Generous hosts: Why the larvae of greater wax moth, Galleria mellonella is a perfect infectious host model?

Figures & data

Figure 1. Collection and chemical analyses of Galleria mellonella. A: Haemolymph was collected by first puncturing the larvae behind the first set of thoracic (true) legs, in the inter-spiracle space, with a 0.5 mL BD fine insulin syringe. For each replicate, haemolymph from five larvae was collected. From 5 to 7 µL of pure haemolymph was collected from each larvae using a 10 µL capillary tube and was ejected into a 0.2 ml tube containing 50 µL of solvent (8:1:1 methanol:chloroform:water) to prevent melanization. Portions from the replicates were derivatized using TMS, MCF, or BF3 prior to the GC-MS analysis. B: GC-MS total ion chromatogram of G. mellonela haemolymph after TMS derivatization. The numbers above the peaks refer to the numbers listed in Table 1 Is: internal standard used to calculate the absolute amount of each compounds.

Table 1. Concentration (mM) of metabolites of Galleria mellonella haemolymph derivatized by trimethylsilylation (TMS) and analyzed by GC-MS. Means ± standard deviation shown were generated from total ion chromatograms (TIC) peaks from duplicate injections from five biological samples (n = 10). Peak areas were converted to mM concentration by use of linear calibration curves of standard reference compounds treated the same as samples.

Peak No.	LRI	TMS Derivative	Mean ± SD	[%] mM
1	1085	L-Alanine	1.24 ± 0.63	0.79
2	1223	L-Valine	1.15 ± 0.62	0.74
3	1278	Phosphoric acid	6.65 ± 2.43	4.25
4	1296	L-Isoleucine	0.74 ± 0.36	0.47
5	1300	L-Proline	15.2 ± 9.39	9.72
6	1308	Glycine	4.80 ± 2.34	3.07
7	1369	L-Serine	3.45 ± 3.13	2.21
8	1395	L-Threonine	0.37 ± 0.06	0.23
9	1447	β-Alanine	0.24 ± 0.08	0.15
10	1515	Malic acid	1.47 ± 0.80	0.94
11	1538	L-Aspartic acid	0.27 ± 0.02	0.17
12	1541	Pyroglutamic acid	5.43 ± 2.34	3.47
13	1580	2-Ketoglutaric acid	0.92 ± 0.15	0.59
14	1637	L-Glutamic acid	0.16 ± 0.00	0.10
15	1641	L-Phenylalanine	0.22 ± 0.03	0.14
16	1705	L-Asparagine	0.41 ± 0.08	0.27
17	1752	Putrescine	88.7 ± 41.8	56.67
18	1771	α-Glycerophosphate	3.38 ± 0.98	2.16
19	1786	o-Ethanolamine phosphate	2.75 ± 2.80	1.76
20	1819	Citric acid	5.03 ± 1.33	3.21
21	1894;1906	Fructose	0.91 ± 0.18	0.58
22	1916;1929	Glucose	0.88 ± 0.25	0.56
23	1945	Glucitol	0.22 ± 0.02	0.14
24	2013	Glucaric acid	0.09 ± 0.04	0.06
25	2484	Unknown Disaccharide 1	0.10 ± 0.01	0.06
26	2502	Unknown – m/z 456/513	0.31 ± 0.13	0.20
27	2545	Unknown Disaccharide 2	0.20 ± 0.04	0.13
28	2577	Sucrose	0.36 ± 0.09	0.23
29	2651	Trehalose	10.8 ± 4.20	6.92
		Total	156.5 ± 74.3	100.00

^a Compounds were confirmed using derivatized reference substances.

^b Compounds tentatively identified using mass spectral databases (NIST 2011, Wiley 9^th Ed.) or online Golm Metabolome Database (http://gmd.mpimp-golm.mpg.de/).

Table 2. Concentration (mM) of metabolites of Galleria mellonella haemolymph derivatized by methyl chloroformate (MCF) and analyzed by GC-MS. Means ± standard deviation shown were generated from total ion chromatograms (TIC) peaks from duplicate injections from five biological samples (n = 10). Peak areas were converted to mM concentration by use of linear calibration curves of standard reference compounds treated the same as samples.

Peak No.	LRI	MCF Derivative	Mean ± SD	[%] mM
1	603	Fumaric acid	0.22 ± 0.02	0.20
2	608	Maleic acid	0.22 ± 0.02	0.20
3	614	Succinic acid	0.69 ± 0.08	0.63
4	1116	Glycine	5.16 ± 0.93	4.74
5	1116	L-Alanine	10.4 ± 1.76	9.52
6	1286	L-Valine	7.17 ± 1.40	6.59
7	1382	L-Leucine	5.34 ± 1.21	4.91
8	1397	L-Isoleucine	2.50 ± 0.57	2.30
9	1406	L-Threonine	0.41 ± 0.12	0.38
10	1423	Malic acid	1.43 ± 0.21	1.31
11	1433	L-Proline	32.0 ± 4.42	29.85
12	1440	L-Asparagine	2.71 ± 1.28	2.49
13	1489	Quinic acid	5.12 ± 2.86	4.70
14	1514	L-Aspartic acid	0.34 ± 0.24	0.31
15	1527	Citric acid	7.05 ± 2.06	6.48
16	1584	L-Serine	5.58 ± 1.38	5.13
17	1627	L-Glutamine	7.58 ± 2.68	6.96
18	1637	L-Glutamic acid	0.49 ± 0.30	0.45
19	1650	L-Methionine	0.21 ± 0.06	0.19
20	1733	l-Cysteine	0.01 ± 0.01	0.01
21	1755	L-Phenylalanine	1.58 ± 0.35	1.45
22	1765	Myristic acid (C14:0)^,	0.05 ± 0.02	0.05
23	1850	Pentadecanoic acid (C15:0)^,	0.01 ± 0.01	0.01
24	1942	Palmitic acid (C16:0)^,	0.57 ± 0.30	0.52
25	2009	L-Lysine	5.43 ± 1.36	4.99
26	2054	L-Histidine	1.03 ± 0.26	0.95
27	2072	Linoleic acid (C18:2)^,	0.32 ± 0.15	0.29
28	2078	Oleic acid (C18:1)^,	0.22 ± 0.06	0.20
29	2097	Stearic acid (C18:0)^,	0.50 ± 0.38	0.46
30	2133	L-Tyrosine	3.35 ± 0.69	3.08
31	2266	L-Tryptophan	0.71 ± 0.45	0.65
		Total	108.9 ± 25.6	100.00

^a Compounds were confirmed using derivatized reference substances.

^b Compounds tentatively identified using mass spectral databases (NIST 2011, Wiley 9^th Ed.).

^c Results from boron trifluoride derivatization.