pubmed.ncbi.nlm.nih.gov

Infection of Tribolium castaneum with Bacillus thuringiensis: quantification of bacterial replication within cadavers, transmission via cannibalism, and inhibition of spore germination - PubMed

Infection of Tribolium castaneum with Bacillus thuringiensis: quantification of bacterial replication within cadavers, transmission via cannibalism, and inhibition of spore germination

Barbara Milutinović et al. Appl Environ Microbiol. 2015 Dec.

Abstract

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PubMed Disclaimer

Figures

FIG 1
FIG 1

Survival of T. castaneum larvae after exposure to fluorescent beads and spores. To estimate bacterial replication within the larvae, an assay was used that utilized ingestion of fluorescent beads and their subsequent counting in the larvae after death occurred and the bacterial sporulation process had completed. Larvae were exposed to a mixture of spores and fluorescent beads, after which both were counted in a flow cytometer. The figure shows the survival of T. castaneum larvae 3 days after exposure for 2.5 h to beads, spores, or a mixture of spores and beads in the diet. The spore concentration was adjusted to 5 × 109 ml−1. The sample size for each treatment was as follows: beads, n = 96; spores, n = 238; spores + beads, n = 244.

FIG 2
FIG 2

Measurement of spore germination via flow cytometry. Flow cytometric density plot of Bacillus thuringiensis spores before inoculation (A) and 2 h after inoculation into LB nutrient medium (B).

FIG 3
FIG 3

Spore ingestion and Bacillus thuringiensis reproduction in larvae of the red flour beetle. Larvae of T. castaneum were exposed to a spore-containing diet mixed with fluorescent beads. The number of initially ingested spores was extrapolated from the number of ingested beads for larvae that died (left) or survived (right) the exposure to spores (filled circles). The number of spores derived from each cadaver (final spore load; open circles) in relation to the number of ingested spores was used to estimate the number of bacterial generations within the host (left graph, right axis, × symbol).

FIG 4
FIG 4

Growth and germination of cadaver-derived spores compared to spores from the stock culture. (A) Increases in the optical density (OD590), as a proxy of bacterial growth. Spore suspensions were adjusted to 1 × 108 ml−1 in PBS, and 10 μl was added to 90 μl of LB broth in a 96-well plate. (B) The initial decrease in the OD590 of the starting spore suspension was used as a proxy for spore germination. Nutrient medium (LB) was inoculated with cadaver-derived spores, and the optical density of the medium was compared to that of medium containing spores that were cultured from stock cultures, i.e., which had not been propagated through the host (stock). (C) Reduced spore germination was confirmed via flow cytometric measurement. Nutrient medium (LB) was inoculated with spores from cadavers or spores from stock cultures, and the numbers of spores and vegetative cells were quantified at time point 0 (before the inoculation) and at 2, 4, and 6 h after inoculation. Shown is the proportion of vegetative Bt as a measure of spore germination. Bars show ±1 standard error.

FIG 5
FIG 5

Influence of cell-free cadaver homogenates on spore germination. (A) Spores of B. thuringiensis from fresh stock cultures were mixed with germinants and cell-free cadaver homogenates, which were obtained from infected cadavers (homogenate inf. + germinants) or with PBS + germinants. For negative controls, spores were mixed with either homogenates from infected cadavers (homogenate inf.) or with PBS only. (B) Spores of Bacillus thuringiensis from fresh stock cultures were mixed with germinants and cell-free cadaver homogenates obtained from uninfected cadavers (homogenate uninf. + germinants) or infected cadavers (homogenate inf. + germinants). As a positive control, spores were mixed with germinants and PBS. Bars show ±1 standard error.

FIG 6
FIG 6

Reduced killing rate of cadaver-derived spores and successful transmission via cannibalism. (A) Survival of T. castaneum larvae 3 days after constant exposure to cadaver-derived B. thuringiensis bv. tenebrionis spores in flour (n = 113) or spores that were not propagated through the host, i.e., that were cultured from a glycerol stock (n = 57). The spore concentration for both treatments was adjusted to 5 × 109 ml−1. Larvae from the control treatment (naive; n = 60) were treated in the same way, but without the addition of spores. (B) Survival of T. castaneum larvae 7 days after exposure to an uninfected (n = 123) or an infected (n = 123) cadaver as the only food source. A starvation control was used to control for food avoidance or nutrient deficiency (n = 123). Note that during the first 2 days, all of the cadavers were eaten (data not shown).

Similar articles

Cited by

References

    1. Schmid-Hempel P. 2011. Evolutionary parasitology: the integrated study of infections, immunology, ecology, and genetics, 1st ed Oxford University Press, Oxford, United Kingdom.
    1. Ebert D. 1994. Virulence and local adaptation of horizontally transmitted parasite. Science 265:1084–1086. doi:10.1126/science.265.5175.1084. - DOI - PubMed
    1. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806. - PMC - PubMed
    1. de Maagd RA, Bravo A, Crickmore N. 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199. doi:10.1016/S0168-9525(01)02237-5. - DOI - PubMed
    1. Soberon M, Gill S. 2009. Signaling versus punching hole: how do Bacillus thuringiensis toxins kill insect midgut cells? Cell Mol Life Sci 66:1337–1349. doi:10.1007/s00018-008-8330-9. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources