shown in  = 2.7; P = 0.1] were observed between the two groups. Therefore, it can be concluded that in the passive avoidance test, TgIE96 animals present a significant memory deficit as compared with their wild-type littermates.We determined the putative differences in the passive avoidance test for the two experimental groups by measuring the time taken by a mouse to enter the dark compartment after door opening . For theAccording to the present results, TgIE96 mice have marked deficits in spatial learning  as shown by their poor performance in the water maze test. In contrast, they performed the object recognition test ― another task involving the participation of hippocampal circuits ― similarly to controls. Moreover, they also presented significant deficits in two tests (prepulse inhibition and passive avoidance) more directly related with selected cerebral  cortical and subcortical  structures. As already reported for Lurcher mice pcd (Purkinje cell degeneration) mice pcd mice also present affected inferior olivary neurons and some other neuronal groups located far from the cerebellum , the reported deficits cannot be confidently ascribed to cerebellar circuits. As shown in a recent study using classical conditioning of eyelid responses and sumultaneous recordings of multiunitary activity in the red nucleus and in the cerebellar interpositus nucleus The classical conditioning of eyelid responses allowed us to test the effects of cerebellar damage on the acquisition of this type of associative learning. Neural circuits involved in the generation of reflexively evoked eyeblinks were functionally active in TgIE96 animals generating both components (R1 and R2) of the reflex response following electrical stimulation of the supraorbitary nerve As already described in Lurcher mice The involvement of cerebellar circuits in object recognition tasks has been shown using functional MRI The presence of noticeable deficits in the proper execution of water maze, prepulse inhibition, and passive avoidance tasks in TgIE96 animals raises interesting questions regarding cerebellar contributions to those different learning tasks. Indeed, these three learning tests involve the participation of many different cortical  and subcortical  structures TgIE96 animals presented a noticeable learning and memory deficit for acquisition and retention of the Morris water maze task, a fact that cannot be ascribed to their ataxic syndrome, since they were capable of swimming like their littermate controls, as also observed in other cerebellar-deficient mice Although the startle response to a severe tone Neural mechanisms involved in emotional reactivity, such as those mediating passive avoidance tasks, are classically assumed to take place in amygdalar circuits Finally, it can be indicated that in order to properly determine the contribution of cerebellar structures not only to the improvement of motor skills, but also to the acquisition of new motor and cognitive abilities, it will be necessary to use many different associative and non-associative learning tasks as shown by the present study. In addition, it has been recently reported that the cerebellum contributed to the ongoing functional states


Bovine tuberculosis (BTB) today primarily affects developing countries. In Africa, the disease is present essentially on the whole continent; however, little accurate information on its distribution and prevalence is available. Also, attempts to evaluate diagnostic tests for BTB in naturally infected cattle are scarce and mostly complicated by the absence of knowledge of the true disease status of the tested animals. However, diagnostic test evaluation in a given setting is a prerequisite for the implementation of local surveillance schemes and control measures.Mycobacterium bovis.We subjected a slaughterhouse population of 954 Chadian cattle to single intra-dermal comparative cervical tuberculin (SICCT) testing and two recently developed fluorescence polarization assays (FPA). Using a Bayesian modeling approach we computed the receiver operating characteristic (ROC) curve of each diagnostic test, the true disease prevalence in the sampled population and the disease status of all sampled animals in the absence of knowledge of the true disease status of the sampled animals. In our Chadian setting, SICCT performed better if the cut-off for positive test interpretation was lowered from >4 mm (OIE standard cut-off) to >2 mm. Using this cut-off, SICCT showed a sensitivity and specificity of 66% and 89%, respectively. Both FPA tests showed sensitivities below 50% but specificities above 90%. The true disease prevalence was estimated at 8%. Altogether, 11% of the sampled animals showed gross visible tuberculous lesions. However, modeling of the BTB disease status of the sampled animals indicated that 72% of the suspected tuberculosis lesions detected during standard meat inspections were due to other pathogens than Our results have important implications for BTB diagnosis in a high incidence sub-Saharan African setting and demonstrate the practicability of our Bayesian approach for diagnostic test evaluation. