Galleria mellonella (Linnaeus, 1758)

Hazir, Selçuk, Kaya B, Harry K., B, Mustapha Touray, B, Harun Çimen & B, David Shapiro-Ilan, 2022, Basic laboratory and field manual for conducting research with the entomopathogenic nematodes, Steinernema and Heterorhabditis, and their bacterial symbionts, Turkish Journal of Zoology 46 (4), pp. 305-350 : 324-328

4.1. Isolation from infected Galleria mellonella hemolymph

When the IJs infect insects, they proceed to the hemocoel to release the symbiont bacteria. Xenorhabdus or Photorhabdus proliferate in the hemolymph causing sepsis and/or toxemia and eventual death of host within 2 to 3 days while simultaneously eliminating other rival microorganisms ( Boemare and Akhurst, 2006). The symbiotic bacteria can be isolated 28–36 h after inoculation (i.e. moribund infected host) by collecting the hemolymph under sterile conditions. The following procedure can be used.

1. Infect 10 G. mellonella larvae in 9-cm Petri dishes lined with two moist filter papers by adding 1 mL nematode suspension containing approximately 2000 IJs. The following steps 2–5 should be conducted under sterile conditions (e.g., laminar flow hood or biosafety cabinet).

2. After 28–36 h, collect live but moribund larvae and disinfect surface by submerging in 70% alcohol for 4 min. Place larva on a sterile filter paper to dry.

3. Place insect between the index and thumb and the middle finger, bend the insect using index and thumb. Be careful not to over squeeze larva. Obtain insect hemolymph by either:

a. cutting one of the insect`s false legs (prolegs) using a pair of sterile mini-dissecting scissors;

b. injecting a 26G sterile syringe intrahemocoelically (just underneath the cuticle at a 25° angle but not too deep) ( Figure 10 ); or

c. cutting the head of insect and squeezing out insect contents with an L-shaped rod.

4. A clear hemolymph sample will ooze out. Do not use murky or cloudy hemolymph as this a sign of contamination with enteric bacteria of the insect.

5. Collect hemolymph sample using a sterile microbiological loop or micropipette and inoculate on appropriate culture medium, preferably, nutrient agar, NBTA (nutrient agar + bromothymol blue + 2,3,5-triphenyltetrazolium chloride) or T7 (see details in frequently used media and reagents section).

6. To increase success of isolation, use three or more insect larvae.

7. Incubate cultured plates in an incubator at 25–28 ℃.

4.2. Direct isolation of bacteria from nematode stages Bacteria can be isolated directly from the infective juveniles (IJs) ( Cimen, 2013) or from gravid females during endotokia matricida ( Ulug et al., 2015).

Procedure:

1. Transfer 1 mL of IJs to a 1.5-mL sterile Eppendorf tube. Allow nematodes to sink to the bottom of tube. Centrifuge at 13,000 rpm for 3 min and decant supernatant, without disturbing IJs. Or collect females as described in subsection 3.1.

2. Surface sterilize nematodes using 0.4% Hyamine solution for 6 min. Mix thoroughly. Discard Hyamine solution after allowing nematodes to sink to the bottom.

3. Wash IJs with 1 mL sterile Ringer`s solution. Repeat twice.

4. Centrifuge at 13,000 rpm for 3 min, discard final Ringer`s solution and add 20 µL of phosphate-buffered saline (PBS). Crush nematodes using a sterile motorized homogenizer.

5. Inoculate 10 µL of homogenate on NBTA, incubate at 25–28 ℃ for 24 h. Collect identified Xenorhabdus or Photorhabdus colonies and subculture in Luria-Bertani (LB) broth. Before storing, identify bacteria (method described below) and verify (phase) on NBTA. Store in 25% glycerol as stock at –80 ℃.

