An experimental test of the independent action hypothesis in virus-insect pathosystems

Abstract

The 'independent action hypothesis' (IAH) states that each pathogen individual has a non-zero probability of causing host death and that pathogen individuals act independently. IAH has not been rigorously tested. In this paper, we (i) develop a probabilistic framework for testing IAH and (ii) demonstrate that, in two out of the six virus-insect pathosystems tested, IAH is supported by the data. We first show that IAH inextricably links host survivorship to the number of infecting pathogen individuals, and develop a model to predict the frequency of single- and dual-genotype infections when a host is challenged with a mixture of two genotypes. Model predictions were tested using genetically marked, near-identical baculovirus genotypes, and insect larvae from three host species differing in susceptibility. Observations in early-instar larvae of two susceptible host species support IAH, but observations in late-instar larvae of susceptible host species and larvae of a less susceptible host species were not in agreement with IAH. Hence the model is experimentally supported only in pathosystems in which the host is highly susceptible. We provide, to our knowledge, the first qualitative experimental evidence that, in such pathosystems, the action of a single virion is sufficient to cause disease.

Figure 1

The IAH-based model for the frequency of dual-genotype infection; it is summarized in a Venn diagram, where Ω is the set of all possible outcomes. Note that for baculoviruses, we define infection as the production of viral transmission stages (OBs) and virus-induced death of the host.

Figure 2

Model predictions under IAH on the frequency distribution of (a) infecting pathogens and (b) the frequency of single- and dual-genotype infections in dead hosts. For all graphs, frequency is on the y-axis. The frequency that zero pathogens infect a host is also host survival (S) and denoted by the dotted bars in (a). A starting population with a 1 : 1 ratio of pathogen genotypes A and B and an equal infection chance (p_A=p_B) between these pathogen genotypes were assumed ((i) S=0.99, (ii) S=0.50, (iiii) S=0.01 and (iv) S=0.0001).

Figure 3

Dose–response data for AcMNPV in S. exigua L3 larvae. On the x-axis is the occlusion body (OB) concentration per ml (log), and on the y-axis the mortality. Bioassay mortality data (squares), with the standard deviation, and the fitted dose–response relationship under IAH (curve) are displayed. See §2 for the number of replicates per concentration. Note that, for simplicity, OB concentration was taken as dose, rather than as an estimate of the number OBs or virions ingested.

Figure 4

Model predictions and experimental data for the frequency of dual-genotype infections. The presence of genotypes A and B was determined at different doses in (a–c) S. exigua L3 (22 larvae per dose), and in different host species in (d–f) L3 and (g–i) L5 larvae (24 larvae per treatment). Model predictions are depicted with black bars. Experimental data are depicted with white bars, and the number of observations noted above each column. OBs ml⁻¹ is the concentration of OBs used in the challenge experiments. S is survival and f(A∩B) is the observed frequency of dual-genotype infection in cadavers. P′(A∩B) is the predicted frequency of dual-genotype infection, given host mortality, under IAH. Binom. p-value is the observed significance of a binomial test comparing f(A∩B) with P′(A∩B). An asterisk marks significant departure from IAH. (a–c) It is demonstrated that IAH is not rejected in the third larval instar of S. exigua, irrespective of dose or mortality. (d–f) It is shown that IAH is not rejected in L3 of S. exigua and T. ni, but is rejected in L3 of semi-permissive M. brassicae. (g–i) Significant departure from IAH in challenge experiments with fifth larval instars in all three species is shown.

Figure 5

Dose–response relationships in semi-permissive larvae and L5. On the x-axes are the concentrations (log) of OBs in the inoculum, and on the y-axes the mortality for single replicates of dose–response bioassay data of (a) T. ni L3, (b) T. ni L5, (c) S. exigua L5, (d) M. brassicae L3 and (e) M. brassicae L5. Experimental data are denoted by squares and the IAH model for dose–response is given by the curve. This is done only for comparative purposes, using nonlinear regression (see §2). Note that only (a) T. ni L3 dual-genotype infection frequency data support the IAH model. The data suggest that in other instances, dose–response curves are shallower. Whether the T. ni L5 or M. brassicae L5 data are really shallower cannot be ascertained from these data alone.