微生物学

Caenorhabditis elegans is a ubiquitous free-living nematode that feeds on bacteria. The organism was introduced into a laboratory setting in the 1970s and has since gained popularity as a model to study host-bacteria interactions. One advantage of using C. elegans is that its intestine can be colonized by the bacteria on which it feeds. Quantifying the bacterial load within C. elegans is an important and easily obtainable metric when investigating host-bacteria interactions. Although quantification of bacteria harbored in C. elegans via whole-worm lysis is not a novel assay, there is great variation between existing methods. To lyse C. elegans, many protocols rely on the use of a hand-held homogenizer, which could introduce systematic error and subsequent variation between researchers performing the same experiment. Here, we describe a method of lysing the intestines of C. elegans to quantify the bacterial load within the intestine. Our method has been optimized for removing exogenous bacteria while maintaining worm paralysis, to ensure no bactericidal agents are swallowed, which could kill bacteria within the intestine and affect results. We utilize and compare the efficiency of two different homogenization tools: a battery-powered hand-held homogenizer, and a benchtop electric homogenizer, where the latter minimizes variability. Thus, our protocol has been optimized to reduce systematic error and decrease the potential for variability among experimenters.

Graphic abstract:

Simplified overview of the procedure used to quantify the bacterial load within C. elegans.

The two different methods are herein described for worm lysis: “Option 1” is a hand-held homogenizer, and “Option 2” is a benchtop homogenizer.

Hookworms are skin penetrating parasites, however in the laboratory the hookworm model Nippostrongylus brasiliensis, the parasite is traditionally administered subcutaneously bypassing the skin (epidermis and dermis). Here, we describe two complementary approaches for infecting mice with N. brasiliensis in order to study the skin immune responses. The first approach employs a skin percutaneous injection that is poorly efficient with the laboratory strain of the parasite in mice, but represents a natural infection. The second approach employs an intradermal injection of the parasite, allowing the controlled delivery of the parasitic larvae and leads to an infection that closely mimics the natural kinetics of parasite migration and development. Both of those infection models allow the investigator to study the skin immune response mounted against the parasite, in addition to detailed investigations of the early immunomodulatory strategies employed by the parasite during skin invasion.

Physical avoidance of pathogens is a crucial defense strategy used by the host to reduce pathogen infection. Hosts display the use of multiple strategies to sense and avoid pathogens, ranging from olfaction to sensing of damage caused by pathogen infection. Understanding various mechanisms of pathogen avoidance has the potential to uncover conserved host defense responses that are important against pathogen infections. Here, we describe protocols for studying pathogen lawn avoidance behavior as well as a change of bacterial preferences in the model nematode Caenorhabditis elegans. Besides, we describe the protocol for measuring preferences for pathogenic and nonpathogenic bacteria after training of the animals on pathogenic bacteria. These assays can be implemented in discovering various mechanisms of host learning that result in the avoidance of pathogens.

The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.

Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.

Plant parasitic nematodes parasitize roots and/or stems of various plants thereby inhibiting absorption of nutrients and moisture. In particular, root-knot nematodes (RKN) are a group of the most devastating pests. Various techniques, such as soil sterilization, cultivation of resistant crops, and chemical application, have been developed to control damage caused by RKN. Among these techniques, diminish by chemicals that induce or activate host defense to RKN is an attractive method because of its potential to reduce the environmental burden caused by crop protection. Sclareol, a diterpene, was identified as a chemical that induces resistance to RKN (Fujimoto et al., 2015). Here we provide a protocol for assessing the impact of sclareol on the penetration of RKNs into tomato and Arabidopsis roots and the direct nematicidal impact of the chemical on nematodes. This protocol can be used for other nematode resistance-inducing chemicals.

This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.

Plant parasitic nematodes are devastating pests on many crops. Juveniles (J2) of cyst nematodes invade the roots to induce a syncytium. This feeding site is their only source of nutrients. Male nematodes leave the roots after the fourth molt to mate with females. The females stay attached to their syncytia throughout their life and produce hundreds of eggs, which are contained in their bodies. When the females die their bodies form the cysts, which protect the eggs. Cysts can survive for many years in the soil until favorable conditions induce hatching of the juveniles.

The beet cyst nematode Heterodera schachtii is a pathogen of sugar beet (Beta vulgaris) but can also complete its life cycle on Arabidopsis roots growing on agar plates under sterile conditions. We present here protocols for a stock culture of H. schachtii and an infection assay on agar plates.