5. Characterization of bacteria (and variants) Xenorhabdus and Photorhabdus belong to the same family, have similar lifestyles, and share most phenotypic characteristics. The culture and phenotypic features of their Phase-I forms need to be correlated with molecular analysis of DNA gene sequences to ensure accuracy in identification and reduce discrepancy in classification ( Stackebrandt et al., 2002; Hazir et al., 2003a; Adams et al., 2006).

Bacteria in the Xenorhabdus genus are gram-negative motile rods with peritrichous flagella and motile. Phase-I forms absorb dye on NBTA and MacConkey agar thus forming dark blue convex, umbonate and mucoid colonies on NBTA that swarm and red colonies on MacConkey. They produce enzymes like lecithinase, proteases, and various other respiratory enzymes. Xenorhabdus spp. are catalase negative (no bubbles will form if a colony is transferred to a slide with H 2 O 2) and they do not reduce nitrate to nitrite, a trait that distinguishes them from other enteric bacteria. Phase-I variants ferment various carbohydrates like glucose into acid but with no gas ( Boemare, 2002; Boemare and Akhurst, 2006). In contrast, Phase-II forms do not absorb dye and do not produce antibiotics or protein inclusions; some forms may be mobile as Phase-I ( Boemare, 2002).

Photorhabdus spp. are also gram-negative rods with peritrichous flagella that are motile. Phase-I form dark green or yellow, convex, umbonate, mucoid and gummy colonies on NBTA, red colonies on MacConkey agar, and hemolysis zones around colonies streaked on sheep or horse blood agar. They are catalase positive but oxidase negative and cannot reduce nitrate to nitrite. The bacteria ferment various carbohydrates (glucose, fructose, maltose, etc.) without gas production. Photorhabdus have the ability to bioluminescence and produces vibrant pink, red, orange, green or yellow pigmented colonies. Phase-II forms are less pigmented or produce pigmented colonies different from Phase-I form. They also do not absorb neutral red dye on MacConkey agar, or produce antibiotics ( Brunel et al., 1997; Boemare, 2002; Boemare and Akhurst, 2006).

Molecular analysis of 16S rRNA and such like housekeeping genes can help distinguish between both bacteria but are now believed to be inept in detailing with the speciation of these bacteria; therefore, detailed and accurate reclassification methods like whole genome sequencing or concatenation and analysis of multiple DNA loci can help further specialize congeneric and homogeneous bacteria, especially in Photorhabdus , thereby resolving discrepancies at the subspecies level ( Brunel et al., 1997; Forst et al., 1997; Adeolu et al., 2016; Machado et al., 2018).

5.1. Phenotypic assessment of bacteria (biochemical tests and enzymatic activities)

Besides phenotypic assessments of Gram stain and colonial morphology, various rapid biochemical and enzymatic tests can provide discernible characteristics of Xenorhabdus spp. and Photorhabdus spp.

Catalase test: This test is used to identify catalaseproducing bacteria by bringing the organism into contact with hydrogen peroxide (H 2 O 2) which will be broken down into water and oxygen. Enteric bacteria, except Xenorhabdus , are all catalase positive.

Dispense 1–2 mL of 3% H 2 O 2 into a test tube. Collect a single colony from a culture that is not more than 24-h-old and immerse in H 2 O 2. Observe for active bubbling which indicates positive for catalase production ( Cheesbrough, 2009).

Indole test: This test is to assess the ability of bacteria to break down tryptophan amino acid to indole using tryptophanase enzyme. Grow Xenorhabdus or Photorhabdus in tryptophan broth for 24 h. Add a few drops of Kovac`s reagent to culture. Observe for color change; presence of a red or red-violet color shows positive result ( Collins et al., 2004).

Carbohydrate fermentation: This test is used to assess whether a bacterium has the ability to use certain types of carbohydrates as an energy source. Acid, gas, or both are produced if bacteria can ferment the tested carbohydrates. Inoculate a pure culture of bacteria into test tubes with fermentation broths of different carbohydrates (glucose, lactose, sucrose, mannitol, etc.), a pH indicator (phenol red) and a Durham tube for gas collection. Incubate at 28 ℃ for 24 h. Color change of phenol from red to yellow and/ or gas in Durham tubes indicate positive results ( Collins et al., 2004).

