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Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses
Immunology Letters 174 (2016) 45–52
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses Gongqing Wu a,b , Li Xu a , Yunhong Yi a,∗ a b
School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Zhongshan 528458, China Guangdong Cosmetics Engineering & Technology Research Center, China
a r t i c l e
i n f o
Article history: Received 7 March 2016 Received in revised form 16 April 2016 Accepted 16 April 2016 Available online 20 April 2016 Keywords: Galleria mellonella Photorhabdus luminescens TT01 Innate immunity
a b s t r a c t Larvae of Galleria mellonella are useful models for studying the innate immunity of invertebrates or for evaluating the virulence of microbial pathogens. In this work, we demonstrated that prior exposure of G. mellonella larvae to high doses (1 × 104 , 1 × 105 or 1 × 106 cells/larva) of heat-killed Photorhabdus luminescens TT01 increases the resistance of larvae to a lethal dose (50 cells/larva) of viable P. luminescens TT01 infection administered 48 h later. We also found that the changes in immune protection level were highly correlated to the changes in levels of cellular and humoral immune parameters when priming the larvae with different doses of heat-killed P. luminescens TT01. Priming the larvae with high doses of heatkilled P. luminescens TT01 resulted in significant increases in the hemocytes activities of phagocytosis and encapsulation. High doses of heat-killed P. luminescens TT01 also induced an increase in total hemocyte count and a reduction in bacterial density within the larval hemocoel. Quantitative real-time PCR analysis showed that genes coding for cecropin and gallerimycin and galiomycin increased in expression after priming G. mellonella with heat-killed P. luminescens TT01. All the immune parameters changed in a dose-dependent manner. These results indicate that the insect immune system is capable of sensing the extent of priming agent and mounting a proportionate immune response. © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Insects are a large and diverse group of animals that have adapted to extreme environments. They inhabit all ecological niches on the Earth with the exception of the oceans. The success of insects in colonizing such a wide variety of habitats is partly attributed to their effective immune response. Although insects do not have the adaptive immune machinery such as clonal expansion of antigen-specific lymphocytes, to generate a broad defence capability [1], they possess a robust innate immune system and that equips them with the ability to fight off infection. The innate immune system of insects consists of cellular and humoral defense responses. The cellular immune response, mediated by different hemocyte types, clears pathogenic microorganisms by phagocytosis or nodulation and kills large intruder (e.g., an egg of a parasitic wasp, nematode) by encapsulation [2–4]. The humoral response refers to effector molecules including complement-like proteins,
∗ Corresponding author. E-mail address: [email protected] (Y. Yi).
antimicrobial peptides (AMPs), and products generated by the phenoloxidase (PO) pathway [5]. The greater wax moth, Galleria mellonella has become a favourite model host for many insects and human pathogens for investigating the interactions between pathogens and innate immune systems [6,7]. It has well been documented that the insect immune response manifests a number of similarities with the innate immune response of mammals. For example, insect hemocytes and mammalian neutrophils have been shown to phagocytose and kill pathogens in a similar manner [8,9]. Furthermore, the Toll and IMD signalling pathways that mediate the synthesis of AMPs in insects are similar to the Toll-like receptor (TLR) and TNF␣ pathways of mammals, respectively [10]. Although insects do not have the adaptive immune response of mammals and cannot generate antibodies, they show some elements of similarity to the function of the adaptive immune response of mammals. Recent studies have documented that a primary exposure of insects to elicitors (e.g., fungi, bacteria, viruses, protozoa, and the cell wall components of pathogens) increased their resistance to a later pathogenic challenge, a phenomenon called “immune priming” [11–13].
http://dx.doi.org/10.1016/j.imlet.2016.04.013 0165-2478/© 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
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Fig. 1. Killing of Galleria mellonella caterpillars by viable Photorhabdus luminescens TT01 depends on the priming dose of heat-killed P. luminescens TT01. Kaplan–Meier plots of the PBS (group 1), 1 × 103 (group 2), 1 × 104 (group 3), 1 × 105 (group 4) or 1 × 106 (group 5) cells of heat-killed P. luminescens TT01 primed Galleria mellonella larvae survival after infection with viable P. luminescens TT01.
Previous studies demonstrated that the immune priming response provides individual insects with enhanced immunity [11,14,15]. Functionally, the priming effect enhances insect survival upon secondary infection and can be long lasting—even persisting across life stages and generations [16]. However, the proximate mechanisms governing the long term and even trans-generational induction of immune priming currently continue to elude us. It has been documented that the primed response can be pathogenspecific, even to different strains of the same pathogen species [15]. In other words, insect immune responses can discriminate between different challenges. However, whether insects have the ability to mount a proportionate cellular (e.g., total hemocyte counts, phagocytosis and encapsulation abilities of the hemocytes) and humoral (e.g., the levels of AMPs and antbacterial activity of the hemolymph) defense responses according to different priming doses of Photorhabdus luminescens TT01 are still not fully understood. P. luminescens is an enterobacterium that is symbiotic with soil entomopathogenic nematodes and pathogenic to a wide range of insects [17]. In the work presented here, we sought to establish whether there was a positive correlation between a dose of heatkilled P. luminescens TT01 and the degree of enhanced immune protection and whether the immune system of G. mellonella can also differentiate between different amount of an encountered immune priming agent and respond accordingly.
estimated using a hemocytometer and confirmed by plating a known volume of the injected suspension onto nutrient bromothymol blue-triphenyl tetrazolium chloride agar (NBTA) plates. For both priming and challenge, a 50-l Hamilton syringe was used to inject 10-l aliquots of the inoculum into the hemocoel of each caterpillar via the last left proleg. Before injection, the area was cleaned using an alcohol swab. After injection, caterpillars were reared at the same condition and incubated in plastic containers. 2.2. G. mellonella killing assay Five groups (approximately 35 larvae per group) of G. mellonella larvae were immune-primed by hemocoel injection of 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 of heatkilled P. luminescens TT01 cells per larva, respectively. At 48 h after immune priming, each larva was infected by injection with 10 l of a PBS suspension containing approximately 50 viable P. luminescens TT01 cells. The number of dead caterpillars was scored after infection. Caterpillars were considered dead when they displayed no movement in response to touch. For all the killing assays, we plotted killing curves and analysed differences in survival with the KaplanMeier method using STATA 6 statistical software. A P-value of less than 0.05 was considered significant. Three independent trials were performed for all treatments.
2. Materials and methods 2.1. Insects and bacteria
2.3. Hemolymph collection for measurement of immune parameters and RNA isolation
G. mellonella (Lepidoptera: Pyralidae) larvae were reared on an artificial diet and the bacteria P. luminescens TT01 used for priming were prepared as described previously [18]. Last instar G. mellonella larvae were chosen for all experiments. The bacterial density was estimated using a hemocytometer and adjusted to the required concentration with phosphate buffered saline (PBS: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2 HPO4 , and 0.24 g of KH2 PO4 in 1000 ml of distilled water, pH 7.2). The numbers of viable P. luminescens TT01 cells injected were
For each repeat of the experiment, five groups of G. mellonella larvae were immune-primed by hemocoel injection of 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 of heat-killed P. luminescens TT01 cells per larva, respectively. At 48 h after the immune priming, fifteen larvae were sampled from each group for hemolymph collection. All experiments were performed on three independent occasions. In each group, 40 l of fresh hemolymph from each larva was collected from punctured prolegs. The hemolymph was stored in
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10 l of the hemolymph was immediately added to 20 l of anticoagulant buffer for the phagocytosis assays; and 3, The remaining hemolymph was centrifuged at 500 g for 5 min at 4 ◦ C and the hemocytes were kept at −70 ◦ C for RNA isolation. 2.4. Total hemocyte counts (THC) To determine the hemocyte counts, aliquots of the hemolymph suspension prepared as described above were transferred to a Neubauer hemocytometer. The cells were counted using a phase contrast microscope. The hemocyte counts were expressed as the number of cells per milliliter of hemolymph. 2.5. In vitro phagocytosis assay
Fig. 2. Changes in total hemocyte counts (THC) of Galleria mellonella larvae at 48 h after priming with different doses of heat-killed Photorhabdus luminescens TT01. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
micro-tubes on ice to minimize cell clumping. The hemolymph from each individual was divided into several aliquots and processed as follows: (1) A total of 20 l of the hemolymph was diluted in five volumes of anticoagulant solution (93 mM NaCl, 100 mM glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM Na2 EDTA, and a few crystals of phenylthiourea (PTU), pH 4.6) and used immediately for the total hemocyte count assay; (2) A total of
To prepare fluorescently labelled bacteria, heat-killed P. luminescens TT01 cells were resuspended in carbonate buffer (0.2 M Na2 CO3 , 0.2 M NaHCO3 , pH 9.4) containing fluorescein isothiocyanate (FITC, 0.1 mg/ml). The suspension was then incubated for 30 min in the dark at 28 ◦ C and mixed at 200 rpm on a rotary mixer, washed 3 times with PBS to remove all traces of free FITC, and finally resuspended in Grace’s insect medium at a concentration of 109 cells per milliliter. The in vitro phagocytosis quenching assay was performed according to the method described previously [19]. The number of cells which phagocytosed FITC-labelled P. luminescens TT01 was determined under fluorescence microscope (Nikon Eclipse TE2000U). To quantify phagocytic rate for each cover slip, five fields containing at least 100 hemocytes were examined and the phago-
Fig. 3. (A) Bacterial presence in the hemolymph of Galleria mellonella primed with 0 (a) 1 × 103 (b) 1 × 104 (c) 1 × 105 (d) or 1 × 106 (e) cells of heat-killed Photorhabdus luminescens TT01 and followed by 50 cells of viable P. luminescens TT01 infection at 48 h after priming. The hemolymph was collected at 24 h after larval infection with viable P. luminescens TT01. Black arrows indicate bacterial cells. (B) Bacterial density in the hemolymph of G. mellonella primed with different doses of heat-killed P. luminescens TT01. Scale bar = 5 m. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