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB) and belongs to the Mycobacterium tuberculosis complex (MTBC) of bacteria M. bovis infections are of public health concern due to the pathogen's zoonotic potential M. bovisBTB control and surveillance is scarce in sub-Saharan Africa and mostly limited to abattoir meat inspections. However, the performance of meat inspection is rather poor and depends on the disease stage in which infected animals reside, the accuracy of the carcass examination and the presence of other lesion causing pathogens M. bovis antibodies has been described recently Current ante mortem diagnosis of BTB mainly relies on the single intra-dermal comparative cervical tuberculin (SICCT) test, which, although imperfect, could not yet be replaced by any other more accurate diagnostic method Attempts to evaluate diagnostic tests for BTB in naturally infected cattle in Africa are scarce but a prerequisite for the implementation of surveillance schemes and control measures. Gobena et al. have used detailed post mortem examination to define the BTB disease status of Ethiopian cattle for the evaluation of SICCT in this setting Choi et al. A total number of 954 sequentially selected slaughter animals from Southern Chad were subjected to multiple tests for the diagnosis of BTB. Three ante-mortem tests with continuous numerical outcome values (continuous outcome) were used, namely, SICCT and two recently developed FPA tests termed SENTRY 100 and GENios Pro Before slaughter, blood samples were collected and animals underwent SICCT testing. Altogether, 8% (CI: 6%–10%) of the animals tested, reacted positively to SICCT when the official OIE cut-off >4 mm;  was usedBased on the same data, we have previously reported the evaluation of SICCT, SENTRY 100 and GENios Pro using a subset of animals with either PCR confirmed MTBC infections or no visible lesions Model estimates for tests with binary outcome were highly sensitive to the priors. We therefore decided to consider solely tests with continuous outcome for Bayesian modeling .Based on the estimates for the means and variance-covariances of SICCT, SENTRY 100 and GENios Pro test results for the diseased and non-diseased animals in model 2A see , ROC curM. bovis infected animals  and false positive (CFP) diagnosis, which we were not able to accurately do. However, the cost of a false negative diagnosis is likely to exceed the cost of a false positive result by several folds as disease transmission amplifies the total economical losses due to BTB. We found that, assuming a disease prevalence of 8.4% (10.0%), a cut-off >2 mm would be ideal if CFN/CFP lies between 8 and 16 (7 and 13). This suggests that our chosen cut-off values may be acceptable for a broad range of reasonable CFN/CFP ratios.Our results indicated that the most appropriate cut-off for positive SICCT test interpretation was significantly lower then the OIE suggested standard cut-off (>2 mm versus >4 mm). However, our criteria for cut-off selection attributed equal weights to sensitivity and specificity and did not consider the disease prevalence and the cost of misclassifications. As an alternative approach for cut-off selection, the misclassification-cost term (MCT) can be calculated for each point of the ROC curve. The point with the lowest MCT value would then be most appropriate for positive test interpretation A cut-off >2 mm was also found to be most appropriate for positive SICCT test interpretation in a recent study in Ethiopia M. bovis infection and visible lesions (N = 30), 9 or 19 (30% or 63%) did not show a positive reaction to SICCT depending on whether a cut-off >2 mm or >4 mm was applied, respectively. This indicates a considerable proportion of SICCT anergic animals . Unfortunately, our sample size was too small to conclusively assess the ability of the FPA tests to detect such animals.SICCT showed a relatively low sensitivity irrespective of whether our suggested or the OIE cut-off was used . ComparaM. bovis. For 72% of the animals in which lesions have been detected, no M. bovis infection was modeled. This finding was in line with the relatively low amount of MTBC strains detected in animals with lesions . This observation could suggest that Mbororo breeds in fact, are not more likely to be infected with M. bovis but more often develop advanced stages of the disease. Host genetic factors as well as environmental factors or animal husbandry could account for this observation.In a previous study on BTB in Chadian cattle we have reported that the prevalence of BTB was significantly higher in Mbororo zebus than in Arab zebus th breed . NeverthM. bovis.In summary, the present study shows the practicability of a Bayesian method for the evaluation of multiple tests for the diagnosis of BTB in naturally infected cattle and in absence of knowledge of the true disease status of the animals. Our model allowed us to compute the disease status of each sampled animal and the modeling results supported our previous observation that the cut-off for positive SICCT interpretation should be lowered to >2 mm in many countries of sub-Saharan Africa. Moreover, we provide evidence that an unexpectedly high proportion of BTB suspect lesions detected during slaughterhouse meat inspection was due to other pathogens than The animal population subjected to this study has previously been described, in detail All 954 animals were physically examined before slaughter. Body condition was assessed by assigning one of the following three scores: 1 – good body condition, 2 – bad body condition, 3 – very bad body condition Valid SICCT testing results were available for 930 animals. SICCT testing and reading was carried out as explained previously and according to standard protocols Valid SENTRY 100 and GENios Pro FPA results were available for 953 and 954 animals, respectively. The methods have been previously described in detail After slaughter, all 954 animals underwent meat inspection, which included organ and lymph node palpation, visual inspection and incision of organs and lymph nodes according to standard procedures Specimens from all 108 animals with lesions were subjected to direct microscopy and processed as previously described Specimens of lesions from altogether 102 animals were subjected to culture and microscopy. Decontaminated samples were inoculated into two Middlebrook 7H9 medium flasks containing OADC and PANTA and either glycerol (0.75%) or pyruvate (0.6%) AFB containing cultures from 50 