Ben Raymond

Bacillus thuringiensis: an impotent pathogen?

Bacillus thuringiensis (Bt) is an insecticidal bacterium that has successfully been used as a biopesticide for many years. It is usually referred to as a soil-dwelling organism, as a result of the prevalence of its spores in this environment, but one that can act as an opportunistic pathogen under appropriate conditions. Our understanding of the biology of this organism has been challenged further by the recent publication of two reports that claim that Bt requires the co-operation of commensal bacteria within the gut of a susceptible insect for its virulence. It is our opinion that Bt is not primarily a saprophyte and does not require the assistance of commensal bacteria but is a true pathogen in its own right and furthermore that its primary means of reproduction is in an insect cadaver.

The Dynamics of Cooperative Bacterial Virulence in the Field

Twin Tales of Two Toxins The luminescent bacterium, Photorhabdus luminescens, is carried in the gut of an insect-parasitic nematode as a stealth weapon. By using an allele swapping technique, Somvanshi et al. (p. 88) investigated the promoter-switching mechanism that flips the bacterium from the almost dormant M forms, which stick to the adult nematodes posterior gut, into the motile, luminous P forms, which are armed with the toxic virulence factors needed to overcome the insect prey of the nematode. Similar switches may operate in bacteria that flip between harmless commensals and lethal pathogens. The bio-control agent Bacillus thuringiensis also kills insects by means of a crystal toxin, which allows the bacteria to penetrate the host gut and access nutrients. Release of nutrients also allows bacterial cheats that do not make toxin, to grow and outcompete the toxin-producing colonizers. In field experiments, Raymond et al. (p. 85) found that, consequently, the bacterial population becomes less virulent. Because these type of virulence factors are secreted from the cell and are widespread in pathogens, such social interactions may affect the fitness and constrain the virulence of many toxin-producing bacteria. Toxin-producing individuals enable Bacillus thuringiensis to invade its host; once inside, nonproducing cheaters take over. Laboratory experiments have shown that the fitness of microorganisms can depend on cooperation between cells. Although this insight has revolutionized our understanding of microbial life, results from artificial microcosms have not been validated in complex natural populations. We investigated the sociality of essential virulence factors (crystal toxins) in the pathogen Bacillus thuringiensis using diamondback moth larvae (Plutella xylostella) as hosts. We show that toxin production is cooperative, and in a manipulative field experiment, we observed persistent high relatedness and frequency- and density-dependent selection, which favor stable cooperation. Conditions favoring social virulence can therefore persist in the face of natural population processes, and social interactions (rapid cheat invasion) may account for the rarity of natural disease outbreaks caused by B. thuringiensis.

Genes and environment interact to determine the fitness costs of resistance to Bacillus thuringiensis

Genes which provide resistance to novel challenges such as pesticides, toxins or pathogens often impose fitness costs on individuals with a resistant phenotype. Studies of resistance to Bacillus thuringiensis and its insecticidal Cry toxins indicate that fitness costs may be variable and cryptic. Using two field populations (Karak and Serd4) of the diamondback moth, Plutella xylostella, we tested the hypothesis that the costs associated with resistance to the B. thuringiensis toxin Cry1Ac would be evident when insects were grown under poor environmental conditions, namely limited or poor quality resources. On a poor quality resource, a cultivar of Brassica oleracea var. capitata with varietal resistance to P. xylostella, only one resistant population, Karak, showed reduced fitness. Conversely, when we limited a high quality resource, Brassica pekinensis, by imposing larval competition, only resistant Serd4 insects had reduced survival at high larval densities. Furthermore, Cry1Ac resistance in Serd4 insects declined when reared at high larval densities while resistance at low densities fluctuated but did not decline significantly. These results confirm the hypothesis that resistance costs can appear under stressful conditions and demonstrate that the fitness cost of resistance to Bacillus thuringiensis can depend on the particular interaction between genes and the environment.

Environmental Factors Determining the Epidemiology and Population Genetic Structure of the Bacillus cereus Group in the Field

Bacillus thuringiensis (Bt) and its insecticidal toxins are widely exploited in microbial biopesticides and genetically modified crops. Its population biology is, however, poorly understood. Important issues for the safe, sustainable exploitation of Bt include understanding how selection maintains expression of insecticidal toxins in nature, whether entomopathogenic Bt is ecologically distinct from related human pathogens in the Bacillus cereus group, and how the use of microbial pesticides alters natural bacterial populations. We addressed these questions with a MLST scheme applied to a field experiment in which we excluded/added insect hosts and microbial pesticides in a factorial design. The presence of insects increased the density of Bt/B. cereus in the soil and the proportion of strains expressing insecticidal toxins. We found a near-epidemic population structure dominated by a single entomopathogenic genotype (ST8) in sprayed and unsprayed enclosures. Biopesticidal ST8 proliferated in hosts after spraying but was also found naturally associated with leaves more than any other genotype. In an independent experiment several ST8 isolates proved better than a range of non-pathogenic STs at endophytic and epiphytic colonization of seedlings from soil. This is the first experimental demonstration of Bt behaving as a specialized insect pathogen in the field. These data provide a basis for understanding both Bt ecology and the influence of anthropogenic factors on Bt populations. This natural population of Bt showed habitat associations and a population structure that differed markedly from previous MLST studies of less ecologically coherent B. cereus sample collections. The host-specific adaptations of ST8, its close association with its toxin plasmid and its high prevalence within its clade are analogous to the biology of Bacillus anthracis. This prevalence also suggests that selection for resistance to the insecticidal toxins of ST8 will have been stronger than for other toxin classes.