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Fig. 4. (A) Hemocytes phagocytosis images of Galleria mellonella larvae taken with a differential interference contrast microscope and a fluorescence microscope at 48 h after priming with 0 (a), 1 × 103 (b), 1 × 104 (c), 1 × 105 (d) or 1 × 106 (e) cells of heat-killed Photorhabdus luminescens TT01. (B) Changes in the phagocytic rate of hemocytes collected from G. mellonella larvae at 48 h after priming with different doses of heat-killed P. luminescens TT01. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
cytic rate was determined using the following formula: Phagocytic rate = [(phagocytic hemocytes)/(total hemocytes)] × 100%. 2.6. In vivo encapsulation assay An in vivo encapsulation assay was performed according to a modification of a method of Hu et al. [20]. Sephadex DEAE A-25 chromatography beads (Pharmacia) were used as encapsulation targets. To facilitate identification of the beads in vivo, they were stained in a 0.1% Congo red solution for 2 h. The beads were dried under UV light, and resuspended in PBS solution. Five groups of G. mellonella larvae were immune-primed by hemocoel injection of 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 of heat-killed P. luminescens TT01 cells per larva. At 48 h after the immune priming, 10 larvae were selected from each treatment, and approximately 30 beads were injected into each larval hemocoel using a microsyringe. The larvae were dissected 24 h post-injection, and the beads were examined under a microscope (as shown in Fig. 5A). For each bead, the radius (R) of the bead and the thickness (T) of the capsule were measured. To compare the extent of encapsulation of the injected beads, the bead capsules were classified into four grades according to their thickness as follows: 1, beads with adherent cells, but without a clear capsule were counted as unencapsulated; 2, beads with a clear capsule, but 0 < T/R < 0.5; 3, 0.5 < T/R < 1; and 4, T/R > 1 (as shown in Fig. 3A). The extent of encapsulation was estimated by weighted sum as follows: weighted sum = (P × A), where P represents the percent of encapsulated beads of every grade to the total beads, and A represents the grade number of encapsulated beads (1–4).
2.7. Real-time quantitative RT-PCR Total RNA from the hemocytes was isolated using a Trizol Extraction Kit (Invitrogen). Followed by DNase treatment, the firststrand cDNA was synthesized by iScriptTM cDNA Synthesis Kit (Bio-Rad). The qPCR was performed using a Bio-Rad IQ5 Real-Time PCR system with SYBR-Green detection (SYBR Premix, TIANGEN) according to the manufacturer’s instructions. The primers for S7e, gallerimycin, cecropin and galiomycin genes were chosen as described previously [21]. The primers used for PCR were as follow: S7e forward, 5 -ATG TGC CAA TGC CCA AGT TG-3 , S7e reverse, 5 GTG GCT AGG CTT GGG AAG AAT-3 ; gallerimycin forward, 5 -TAT CAT TGG CCT TCT TGG CTG-3 , gallerimycin reverse, 5 -GCA CTC GTA AAA TAC ACA TCC GG-3 ; cecropin forward, 5 -ATT TGC CTG CAT CGT AGC G-3 , cecropin reverse, 5 -CTT GTA CTG CTG GAC CAG CTT TT-3 ; galiomycin forward, 5 -TCG TAT CGT CAC CGC AAA ATG3 , galiomycin reverse, 5 -GCC GCA ATG ACC ACC TTT ATA-3 . All the products were of 131 bp length. The real-time qPCR conditions were: 95 ◦ C for 3 min, 44 × (95 ◦ C for 15 s, 60 ◦ C for 15 s, 72 ◦ C for 1 min). Each reaction was run in triplicate, after which the average threshold cycle (Ct) was calculated per sample. The amount of mRNA detected was normalized to ribosomal protein S7e mRNA values (a housekeeping gene, whose level was constant in all the conditions tested).
2.8. Bacterial infection of G. mellonella (in vivo bacterial growth) Five groups of G. mellonella larvae were immune-primed with 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or
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Fig. 5. (A) Encapsulation of Sephadex A-25 beads by Galleria mellonella larvae hemocytes in vivo: (a) grade 1; (b) grade 2; (c) grade 3; (d) grade 4; (for details see Section 2.5). (B) Encapsulation index of hemocytes in G. mellonella larvae primed with different doses of heat-killed Photorhabdus luminescens TT01. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
1 × 106 of heat-killed P. luminescens TT01 cells per larva, respectively. At 48 h after the immune priming, 10 l of PBS containing approximately 50 cells of viable TT01 were injected into the hemocoel cavity of each G. mellonella larva. In all the experiments larvae were bled 24 h after the injection. Hemolymph was obtained from punctured prolegs of the larvae; whole hemolymph samples were then processed by two low speed centrifugations (700 g for 10 min, at 4 ◦ C) to remove hemocytes and tissue debris. The plasma was diluted in PBS solution. Aliquots of cell-free fractions of supernatants were placed on an NBTA plate. Petri dishes were incubated for 48 h at 28 ◦ C, and bacteria were quantified by Colony Forming Units (CFU) count. In addition, in order to examine the presence of growing bacteria in the host hemocoel cavity, five groups of
G. mellonella larvae were slected and treated as described above. Hemolymph samples in each group were diluted in five volumes of Grace’s insect medium and assayed visually by light microscope in microwells cultures.