animals were subjected to molecular characterization. Heat inactivation of the cultures was carried out as previously explained M. bovis infection status of all sampled animals. The model combined the results of the continuous as well as the binary diagnostic tests for BTB , applied to the same animal population without considering a gold standard. It also allowed the estimation of the true disease prevalence in the sampled population as well as the sensitivities and specificities of the diagnostic tests. A mathematical description of the model and the WinBUGS code are provided in A Bayesian model was developed to estimate the true M. bovis infection status were identified by univariate and multiple logistic regression analysis in Stata (Stata/IC v10.0). Association between lesion localisation and modeled M. bovis infection was assessed by the Fisher's exact test in Stata.Risk factors for the modeled M. bovis infected animals  and tried to directly model their sensitivities and specificities . The mathematical description of the model and the WinBUGS code are shown in We assumed that the distribution of the test values of SICCT, SENTRY 100 and GENios Pro was trivarite normal with means and variance-covariances separately estimated for the diseased and the non-diseased animals. The normality assumption was verified via the shape of the histogram of the test values. In an initial model we have included the data from the multiple post mortem tests for the detection of k , respectively; k, respectively.From the estimates of the means and variance-covariances of the multivariate normally distributed continuous test values of SICCT, SENTRY 100 and GENios Pro for the diseased and non-diseased animals, a ROC curve was calculated in Stata. Pairs of 1-specificity and sensitivity were calculated and plotted for all possible cut-off points according to the following formula:FN/CFP) P (1−Se)+(1−P) (1−Sp)] was chosen, with CFN and CFP being the cost of false negative and false positive diagnosis, respectively and P being the disease prevalence in the target population FN/CFP but the cost of false-negative diagnosis is likely to exceed the cost of false positive diagnosis. Therefore, MCT values for each possible cut-off point and different ratios of CFN/CFP were calculated and compared, assuming a disease prevalence of 8.4%, as estimated by our model. In addition, MCT values for different CFN/CFP ratios were calculated for a 10.0% disease prevalence.We considered the point of the ROC plot with the greatest distance from the diagonal line (sensitivity = 1−specificity) as the best cut-off; this corresponds to the point with the largest Youden index (J = sensitivity+specificity−1) Table S1d: Mean diagnostic value for non-diseased (d = 0) and diseased (d = 1) animals, respectively. τd: precision of the diagnostic values for non-diseased (d = 0) and diseased (d = 1) animals, respectively. mS/σS, mC/σC, mπ/σπ : Mean and standard deviation of the test sensitivity, specificity and true disease prevalence, respectively. The normal distribution is parametrized in terms of mean and variance. The Gamma distribution is parametrized in a non-conventional way in terms of mean and variance instead of the shape and scale parameters. Model-based estimates correspond to the posterior mean and standard deviation in brackets.Priors and model estimates for different parameters. μ(0.04 MB XLS)Click here for additional data file.Text S1Mathematical model description(0.12 MB DOC)Click here for additional data file.Text S2WinBUGS code(0.03 MB DOC)Click here for additional data file.


Ureaplasmas and urogenital tract disease in humans. Since healthy humans can be colonized with Ureaplasmas, its role as a pathogen remains controversial. In order to begin to define the role of the host in disease, we developed a rodent model of urinary tract infection (UTI) using Fischer 344 (F344) rats. Animals were inoculated with sterile broth, 101, 103, 105, 107, or 109 log CFU of a rat-adapted strain of Ureaplasma parvum.Epidemiologic studies show a strong association between U. parvum affected the incidence of UTI, and 50% to 57% of animals inoculated with ≥ 107 CFU of U. parvum remained infected (p < 0.04). However, inoculum dose did not influence immune response to U. parvum. Asymptomatic UTI was characterized by a minimal immune response that was predominantly monocytic and lymphocytic, with limited lesions, and elevated urinary levels of IFN-γ, IL-18 and MCP-1 (P ≤ 0.02). UTI complicated with struvite formation was characterized by an exaggerated immune response that was mostly neutrophilic (P ≤ 0.0001), with lesions that showed extensive uroepithelial hyperplasia (P ≤ 0.0001), and a predominance of IL-1α, IL-1β, and GRO/KC in the urine (P ≤ 0.02). Animals with asymptomatic UTI also had a significantly high rate of kidney infection (P ≤ 0.0005).Infected animals exhibited two distinct profiles, asymptomatic UTI and UTI complicated with struvite urolithiasis. Inoculum dose of U. parvum infection are primarily dependent upon host-specific factors rather than Ureaplasma microbial load. The immune response in F344 rats is similar to that which occurs in humans with ureaplasmal associated disease. Therefore, this model of infection is a useful tool for elucidating U. parvum-host interactions that confer UTI and disease.Complications associated with  Ureaplasma species are among the most common isolates from the human urogenital tract . All animals within the Struvite group had bladder stones, which were composed of 89 to 95% magnesium ammonium phosphate (struvite), 1 to 5% calcium phosphate (carbonate apatite) and 3 to 10% protein and blood. No animal had macroscopic evidence of kidney stones at time of necropsy. Although not statistically significant (P ≤ 0.11), animals inoculated with 107 or