Intraguild predators and the spatial distribution of a parasitoid

Abstract An experimental plot of the aphid Aphis fabae on various host plant species was colonized by natural populations of the aphidiine parasitoid Lysiphlebus fabarum and insect predators, especially coccinellids. Parasitism of A. fabae by L. fabarum was significantly depressed on plants bearing coccinellids. The number of parasitized aphids increased with aphid abundance on three plant species (Papaver dubium, Rumex obtusifolius, Vicia faba), but not on the plant species (Chenopodium album) which bore very high numbers of coccinellids. In complementary laboratory experiments, L. fabarum offered a choice between odours of plants infested with A. fabae and/or coccinellids selected the odour fields from coccinellid treatments at significantly lower frequency than the odour fields of other treatments. It is concluded that avoidance of coccinellids by L. fabarum contributes to the negative association between the abundance of coccinellids and parasitoids in the field.

Genetic and biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin Cry1Ac in the diamondback moth, Plutella xylostella

ABSTRACT The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.

Host plant and population determine the fitness costs of resistance to Bacillus thuringiensis

Novel adaptations often cause pleiotropic reductions in fitness. Under optimal conditions individual organisms may be able to compensate for, or reduce, these fitness costs. Declining environmental quality may therefore lead to larger costs. We investigated whether reduced plant quality would increase the fitness costs associated with resistance to Bacillus thuringiensis in two populations of the diamondback moth Plutella xylostella. We also measured the rate of decline in resistance on two host-plant (Brassica) species for one insect population (Karak). Population×plant species interactions determined the fitness costs in this study. Poor plant quality increased the fitness costs in terms of development time for both populations. However, fitness costs seen in larval survival did not always increase as plant quality declined. Both the fitness and the stability experiment indicated that fitness costs were higher on the most suitable plant for one population. Theoretically, if the fitness cost of a mutation interacts additively with environmental factors, the relative fitness of resistant insects will decrease with environmental quality. However, multiplicative costs do not necessarily increase with declining quality and may be harder to detect when fitness parameters are more subject to variation in poorer environments.

Host-shifting by Operophtera brumata into novel environments leads to population differentiation in life-history traits

Abstract.  1. Operophtera brumata L. (Lepidoptera: Geometridae), a polyphagous herbivore usually associated with deciduous trees such as oak Quercus robur L., has expanded its host range to include the evergreen species heather Calluna vulgaris (L.) Hull and, most recently, Sitka spruce Picea sitchensis (Bong.) Carrière.

A mid-gut microbiota is not required for the pathogenicity of Bacillus thuringiensis to diamondback moth larvae

The mode of action of the entomopathogenic bacterium Bacillus thuringiensis (Bt) remains a matter of debate. Recent reports have claimed that aseptic lepidopteran hosts were not susceptible to Bt and that inoculation with mid-gut bacteria restores pathogenicity. These claims are controversial because larvae were rendered aseptic by consuming antibiotics, although the effect of these antibiotics on Bt was not examined. We tested the generality of the mid-gut bacteria hypothesis in the diamondback moth, Plutella xylostella using properly controlled experiments that investigated the effect of antibiotic consumption and absence of gut microbiota separately. We found that purified Bt toxin and spore/toxin mixtures were fully pathogenic to larvae reared aseptically. Persistence of antibiotics in larval tissues was implicated in reducing host mortality because larval consumption of the antibiotic rifampicin reduced the pathogenicity of rifampicin-sensitive Bt strains but not rifampicin-resistant strains. Inoculating larvae with Enterobacter sp. Mn2 reduced the mortality of larvae feeding on Bt HD-1 and the presence of a culturable gut microbiota also reduced the pathogenicity of the Bt toxin Cry1Ac, in agreement with other studies indicating that an intestinal microbiota can protect taxonomically diverse hosts from pathogen attack. As ingestion of antibiotics suppresses host mortality the vegetative growth of Bt in the host must be important for its pathogenicity. Furthermore, claims that aseptic larvae are not susceptible to Bt must be supported by experiments that control for the effect of administering antibiotics.

Antagonistic competition moderates virulence in Bacillus thuringiensis.

Classical models of the evolution of virulence predict that multiple infections should select for elevated virulence, if increased competitiveness arises from faster growth. However, diverse modes of parasite competition (resource-based, antagonism, immunity manipulation) can lead to adaptations with different implications for virulence. Using an experimental evolution approach we investigated the hypothesis that selection in mixed-strain infections will lead to increased antagonism that trades off against investment in virulence. Selection in mixed infections led to improved suppression of competitors in the bacterial insect pathogen Bacillus thuringiensis. Increased antagonism was associated with decreased virulence in three out of four selected lines. Moreover, mixed infections were less virulent than single-strain infections, and between-strain competition tended to decrease pathogen growth in vivo and in vitro. Spiteful interactions among these bacteria may be favoured because of the high metabolic costs of virulence factors and the high risk of mixed infections.