2.9. Data processing and statistical analysis Differences between mean values were analyzed with one-way analysis of variance (ANOVA) by using the least significant difference (LSD) test and considered significant when P < 0.05. All experiments were replicated at least three times. Data were processed with DPS package.
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3. Results 3.1. The degree of immune protection of G. mellonella against viable P. luminescens TT01 infection depended on the doses of heat-killed P. luminescens TT01 with which they were inoculated As shown in Fig. 1, the larvae of the control group were killed significantly faster compared to larvae primed with 1 × 104 (P = 0.0001), 1 × 105 (P < 0.0001) or 1 × 106 (P < 0.0001) cells of heatkilled P. luminescens TT01 48 h prior to the viable P. luminescens TT01 infection. In addition, caterpillar killing speed depended on the dose of heat-killed P. luminescens TT01 with which they were inoculated. For example, larvae primed with 1 × 103 cells of heatkilled P. luminescens TT01 were killed significantly faster compared to larvae that received higher doses (1 × 105 or 1 × 106 cells/larva) of heat-killed P. luminescens TT01 priming when the larvae were challenged with viable P. luminescens TT01 48 h after priming (P < 0.0001). There was no significant difference in killing speed between the 1 × 103 cells of heat-killed P. luminescens TT01 priming group and the control group. 3.2. Administration of heat-killed P. luminescens TT01 leads to alterations in THC and bacterial density in the larval hemolymph In this work, the THC of larvae that had been inoculated with different doses of heat-killed P. luminescens TT01 was ascertained at 48 h after inoculation. The results (Fig. 2) indicated that after 48 h, THC of larvae that received a dose of 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 heat-killed P. luminescens TT01 per larva had increased to approximately 19.3 × 106 , 20.8 × 106 , 30.0 × 106 , 32.9 × 106 cells per larva, respectively and was significantly higher than the THC of the larvae inoculated with PBS (P < 0.05). The bacterial density in the larval hemolymph was also examined at 24 h after infection with viable P. luminescens TT01 in each primed group and the control group. Those larvae that had been primed with different doses (1 × 104 , 1 × 105 and 1 × 106 cells/larva) of heat-killedP. luminescens TT01 48 h prior to infection showed significantly lower bacterial densities (32.5 × 107 , 10.6 × 107 and 3.1 × 107 cfu/ml, respectively) at 24 h after viable P. luminescens TT01 infection than the control group (Fig. 3A, B). In contrast, the larvae that had been primed with the lower dose (1 × 103 cells/larva) of heat-killed P. luminescens TT01 showed significantly higher bacterial density than those larvae that had been primed with the higher doses of heat-killed P. luminescens TT01 (1 × 104 , 1 × 105 and 1 × 106 cells/larva) at 24 h after being challenged with viable P. luminescens TT01, and they displayed no significant difference from the control group. 3.3. Priming G. mellonella with heat-killed P. luminescens TT01 caused dose-dependent elevation in phagocytosis and encapsulation abilities of the hemocytes The phagocytic images (Fig. 4A) generated by merging images taken with a differential interference contrast microscope and a fluorescence microscope clearly reveal changes in phagocytic activities 48 h after priming the larvae with different doses of heat-killed P. luminescens TT01. Phagocytic activities of hemocyte collected from larvae primed with 1 × 104 , 1 × 105 or 1 × 106 cells of heatkilled P. luminescens TT01 increased to 50.5%, 56.4% and 61.8%, respectively and were significantly higher than that of the control group (Fig. 4B). In contrast, there was no significant difference between the 1 × 103 cells of heat-killed P. luminescens TT01 per larva priming group and the control group at 48 h after-priming. We also observed similarly changed patterns in the encapsulation index. Compared with the control, the larvae primed with the higher doses (1 × 105 or 1 × 106 cells/larva) of heat-killed P. lumi-
Fig. 6. Quantitative RT-PCR analysis of cecropin, galiomycin, and gallerimycin transcripts in total RNA isolated from the hemocytes of Galleria mellonella larvae at 48 h after priming with different doses of heat-killed Photorhabdus luminescens TT01. Expression of each gene was normalized to the expression of S7e ±SD. Values for the same antimicrobial peptide gene followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