greater CFU tended to have the highest frequency of struvite uroliths.Animals inoculated with us Table . The NegU. parvum did not impact the distribution of animals that developed either UTI or struvite uroliths . However, there was a significant difference (P ≤ 0.006) in the inflammatory cell type score and inoculating dose of U. parvum . There also was no correlation between the total area affected score and clinical profile , additional file Scoring of kidney tissues on the basis of total area affected did not reveal any patterns that could be correlated to local/active infection or clinical profile. Specifically, there was no correlation between the total area affected score and U. parvum were examined by Spearman Correlation analysis. There were no correlations between urine cytokine levels and degree of inflammation score among animals inoculated with sterile 10B broth (data not shown). However, there was a significant correlation . Control animals had significantly higher levels of MCP-1 than animals in either the Negative or UTI groups Figure . AnimalsIn order to identify distinctive chemokine/cytokine patterns between clinical profiles associated with active infection, samples from animals within the Negative group were excluded from this analysis. Each urine cytokine multiplex from each animal was normalized prior to analysis by one-way ANOVA using row by row modeling and Fischer C correction for multiple comparisons. Figure U. parvum (P ≤ 0.05). Figure Urine cytokine data from control and culture negative animals was normalized prior to statistical analysis and analyzed as described above. There was a significant difference in the overall pattern of IL-2, IL-4, IL-10, TNF-α, IFN-γ, and GRO/KC in the urine of culture negative animals that received different inoculating doses of Ureaplasmas is confounded by the isolation of the microbe from the lower urogenital tract of normal, asymptomatic individuals. In addition, the severity of disease for most mycoplasmal infections depends on the host immune response. Therefore, experimental infections in genetically defined animal models will be critical to unraveling the key interactions in the host/parasite relationship that contribute to disease severity. By using a combined approach involving histopathology and cytokine profiling, we were able to further characterize the immune response associated with asymptomatic UTI and UTI complicated with struvite formation.Ureaplasmas are an underappreciated pathogen of the urogenital tract. Despite strong epidemiological evidence and even experimental infections in humans that fulfilled Koch's postulates , the etiU. parvum [U. parvum significantly affected the frequency of animals that remained colonized two weeks after inoculation. Therefore, the initial microbial load is important in establishing infection. However, once infected, the proportion of animals that developed complicated UTI in response to varying inocula of U. parvum did not show a definitive dose response effect. More importantly, the immune response to infection in this group of animals with complicated UTI was consistent, regardless of initial inoculating dose. For example, the cytokine profile and urinary tract pathology of a struvite positive animal that was inoculated with 101 CFU was indistinguishable from a struvite positive animal that was inoculated with 109 CFU. This suggests that the initial microbial load of U. parvum is not a critical factor in the development of complicated UTI in this rat strain. Further, it supports the concept that, once infection is established, the host inflammatory response is a key determinant of lesion severity in the urinary tract.The F344 rat strain is highly susceptible to development of complicated UTI following experimental inoculation with . parvum . In thisU. parvum, and 65% exhibited an ascending infection into the kidneys. Further, the microbial load of U. parvum in animals with asymptomatic UTI was equivalent to animals in the Struvite group. Another intriguing feature in animals with asymptomatic UTI was the overall lack of uroepithelial proliferation that was present in varying degrees in the Negative group as well as the Struvite group. A primary defense mechanism of uroepithelium exposed to bacteria involves desquamation, necrosis or apoptosis followed by proliferation [U. parvum.As previously reported , animalsferation . TherefoF344 rats with struvite uroliths had a similar clinical profile to what we have previously described . SpecifiU. parvum infected animals. Therefore, these cytokines may be critical in directing a more efficient immune response that leads to bacterial clearance with minimal tissue damage. The second cytokine cluster in the negative group included IFN-γ and GRO/KC, which are significant cytokines in the UTI and Struvite groups, respectively. Except for animals that were inoculated with log 9 CFU of U. parvum, the expression pattern of IFN-γ and GRO/KC was not inversely related as they were in culture positive animals. This may be reflecting a more balanced immune response than what is seen in U. parvum positive animals, and we suggest that this balanced response may be critical to resolution of infection and prevention of severe disease.The immune response of animals within the negative group was highly variable and most likely comprises a mixed population of rats, including animals that never became colonized as well as animals that cleared the infection at various time points post-inoculation. Therefore, interpretation of data from this group of animals is difficult and is done with caution. In spite of this limitation, profiling urine cytokine data and bladder lesion scores by inoculum dose was informative. The threshold dose for successful colonization appears to be between log 5 and log 7 CFU, since 64% and 