Lecithinase activity: The purpose of this test is to check for the ability to produce lecithinase enzyme which digests lecithin protein found in most animal tissues. Streak a loopful of bacteria on egg yolk agar and incubate for 72 h. Lecithinase activity is confirmed by opaque zone around inoculum ( Collins et al., 2004).

Casein hydrolysis: This test is used to identify proteaseproducing bacteria by assessing their ability to degrade casein protein. Inoculate bacteria in a straight line on skim milk agar and incubate for 3 days. Proteolysis is indicated by clear zone around inoculum ( Collins et al., 2004).

Motility: Spot inoculate 5 µL of bacteria culture broth in the center of motility agar (LB broth supplemented with 0.3% agar). Incubate at 28 ℃ for 24 h and check for swarming ( Easom and Clarke, 2008; Cimen, 2013).

Bioluminescence: Grow Photorhabdus on any suitable agar medium [LB, protease agar or Trypic soy broth (TSB), etc.] for 24–72 h depending on species or isolate. Or infect G. mellonella with an overnight bacteria suspension and incubate for 24–72 h. Bioluminescence activity can be seen with the naked eye under totally dark conditions. Measure luminescence activity using a bioluminescence imaging like IVIS Spectrum ( Stock and Goodrich-Blair, 2012).

Antibiotic production: Inoculate a loopful of Xenorhabdus or Photorhabdus from LB or TSB agar to broth medium and incubate overnight. Spot inoculate 5 µL of overnight culture on Mueller Hinton agar and incubate at 28 ℃ for 3–5 days; place Petri dishes under UV light for 5–10 min to kill the bacteria. Separately prepare Micrococcus luteus broth culture as an indicator, by adding 1% overnight bacterial culture to LB or TSB with 0.75% agar and mix homogenously, then overlay suspension on spot inoculated Xenorhabdus or Photorhabdus . Incubate at 30 ℃ overnight and check for zone of inhibition ( Donmez Ozkan et al., 2019).

Pathogenicity: Incubate LB broth with pure cultures of Xenorhabdus or Photorhabdus for 24 h at 28 ℃. Wash this overnight culture in PBS, then adjust suspension to OD 600 to 1 (4 × 108 cfu/mL) (cfu = colony-forming units). Dilute 10 times to 2 × 108 cfu/mL. Inject 10 µL of diluted suspension into the hemocoel of a G. mellonella larva. Infect at least 10 larvae and as negative control inject with equal amount of PBS. Store larvae at room temperature (23–24 ℃) in the dark. Check for mortality 48–72 h later ( Easom and Clarke, 2008).

Oxidase test (Cytochrome oxidase): The principle of this test is to assess if a bacterium produces cytochrome c oxidases, an enzyme of the bacterial electron transport chain. Add 2 or 3 drops of oxidase reagent (tetramethylp-phenylenediamine dihydrochloride) to a piece of filter paper placed on a clean Petri dish. Smear a single colony of bacteria on filter paper. A deep blue color change on inoculated area on paper indicates positive results ( Collins et al., 2004, Cheesbrough, 2009).

Lipolytic activity: This test is to check lipase enzyme production. Inoculate bacteria on nutrient agar supplemented with Tween-80, -60, or -20 (0.5% v/v). Incubate for 48 h at 28 °C. Lipase producing bacteria will have a clear zone around their colonies ( Cimen, 2013).

Analytical profile index (API): The API identification systems (bioMerieux Inc. Hazelwood MO) are miniature biochemical test kits that can be used for the identification of bacteria by evaluation of enzymatic activity on or the fermentation of various dehydrate substrates on strips. They are easy to use and provide rapid results. For Xenorhabdus and Photorhabdus, API 20E or API Rapid 20E can be used for identification according to manufacturer`s instructions ( Aslanzadeh, 2006).