nescens TT01 significantly enhanced the encapsulation index of the hemocyte, while no significant differences could be observed in the other treatment groups (Fig. 5B). 3.4. Administration of heat-killed P. luminescens TT01 results in increased gene expression levels of cecropin and gallerimycin and galiomycin in the hemocytes of G. mellonella larvae We studied the appearance of transcripts for AMPs in the hemocytes of the larvae primed with different doses of heat-killed P. luminescens TT01 cells and noticed a gradual increase in the amount of transcripts for cecropin and gallerimycin and galiomycin as the priming dose of heat-killed P. luminescens TT01 increased. In other words, at 48 h after immune-priming, the expression of each tested gene was significantly higher in the hemocytes of the larvae preexposed to the higher doses of heat-killed P. luminescens TT01 (1 × 104 , 1 × 105 or 1 × 106 cells/larva) in comparison to the nonprimed (PBS control) larvae (Fig. 6). 4. Discussion Here we showed that priming G. mellonella larvae with different doses of heat-killed P. luminescens TT01 protects the insects from a challenge 48 h later with a lethal dose of viable P. luminescens TT01. As a response to the priming with heat-killed P. luminescens TT01 the larvae also showed elevated levels of humoral (expression of genes coding for AMPs) and cellular (hemocyte number, phagocytosis and encapsulation abilities of hemocytes) immune parameters 48 h after priming, offering a potential explanation for the immune protection. Previous work has demonstrated that sub-lethal doses of Aspergillus fumigatus, Candida albicans or Saccharomyces cerevisiae have a similar immune priming effect on inoculated G. mellonella larvae [12,22]. Although our previous work revealed that the heat-killed P. luminescens TT01 cells can elicit immune priming in G. mellonella larvae [18], the work presented here demonstrated for the first time a correlation between the dose of heat-killed P. luminescens TT01 inoculated and the levels of change in cellular and humoral immune parameters evident in G. mellonella larvae. Priming the larvae with doses of 1 × 104 , 1 × 105 or 1 × 106 heat-killed P. luminescens TT01 cells per larva was sufficient to render the larvae resistant to viable P. luminescens
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TT01 infection and provoked the increases in humoral and cellular immune parameters found at 48 h. In contrast larvae primed with heat-killed P. luminescens TT01 at a dose of 1 × 103 cells per larva showed no significant difference in killing speed relative to that observed in PBS inoculated (control) larvae and no significant increase in humoral and cellular immune parameters or decrease in the bacterial density. These results indicated that G. mellonella mount an immune response proportionate to the size of the inoculums to reduce the immunological cost to the larva. Previous work demonstrated that the hemocyte number of insects is altered by the presence of pathogenic stimuli or by microbial cell wall components [13,23]. Our results showed that the hemocyte number of the larvae increases as priming dose of heatkilled P. luminescens TT01 cells increases. A Previous study showed that the elevated levels of hemocytes were possibly due to the release of hemocytes attached to the internal organs within the insect [24]. In another study, the higher number of hemocytes correlated positively to a greater ability to fight invaders [25]. In this study we found that the higher level of hemocyte density in larvae primed with 1 × 106 heat-killed P. luminescens TT01 cells correlated positively with increased resistance to subsequent infection. Therefore, we can conclude that the higher level of hemocyte density in the larvae benefits to phagocytosing and killing invaded pathogens. We also observed a high correlation between the protection level and the increase in both phagocytosis and encapsulation abilities of the hemocytes in the heat-killed P. luminescens TT01 primed larvae. The larvae primed with the highest dose (1 × 106 cells/larva) of heat-killed P. luminescens TT01 showed significantly higher phagocytosis rates and encapsulation indices than the larvae that received the lower doses (1 × 103 , 1 × 104 cells/larva) of heat-killed P. luminescens TT01. These result is in line with a previous study showing that pre-exposure of juvenile shrimp to a formalin-inactivated pathogenic vibrio results in an increased phagocytic uptake of this bacterium by the hemocytes [26]. Phagocytosis and encapsulation represent the main elements of insect cellular responses that play key roles against invading pathogens. The up-regulation activity of phagocytosis and encapsulation following immune priming is of great benefit to resisting a subsequent lethal P. luminescens TT01 infection—but it does impose a cost on the insect and can even be fatal [27]. Therefore it is reasonable that entry of a low level of pathogen or pathogen associated material would appear to activate a response designed to eliminate the threat. The work presented here demonstrates that administration of heat-killed P. luminescens TT01 induces immune priming, and it is clear that the amount of heat-killed P. luminescens TT01 administered to the insect determines the degree of the immune response. Analysis of the expression of genes coding for antimicrobial peptides in the hemocytes of the larvae after immune-priming and examination of bacterial density in the hemolymph of immuneprimed larvae after viable P. luminescens TT01 infection were performed to establish whether there were correlations with decreased larval mortality following priming with heat-killed P. luminescens TT01 bacteria. Our