43% of rats respectively were culture negative 2 weeks post-inoculation. The animals within the log 5 and log 7 CFU inoculation groups also had the greatest flux in both pro-inflammatory  and anti-inflammatory  cytokines. Interestingly, pattern analysis of urine cytokine data showed two distinct clusters. The first cluster identified in the negative group included IL-2, IL-4, IL-10, and TNF-α; these cytokines were notable as they were not part of the cytokine cluster groupings of U. parvum in the F344 rat is an interesting phenomenon since this is an inbred strain. In this study, both genetic and environmental influences on disease were minimized to the extent possible. All of the animals in this study originated from the same colony. Further, rats were housed under the same barrier maintained conditions in order to minimize environmental variability. Despite our efforts, it was common to find that a rat that developed asymptomatic UTI had co-habited the same cage with a rat that developed struvites or was culture negative at time of necropsy. Therefore, external environment could not account for the varying clinical outcome in our study. However, our experimental inoculation procedure may be a critical source of variability. Although attempts were made to reduce mechanical trauma caused by catherization, it is possible that the trauma may have been sufficient to shift the immune response towards a pro-inflammatory profile in a subset of animals. Once this occurred, the pro-inflammatory cycle progressed until infection was resolved (Negative group) or the study was terminated (Struvite group). Another possible explanation for our findings may involve the actual placement of ureaplasmas within the urinary tract at time of inoculation. For example, if the catheter disrupted the uroepithelial barrier so that a sufficient number of ureaplasmas were deposited into the submucosa instead of the mucosal surface, this could elicit a different inflammatory response cascade than what would normally occur if the microorganisms were only present on the mucosal surface of the bladder. The results of this study were similar to what we have previously reported, thus showing the consistency and reproducibility of this model of infection. Moreover, the clinical outcome to ureaplasmal infection in the F344 rat is similar to what occurs in humans. The complex interactions between most mucosal pathogens and the host that lead to uncomplicated colonization versus inflammation and disease are largely unknown. Therefore, this model may be particularly useful for identifying the molecular events that confer asymptomatic infection, complicated infection as well as resolution of infection with an opportunistic pathogen of the urogenital tract.The variable clinical outcome to experimental inoculation with Ureaplasma and the host that lead to uncomplicated colonization versus inflammation and disease are largely unknown. We characterized the F344 rat immune response in the urinary tract to varying inoculum concentrations of U. parvum. Establishment of UTI was influenced by microbial load, but the host immune response was independent of microbial load. Two distinct innate immune profiles were identified with two different clinical outcomes: asymptomatic UTI and complicated UTI with struvite formation. Asymptomatic UTI was characterized by a minimal immune response that was predominantly monocytic and lymphocytic and was accompanied by a significantly high rate of kidney infection. UTI complicated with struvite formation was characterized by an exaggerated immune response that was predominantly neutrophilic and was accompanied by uroepithelial hyperplasia and extensive tissue damage.The complex interactions between The authors declare that they have no competing interests.LR designed and executed animal infection studies, data analysis and manuscript preparation. MR performed histopathologic evaluation of tissues and developed the lesion scoring system implemented in this study. MBB participated in the design and coordination of the study, and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/9/prepubU. parvumDistribution of kidney lesion scores in F344 rats inoculated with varying doses of . Lesion score analysis of kidney tissue was based on total area affected. The data provided represents the raw lesion scores for each biological replicate. Horizontal bars demarcate the median value for each clinical profile group. Lesion scoring of each sample was performed without prior knowledge of the clinical profile. Data is a summation of 5 separate experiments. Nonparametric lesion scores were grouped according to the clinical profile and analyzed by Kruskall-Wallis test .Click here for file


Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in  Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, and Enterococcus faecalisBacillus thuringiensis (Bt) are a large family of PFTs Caenorhabditis elegansPore-forming toxins (PFTs) are bacterial toxins that damage the plasma membrane of host cells and play important roles in the pathogenesis of many important human pathogens including C. elegans–Cry toxin interaction system opened up the first, and to date the only, whole-animal genetic model for studying PFTs in vivo and led to the discovery of signal transduction pathways that protect cells against PFTs, including the p38 mitogen-activated protein kinase (MAPK) pathway and the unfolded protein response (UPR) pathway, which are also protective against and/or activated by PFTs in mammalian cells C. elegans against PFTs e.g., modulation of signal transduction cascades) have been recorded, the roles of these responses in coping with PFTs as yet poorly understood This C. elegans and is homologous to the mammalian insulin and IGF-1 signaling pathway C. elegans daf-2 gene encodes