5.2. Genomic DNA extraction from bacteria ( Maniatis et al., 2012)

DNA can be extracted using commercial kits but if kits cannot be procured, the protocol described below can be used:

1. Grow a pure culture of bacteria in LB broth to obtain overnight culture. Pellet at 12,000 rpm for 1 min, decant supernatant to collect pelleted bacteria cells.

2. Resuspend cells in 600 µL of lysis solution (9.34 mL TE buffer, 600 µL of 10% SDS, 60 μL of proteinase K, 20 mg /mL) and vortex.

3. Add 5 µL of RNase. Incubate 37 ℃ for 30 min.

4. Add an equal volume of phenol/chloroform. Mix by inverting tube until two phases mix.

5. Centrifuge at 12,000 rpm for 5 min. Transfer upper phase to a new tube without disturbing the white protein layer of phenol/chloroform interface

6. Add an equal volume of chloroform, mix well and centrifuge for 5 min at 12000 rpm. Transfer aqueous phase to a new tube.

7. Precipitate DNA by adding cold isopropanol (three times the volume of aqueous phase collected in step 5). Incubate at –20 ℃ for 30 min.

8. Centrifuge at 12,000 rpm for 15 min at 4 ℃. Discard supernatant and rinse DNA with 70% ethanol.

9. Centrifuge for 2 min at 12,000 rpm. Discard supernatant and air dry for 5–10 min or overnight until you cannot smell ethanol any longer. Resuspend DNA in sterile distilled water or TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0). Store until further use.

10. For PCR amplification, check subsection 1.2.2.

Table 8 lists some 16S primers used in the amplification of DNA in molecular identification of Xenorhabdus and Photorhabdus .

5.3. Obtaining bacterial growth culture, cell-free supernatant, and pellet

In recent years, there has been a multitude of studies investigating the biological activities of metabolites produced by Xenorhabdus and Photorhabdus . These studies used the bacterial growth culture, cell-free supernatant or pellet to suppress plant pathogenic fungi ( Bock et al., 2014; Shapiro-Ilan et al., 2014b; Hazir et al., 2016; Chacon-Orozco et al., 2020; Cimen et al., 2021), bacteria ( Furgani et al., 2008; Fodor et al., 2010; Donmez-Ozkan et al., 2019), mites ( Bussaman et al., 2012; Eroglu et al., 2019; Cevizci et al., 2020; Incedayi et al., 2021), plant-parasitic nematodes ( Kepenekci et al., 2016, 2018), insects ( Da Silva et al., 2013; Wagutu et al., 2017; Vitta et al., 2018; Shah et al., 2021), etc.

The following method can be used to obtain the bacterial growth culture, cell-free supernatant, or pellet:

1. Inoculate bacteria from stock cultures onto LB (Merck, Darmstadt-Germany) agar plates at 28 °C for 24 h.

2. Transfer a single colony from these plates into flasks containing 10 mL sterile TSB (Merck, Darmstadt, Germany) and incubate the flasks at 30 °C and 150 rpm for 24 h. Measure the cell density of the bacterial culture using a spectrophotometer and adjust turbidity at OD 6OOnm as needed. Use this bacterial growth culture for experiments.

3. To obtain cell-free supernatant transfer this bacterial broth culture to Falcon tubes and centrifuge at 10,000 rpm at 4 °C for 20 min. Then filter the supernatant through a 0.22-μm Millipore filter. These supernatants can be stored at 4 °C for up to 2 weeks prior to use in the experiments ( Hazir et al., 2016, 2017; Cimen et al., 2021).

4. The remaining pellet at the bottom of the Falcon tubes after centrifugation can be resuspended in sterile Ringer’s solution or physiological saline and turbidity can be adjusted as needed ( Vitta et al., 2018).

Bacterial growth culture or cell-free supernatants can be incorporated into agar plates or liquid media to assess biological activity.

Figures

Figure 10. Isolation of Xenorhabdus and Photorhabdus from Galleria mellonella hemolymph.