results showed that when larvae are immune-primed with appropriate doses of heat-killed P. luminescens TT01 there is an increase in the expression of genes coding for antimicrobial peptides, such as gallerimycin, cecropin, and galiomycin. We also observed that the level of transcripts increased in a dose-dependent manner. This is consistent with the changes of bacterial density in the larval hemolymph; and the larvae primed with higher dose of heat-killed P. luminescens TT01 showed significant lower bacterial density when infected with viable P. luminescens TT01. Consequently, we can conclude that activation of defence mechanisms after immune priming is also noticeable at the molecular level. The elevated mRNA levels of genes coding for antimicrobial peptides have previously
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been recorded in larvae challenged with bacteria. For example, gallerimycin and cecropin have been shown to increase in expression when larvae were inoculated with Bacillus thuringiensis [28]. Galiomycin has been shown to be induced in larvae infected with Escherichia coli and to have antifungal as well as antibacterial activities [29]. The production of AMPs, a key component of the humoral response, serves to kill pathogens that have escaped or withstood the cellular immune response. This work indicated that prior exposure to a heat-killed P. luminescens TT01 primes the G. mellonella immune system and allows the larvae to withstand a subsequent viable P. luminescens TT01 infection. This effect is also mediated by the production of elevated levels of AMPs which protect the insect from the second exposure to the pathogen. A dose-dependent immune response in G. mellonella larvae to heat-killed P. luminescens TT01 inoculation indicated that insects can modulate their immune responses depending upon the nature and size of the microbial challenge. Upregulation of immune responses in bumble bees following exposure to low levels of pathogen, but in the absence of compensatory feeding can be fatal [27], thus indicating that immune protection does have a cost for the insect. Elevated immunity following such immune priming offers protection to G. mellonella larvae and would have an obvious survival advantage but does have a cost in terms of utilization of resources [16,30]. Therefore, we can conclude that G. mellonella larvae mount an immune response proportionate to the amount of the priming agent to ensure survival but minimize the use of resources. The present study will increase our knowledge of invertebrate immunity and can be of benefit for understanding the evolution, ecology, and epidemiology of economic invertebrates diseases. Acknowledgements This work was supported by the National Science of Foundation of China (No. 21301034) and the National Science of Foundation of Guangdong Province (S2013040014083). References [1] S.A. Armitage, R. Peuss, J. Kurtz, Dscam and pancrustacean immune memory−a review of the evidence, Dev. Comp. Immunol. 48 (2015) 315–323. [2] C.J. Coates, T. Whalley, J. Nairn, Phagocytic activity of Limulus polyphemus amebocytes in vitro, J. Invertebr. Pathol. 111 (2012) 205–210. [3] C. Anagnostou, E.A. LeGrand, M. Rohlfs, Friendly food for fitter flies?−Influence of dietary microbial species on food choice and parasitoid resistance in Drosophila, Oikos 119 (2010) 533–541. [4] A. Tokura, G.S. Fu, M. Sakamoto, H. Endo, S. Tanaka, S. Kikuta, et al., Factors functioning in nodule melanization of insects and their mechanisms of accumulation in nodules, J. Insect Physiol. 60 (2014) 40–49. [5] A.L. Flores-Villegas, P.M. Salazar-Schettino, A. Cordoba-Aguilar, A.E. Gutierrez-Cabrera, G.E. Rojas-Wastavino, M.I. Bucio-Torres, et al., Immune defence mechanisms of triatomines against bacteria, viruses, fungi and parasites, Bull. Entomol. Res. 105 (2015) 523–532. [6] C.J. Tsai, J.M. Loh, T. Proft, Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing, Virulence (2016) 1–16. [7] J.C. Junqueira, Galleria mellonella as a model host for human pathogens, Virulence 3 (2012) 474–476. [8] N. Browne, M. Heelan, K. Kavanagh, An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes, Virulence 4 (2013) 597–603. [9] J. Renwick, E.P. Reeves, F.B. Wientjes, K. Kavanagh, Translocation of proteins homologous to human neutrophil p47(phox) and p67(phox) to the cell membrane in activated hemocytes of Galleria mellonella, Dev. Comp. Immunol. 31 (2007) 347–359. [10] R.S. Khush, F. Leulier, B. Lemaitre, Drosophila immunity: two paths to NF-kappa B, Trends Immunol. 22 (2001) 260–264. [11] B.M. Sadd, P. Schmid-Hempel, Insect immunity shows specificity in protection upon secondary pathogen exposure, Curr. Biol. 16 (2006) 1206–1210. [12] J.P. Fallon, N. Troy, K. Kavanagh, Pre-exposure of Galleria mellonella larvae to different doses of Aspergillus fumigatus conidia causes differential activation of cellular and humoral immune responses, Virulence 2 (2011) 413–421. [13] P. Mowlds, C. Coates, J. Renwick, K. Kavanagh, Dose-dependent cellular and humoral responses in Galleria mellonella larvae following beta-glucan inoculation, Microbes Infect. 12 (2010) 146–153.