the worm homolog of the insulin/IGF-1 receptor. C. elegans strains that carry reduction-of-function or loss-of-function mutations in daf-2 or the downstream phosphoinositol 3-kinase (PI3K) age-1 are long-lived and are also resistant to a variety of stresses and bacterial pathogens The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor pathway is part of a neuroendocrine system that regulates longevity, metabolism, and development in C. elegans. Unexpectedly, this resistance does not solely rely on the canonical DAF-2 insulin/IGF-1 signaling pathway through AKT/PKB and DAF-16 but that rather, at least in part, deviates form the main pathway at PDK-1 and that includes WWP-1. We demonstrate that WWP-1 may be a novel signaling arm that diverges from PDK-1 and functions in parallel to DAF-16 in the DAF-2 insulin/IGF-1 signaling network. Furthermore WWP-1 is functionally important for the intrinsic cellular defenses (INCED) against pathogenic attacks since loss of this pathway leads to animals hypersensitive to PFTs and pathogenic bacteria Pseudomonas aeruginosa in C. elegans.Here we demonstrate that reduction of the DAF-2 insulin/IGF-1 signaling pathway confers resistance to Bt Cry PFTs in C. elegans against the PFTs, C. elegans daf-2 reduction-of-function mutant daf-2(e1370) were qualitatively compared to wild-type N2 animals in their susceptibilities to the nematicidal PFT, Cry5B  mutant animals were qualitatively healthier and appeared resistant to Cry5B.To address whether the DAF-2 insulin/IGF-1 signaling pathway plays a role in T, Cry5B . Fourth earances . However50  values were obtained (daf-2(e1370) mutant animals are at least an order of magnitude more resistant to Cry5B than wild-type N2 animals (P<0.01). In order to test whether daf-2(e1370) mutant animals were also resistant to another Cry PFT, we also quantitative analyzed the LC50 values of daf-2(e1370) animals to the nematicidal Cry PFT, Cry21A daf-2(e1370) animals are 7X more resistant to Cry21A than wild type N2 animals  . This result implies that Cry21A at least partly might require a different receptor than Cry5B for intoxication of C. elegans.To independently confirm the importance of DAF-2 in PFT responses, DAF-2 function was reduced using RNA interference (RNAi) in the RNAi-sensitive strain (pk1426) . The resdaf-2 are totally suppressed by loss-of-function mutations in daf-16, which encodes a Forkhead transcription factor daf-2(e1370) mutant, the daf-16 null mutant, mu86daf-2(e1370);daf-16(mu86) double mutant were exposed to Cry5B and Cry21A and scored for viability at the single dose of 40 µg/ml purified Cry5B daf-2(e1370) animals are significantly more resistant  to Cry5B than wild-type N2 animals (P<0.01) whereas loss-of-function daf-16(mu86) mutant animals are as sensitive as N2 (daf-2(e1370);daf-16(mu86) double mutant animals had an intermediate phenotype that was statistically more sensitive than daf-2(e1370) animals (P<0.001) but statistically more resistant  than daf-16(mu86) or wild-type animals in response to Cry5B (P<0.001 and P<0.001). Similar results were also seen in the animals treated with 8 µg/ml of the Cry21A. daf-2(e1370);daf-16(mu86) double mutant animals also had an intermediate phenotype that was statistically more sensitive than daf-2(e1370) animals (P<0.05) but statistically more resistant  than daf-16(mu86) or wild-type animals in response to Cry21A (P<0.05 and P<0.05)  , Table 2C. elegans, DAF-2 regulates DAF-16 through the activation of PI3K, encoded by age-1 and aap-1 for the catalytic subunit and the regulatory subunit respectively. PI3K potentiates the activity of PDK-1, which in turn activates the three downstream serine/threonine kinases: the Akt/PKB homologs AKT-1 and AKT-2, and the serum- and glucocorticoid-inducible kinase homolog, SGK-1. These three serine/threonine kinases than inhibit the nuclear translocation and transcriptional activity of DAF-16 by phosphorylating DAF-16 at different serine/threonine residues Caenorhabditis Genetic Center (CGC) and exposed them to E. coli expressing Cry5B in liquid medium. In this Cry5B toxicity assay, the daf-2(e1370), age-1(hx546), aap-1(m889), pdk-1(sa680), pdk-1(sa709), and daf-2(e1370);daf-16(mu86) animals are all statistically resistant to Cry5B compared to the wild-type N2 animals (daf-18(e1375) mutant animals , are hypersensitive to Cry5B compare to wild-type animals. Thus, animals from the insulin-receptor down to PDK-1 behave as expected from what is known about the pathway. However, animals with mutations in the three serine/threonine kinases immediately downstream of PDK-1, including akt-1(sa573), akt-1(ok525), akt-1(mg144), akt-2(ok393), and sgk-1(ok538), were not resistant to Cry5B (akt-1(ok525) and akt-2(ok393)) were hypersensitive to the PFT.In the canonical DAF-2 insulin/IGF-1 signaling pathway for the regulation of lifespan, energy metabolism, and dauer development in  animals , Table 2to Cry5B , Table 2pdk-1, akt-1, akt-2 and sgk-1 genes were quantitatively assessed using the Cry5B dose-dependent mortality assay  and pdk-1(sa709), were statistically more resistant to Cry5B than wild type N2 animals. A gain-of-function mutation in pdk-1(mg142) was, as predicted, sensitive to Cry5B. As above, we found animals with the loss-of-function mutations in akt-1, akt-2 and sgk-1, including akt-1(sa573), akt-1(ok525), akt-2(ok393), and sgk-1(ok538) were either as sensitive or hypersensitive to Cry5B compared to N2 animals  for novel PDK-1 interacting proteins CarboxyMethyltransferase), were identified from high-throughput yeast two-hybrid screens using H42K12.1(PDK-1) as the bait , and human WWP1 and WWP2 pcm-1 gene encodes an L-isoaspartate O-methyltransferase orthologous to human PCMT1 wwp-1 and pcm-1 genes were quantitatively assessed using the Cry5B dose-dependent lethality assay and LC50 values were obtained (wwp-1(ok1102) loss-of-function mutant animals are significantly hypersensitive to Cry5B PFT compared to wild type N2 animals , but the sensitivity to Cry5B PFT of pcm-1(qa201) mutant animals are statistically indistinguishable from N2 animals. Two additional wwp-1 mutant alleles, wwp-1(gk372) and wwp-1(gk397)  , were alry5B PFT , Table 1rrf-3(pk1426);glp-4(bn2) mutant worms were used to knock down the wwp-1 genes by two independent E. coli RNAi feeding clones (8A5 and 8A15) from the Ahringer RNAi library rrf-3(pk1426);glp-4(bn2) animals were used because the rrf-3(pk1426) mutant is hypersensitive to RNAi and the glp-4(bn2) mutant does not produce progeny that would otherwise complicate the assay . We have demonstrated that both mutants have roughly normal response to Cry5B wwp-1 RNAi clones target different sequence regions of the wwp-1 gene , we predict WWP-1 would also play a role in response to P. aeruginosa and be antagonistic to DAF-2. We exposed wwp-1 mutants to the pathogenic bacteria P. aeruginosa strain PA14 . These data also demonstrate that WWP-1 is involved in promoting innate immunity against pathogenic bacteria.The DAF-2 signaling network in ain PA14 . As we pC. elegans. It also has been suggested that signal transductions in the DAF-2 insulin/IGF-1 signaling pathway for longevity and innate immunity can be interrelated C. elegans lifespan, again antagonistic to DAF-2. As predicted, we found that wwp-1(gk372) and wwp-1(ok1102) mutant animals had statistically significant shorter lifespan compared with wild-type N2 animals  2O2 (a toxic insult that kills much more rapidly). All wwp-1 mutants, including wwp-1(gk372), wwp-1(gk397), and wwp-1(ok1102), have the similar sensitivity as wild type to killing by either CuSO4 or H2O2  against PFTs and the innate immune response against pathogenic bacteria in C. elegans.To confirm that the hypersensitivity of WWP-1 mutant animals to aging,  or H2O2 ; Table 4P. aeruginosa results are consistent with WWP-1 acting in the DAF-2 pathway antagonistic to and downstream of DAF-2. If true, then we predict that knock-down of WWP-1 should suppress a DAF-2 mutant phenotype. We therefore knocked down wwp-1 in daf-2(e1370) and daf-2(e1370);daf-16(mu86) mutant animals and exposed these wwp-1 knockdown animals  to purified Cry5B (wwp-1 RNAi can partly suppress the daf-2(e1370) resistant phenotype and that the combination of daf-16 knock out (via mu86 mutation) and wwp-1 knock down (via RNAi) can completely suppress daf-2(e1370) resistance back to a response similar to wild-type . These data are consistent with both daf-16 and wwp-1 acting in parallel daf-2-dependent pathways to mediate resistance to Cry5B PFT. Furthermore, that wwp-1 RNAi can not further sensitize the wwp-1(ok1102) worms in this experiment also confirmed that this mutant allele is a total loss-of-function mutant.The two-hybrid interactome data and the Cry5B/aging/ed Cry5B , Table 3daf-2 PFT defense pathway bifurcates at PDK-1 into DAF-16-dependent and DAFT-16-independent branches. This is the first report of a bifurcation of the daf-2 insulin/IGF-1 pathway at this junction. We furthermore find a protein, WWP-1, that appears to be involved in the DAF-16 –independent branch of the INCED/innate immune response. Loss of WWP-1, a protein in the ubiquitin E3 ligase family, leads to hypersensitivity to the PFT Cry5B and the pathogen P. aeruginosa and that, based on double and triple knock out/knock down analyses with daf-2 and daf-16 mutants, appears to work in parallel with DAF-16. A model summarizing our findings is shown in Here we demonstrate for the first time that the DAF-2 insulin/IGF-1 pathway is involved in intrinsic cellular defenses (INCED) against PFTs. Furthermore, our data suggest that the P. aeruginosa, and aging, and on our genetic pathway data. A similar conclusion is evidenced from a previous genome-wide RNAi screen in which it was shown that knockdown of wwp-1 gene decreased median lifespan by 24% in daf-2(e1370) mutants, 5% in daf-2(e1370);daf-16(mgDf47) mutants, and 9% in wild-type N2 animals wwp-1 gene more significantly shortened the lifespan of daf-2 animals but still decreased the lifespan of daf-2;daf-16 mutant and wild-type N2 animals. This suggested that wwp-1 functions in parallel to daf-16 and converging within the DAF-2 insulin/IGF-1 signaling network to regulate longevity, which is reminiscent of our data. In addition, recently ubiquitin ligase activity has been shown for WWP-1, as well as demonstration that WWP-1 is involved in a daf-16-independent life span extension in response to diet restriction P. aeruginosa, as well as longevity in C. elegans.The assignment of WWP-1 as downstream of the DAF-2 insulin/IGF-1 pathway is based on two-hybrid interactome data with PDK-1, on phenotypic analyses of responses to PFT, daf-2 mutants is completely dependent upon daf-16sgk-1 mutants are long-lived Our results also show that regulation of PFT defense and lifespan can clearly be decoupled. First, we note that lifespan extension of C. elegans dauer development. That Eak-3 mutants have normal lifespan indicates EAK-3 decouples insulin-like regulation of development and longevity daf-2, such as pdk-1 and sgk-1, show wild-type resistance to the human opportunistic pathogen, Pseudomonas aeruginosa strain PA14. However, mutants of akt-1 and akt-2 show enhanced resistance to P. aeruginosa PA14 akt-1 and pdk-1 alleles can uncouple the dauer arrest, adult longevity and stress resistance phenotypes of age-1(mg109) mutants age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. These findings suggested larval and adult phenotypes of DAF-2 signaling are fully separable in these mutants. Finally, the other possible PDK-1 interacting protein identified in the interactome database, PCM-1, has also been demonstrated