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[14] M. Moreno-Garcia, V. Vargas, I. Ramirez-Bello, G. Hernandez-Martinez, H. Lanz-Mendoza, Bacterial exposure at the larval stage induced sexual immune dimorphism and priming in adult Aedes aegypti mosquitoes, PLoS One 10 (2015). [15] G. Wu, M. Li, Y. Liu, Y. Ding, Y. Yi, The specificity of immune priming in silkworm, Bombyx mori, is mediated by the phagocytic ability of granular cells, J. Insect Physiol. 81 (2015) 60–68. [16] J. Moreau, G. Martinaud, J.P. Troussard, C. Zanchi, Y. Moret, Trans-generational immune priming is constrained by the maternal immune response in an insect, Oikos 121 (2012) 1828–1832. [17] E. Duchaud, C. Rusniok, L. Frangeul, C. Buchrieser, A. Givaudan, S. Taourit, et al., The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens, Nat. Biotechnol. 21 (2003) 1307–1313. [18] G. Wu, Z. Zhao, C. Liu, L. Qiu, Priming Galleria mellonella (Lepidoptera: pyralidae) larvae with heat-killed bacterial cells induced an enhanced immune protection against Photorhabdus luminescens TT01 and the role of innate immunity in the process, J. Econ. Entomol. 107 (2014) 559–569. [19] G.Q. Wu, Y.H. Yi, Effects of dietary heavy metals on the immune and antioxidant systems of Galleria mellonella larvae, Comp. Biochem. Phys. C 167 (2015) 131–139. [20] J. Hu, X.X. Zhu, W.J. Fu, Passive evasion of encapsulation in Macrocentrus cingulum Brischke (Hymenoptera: braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenee (Lepidoptera: pyralidae), J. Insect Physiol. 49 (2003) 367–375. [21] I. Wojda, T. Jakubowicz, Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae, J. Insect Physiol. 53 (2007) 1134–1144. [22] D. Bergin, L. Murphy, J. Keenan, M. Clynes, K. Kavanagh, Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by
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Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses Gongqing Wu a,b , Li Xu a , Yunhong Yi a,∗ a b
School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Zhongshan 528458, China Guangdong Cosmetics Engineering & Technology Research Center, China
a r t i c l e
i n f o
Article history: Received 7 March 2016 Received in revised form 16 April 2016 Accepted 16 April 2016 Available online 20 April 2016 Keywords: Galleria mellonella Photorhabdus luminescens TT01 Innate immunity
a b s t r a c t Larvae of Galleria mellonella are useful models for studying the innate immunity of invertebrates or for evaluating the virulence of microbial pathogens. In this work, we demonstrated that prior exposure of G. mellonella larvae to high doses (1 × 104 , 1 × 105 or 1 × 106 cells/larva) of heat-killed Photorhabdus luminescens TT01 increases the resistance of larvae to a lethal dose (50 cells/larva) of viable P. luminescens TT01 infection administered 48 h later. We also found that the changes in immune protection level were highly correlated to the changes in levels of cellular and humoral immune parameters when priming the larvae with different doses of heat-killed P. luminescens TT01. Priming the larvae with high doses of heatkilled P. luminescens TT01 resulted in significant increases in the hemocytes activities of phagocytosis and encapsulation. High doses of heat-killed P. luminescens TT01 also induced an increase in total hemocyte count and a reduction in bacterial density within the larval hemocoel. Quantitative real-time PCR analysis showed that genes coding for cecropin and gallerimycin and galiomycin increased in expression after priming G. mellonella with heat-killed P. luminescens TT01. All the immune parameters changed in a dose-dependent manner. These results indicate that the insect immune system is capable of sensing the extent of priming agent and mounting a proportionate immune response. © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Insects are a large and diverse group of animals that have adapted to extreme environments. They inhabit all ecological niches on the Earth with the exception of the oceans. The success of insects in colonizing such a wide variety of habitats is partly attributed to their effective immune response. Although insects do not have the adaptive immune machinery such as clonal expansion of antigen-specific lymphocytes, to generate a broad defence capability [1], they possess a robust innate immune system and that equips them with the ability to fight off infection. The innate immune system of insects consists of cellular and humoral defense responses. The cellular immune response, mediated by different hemocyte types, clears pathogenic microorganisms by phagocytosis or nodulation and kills large intruder (e.g., an egg of a parasitic wasp, nematode) by encapsulation [2–4]. The humoral response refers to effector molecules including complement-like proteins,
∗ Corresponding author. E-mail address: [email protected] (Y. Yi).
antimicrobial peptides (AMPs), and products generated by the phenoloxidase (PO) pathway [5]. The greater wax moth, Galleria mellonella has become a favourite model host for many insects and human pathogens for investigating the interactions between pathogens and innate immune systems [6,7]. It has well been documented that the insect immune response manifests a number of similarities with the innate immune response of mammals. For example, insect hemocytes and mammalian neutrophils have been shown to phagocytose and kill pathogens in a similar manner [8,9]. Furthermore, the Toll and IMD signalling pathways that mediate the synthesis of AMPs in insects are similar to the Toll-like receptor (TLR) and TNF␣ pathways of mammals, respectively [10]. Although insects do not have the adaptive immune response of mammals and cannot generate antibodies, they show some elements of similarity to the function of the adaptive immune response of mammals. Recent studies have documented that a primary exposure of insects to elicitors (e.g., fungi, bacteria, viruses, protozoa, and the cell wall components of pathogens) increased their resistance to a later pathogenic challenge, a phenomenon called “immune priming” [11–13].
http://dx.doi.org/10.1016/j.imlet.2016.04.013 0165-2478/© 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
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Fig. 1. Killing of Galleria mellonella caterpillars by viable Photorhabdus luminescens TT01 depends on the priming dose of heat-killed P. luminescens TT01. Kaplan–Meier plots of the PBS (group 1), 1 × 103 (group 2), 1 × 104 (group 3), 1 × 105 (group 4) or 1 × 106 (group 5) cells of heat-killed P. luminescens TT01 primed Galleria mellonella larvae survival after infection with viable P. luminescens TT01.