to participate in the repair of age-damaged proteins and overexpression of PCM-1 increases adult life span C. elegans.That DAF-2 signal can be decoupled from the conical pathway is reminiscent of several recent reports. Firstly, it has been demonstrated that EAK-3 (Enhancer of AKT-1 null) functions in parallel to AKT-1 to inhibit the expression of Forkhead transcription factor DAF-16 target genes involved in P. aeruginosa PA14 but also the INCED against PFTs as well as perhaps some yet unknown responses.In summary, we have identified specifically WWP-1 and the DAF-2 insulin/IGF-1 signaling network as components of INCED against PFTs. The biological functions of the DAF-2/DAF-16 signaling pathway in longevity and pathogens resistance have been extensively studied for years Caenorhabditis elegans strains used in this work were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH National Center for Research Resources (NCRR). C. elegans strains were maintained on NG plates using Escherichia coli strain OP50 as the food source daf-2(e1370), age-1(hx546), aap-1(m889), daf-18(e1375), pdk-1(sa680), pdk-1(sa709), pdk-1(mg142), akt-1(ok525), akt-1(sa573), akt-1(mg144), akt-2(ok393), sgk-1(ok538), daf-16(mu86), daf-2(e1370);daf-16(mu86), bre-3(ye28), rrf-3(pk1426), rrf-3(pk1426); glp-4(bn2), pcm-1(qa201), wwp-1(ok1102), wwp-1(gk372), and wwp-1(gk397) were each backcrossed at least 4 times. Bacteria expressing dsRNA directed against bre-3 and wwp-1 were part of a C. elegans RNAi library in E. coli strain HT115 . All RNAi clones have been confirmed by plasmid DNA sequencing. Escherichia coli HT115 transformed with the pAD48 construct, which expresses dsRNA targeting the daf-2 gene, was kindly provided by A. Dillin  Some E. coli-expressed Cry5B as described E. coli that did not express Cry5B (pQE9 vector control) or on plates prepared with E. coli expressing Cry5B (pQE9-Cry5B) 50 values for each toxin. E. coli-expressed Cry5B liquid toxicity assay: N2 wild-type and various daf-2 pathway mutant worms were exposed to E. coli-expressed Cry5B in S media in 48-wll plates to quantitative scored the survival. Similar conditions were used as the quantitative mortality assays described above, except that E. coli JM103 Cry5B expressing bacteria at 0.6 OD600 in stead of purified Cry5B and E. coli OP50 were used in this assay. The survival rate of each well was scored after incubating at 25°C for 6 days.All assays were performed at 25°C unless indicated elsewhere. Qualitative toxicity assays based on visual comparison of worm intoxication were performed on plates with E. coli-expressed Cry5B Plates. E. coli strain HT115 transformed with RNAi plasmids were spread on NG-IC plates [NG plates with 25 µg/ml carbenicillin and 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG)] and incubated at 25°C overnight to induce the dsRNA expression. E. coli HT115 with pL4440, an empty vector, was used as negative control of RNAi. Synchronized rrf-3(pk1426) L1 larvae were obtained using standard protocols pL4440, bre-3, or daf-2 genes dsRNA expressing RNAi-plates at 20°C until L4 stage. These L4 stage worms were transferred to either control plates with E. coli HT115 that did not express Cry toxins (empty vector) or plates prepared with E. coli HT115 expressing either Cry5B or Cry21A ;glp-4(bn2) animals ;glp-4(bn2) RNAi knock down animals were washed out by S medium and transferred to the wells of 48-well plate with S medium containing E. coli HT115 RNAi bacteria at 0.6 OD600 and 20 µg/ml of purified Cry5B. After incubating at 25°C for 6 days, the survival rate of each well was scored.For daf-2(e1370), daf-16(mu86), daf-2(e1370);daf-16(mu86) and wwp-1(ok1102) mutant worms were used. Synchronized L1 animals were cultured on the NG-IC wwp-1 RNAi or pL4440 E. coli HT115 plates at 20°C until L4 stage. L4 stage RNAi knock down animals were washed out by S medium and transferred to the wells of 48-well plate with S medium containing with either wwp-1 RNAi or pL4440 E. coli HT115 bacteria at 0.6 OD600 and 20 µg/ml of purified Cry5B. After incubating at 25°C for 6 days, the survival rate of each well was scored.For C. elegans with hypochlorite/NaOH solution and transferring the resulting eggs to NG agar plates covered with E. coli strain OP50. When these had reached the young adults, ∼150 nematodes were transferred to fresh plates, which also represents the first day of life span analysis. Nematodes were transferred to fresh plates daily during the progeny production period and after that were transferred every second to third day but monitored daily for dead animals. Nematodes that did not respond to gentle prodding and displayed no pharyngeal pumping were scored as dead. Animals that crawled off the plate or died due to internal hatching or protrusion of the gonads through the vulva were censored. Censoring describes an event where partial information on the life span of an individual animal is lost as a consequence of premature death. Thus, censored animals were included in statistical analysis only until the day of the censoring event. Survival analysis was performed using GraphPad Prism 5.0 . The Mantel-Cox logrank test was used to assess statistical significance of difference in survival. Only p-values<0.01 were considered significant to minimize type I errors.Lifespan analysis was conducted according to standard protocols P. aeruginosa killing assay was performed on slow-killing plates as described The 4, E. coli OP50 at an optical density of 0.2–0.25 OD600, and ∼30 L4 larvae were used per well in 48-well plates. Lethality was determined after 6 days of CuSO4 exposure at 25°C.Assays were carried out as described 2O2 exposure at 25°C.Assays were carried out as described 50 values were determined by PROBIT analysis All experiments were performed a minimum of three times. LC