Previous studies demonstrated that the immune priming response provides individual insects with enhanced immunity [11,14,15]. Functionally, the priming effect enhances insect survival upon secondary infection and can be long lasting—even persisting across life stages and generations [16]. However, the proximate mechanisms governing the long term and even trans-generational induction of immune priming currently continue to elude us. It has been documented that the primed response can be pathogenspecific, even to different strains of the same pathogen species [15]. In other words, insect immune responses can discriminate between different challenges. However, whether insects have the ability to mount a proportionate cellular (e.g., total hemocyte counts, phagocytosis and encapsulation abilities of the hemocytes) and humoral (e.g., the levels of AMPs and antbacterial activity of the hemolymph) defense responses according to different priming doses of Photorhabdus luminescens TT01 are still not fully understood. P. luminescens is an enterobacterium that is symbiotic with soil entomopathogenic nematodes and pathogenic to a wide range of insects [17]. In the work presented here, we sought to establish whether there was a positive correlation between a dose of heatkilled P. luminescens TT01 and the degree of enhanced immune protection and whether the immune system of G. mellonella can also differentiate between different amount of an encountered immune priming agent and respond accordingly.
estimated using a hemocytometer and confirmed by plating a known volume of the injected suspension onto nutrient bromothymol blue-triphenyl tetrazolium chloride agar (NBTA) plates. For both priming and challenge, a 50-l Hamilton syringe was used to inject 10-l aliquots of the inoculum into the hemocoel of each caterpillar via the last left proleg. Before injection, the area was cleaned using an alcohol swab. After injection, caterpillars were reared at the same condition and incubated in plastic containers. 2.2. G. mellonella killing assay Five groups (approximately 35 larvae per group) of G. mellonella larvae were immune-primed by hemocoel injection of 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 of heatkilled P. luminescens TT01 cells per larva, respectively. At 48 h after immune priming, each larva was infected by injection with 10 l of a PBS suspension containing approximately 50 viable P. luminescens TT01 cells. The number of dead caterpillars was scored after infection. Caterpillars were considered dead when they displayed no movement in response to touch. For all the killing assays, we plotted killing curves and analysed differences in survival with the KaplanMeier method using STATA 6 statistical software. A P-value of less than 0.05 was considered significant. Three independent trials were performed for all treatments.
2. Materials and methods 2.1. Insects and bacteria
2.3. Hemolymph collection for measurement of immune parameters and RNA isolation
G. mellonella (Lepidoptera: Pyralidae) larvae were reared on an artificial diet and the bacteria P. luminescens TT01 used for priming were prepared as described previously [18]. Last instar G. mellonella larvae were chosen for all experiments. The bacterial density was estimated using a hemocytometer and adjusted to the required concentration with phosphate buffered saline (PBS: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2 HPO4 , and 0.24 g of KH2 PO4 in 1000 ml of distilled water, pH 7.2). The numbers of viable P. luminescens TT01 cells injected were
For each repeat of the experiment, five groups of G. mellonella larvae were immune-primed by hemocoel injection of 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 of heat-killed P. luminescens TT01 cells per larva, respectively. At 48 h after the immune priming, fifteen larvae were sampled from each group for hemolymph collection. All experiments were performed on three independent occasions. In each group, 40 l of fresh hemolymph from each larva was collected from punctured prolegs. The hemolymph was stored in
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10 l of the hemolymph was immediately added to 20 l of anticoagulant buffer for the phagocytosis assays; and 3, The remaining hemolymph was centrifuged at 500 g for 5 min at 4 ◦ C and the hemocytes were kept at −70 ◦ C for RNA isolation. 2.4. Total hemocyte counts (THC) To determine the hemocyte counts, aliquots of the hemolymph suspension prepared as described above were transferred to a Neubauer hemocytometer. The cells were counted using a phase contrast microscope. The hemocyte counts were expressed as the number of cells per milliliter of hemolymph. 2.5. In vitro phagocytosis assay
Fig. 2. Changes in total hemocyte counts (THC) of Galleria mellonella larvae at 48 h after priming with different doses of heat-killed Photorhabdus luminescens TT01. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
micro-tubes on ice to minimize cell clumping. The hemolymph from each individual was divided into several aliquots and processed as follows: (1) A total of 20 l of the hemolymph was diluted in five volumes of anticoagulant solution (93 mM NaCl, 100 mM glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM Na2 EDTA, and a few crystals of phenylthiourea (PTU), pH 4.6) and used immediately for the total hemocyte count assay; (2) A total of
To prepare fluorescently labelled bacteria, heat-killed P. luminescens TT01 cells were resuspended in carbonate buffer (0.2 M Na2 CO3 , 0.2 M NaHCO3 , pH 9.4) containing fluorescein isothiocyanate (FITC, 0.1 mg/ml). The suspension was then incubated for 30 min in the dark at 28 ◦ C and mixed at 200 rpm on a rotary mixer, washed 3 times with PBS to remove all traces of free FITC, and finally resuspended in Grace’s insect medium at a concentration of 109 cells per milliliter. The in vitro phagocytosis quenching assay was performed according to the method described previously [19]. The number of cells which phagocytosed FITC-labelled P. luminescens TT01 was determined under fluorescence microscope (Nikon Eclipse TE2000U). To quantify phagocytic rate for each cover slip, five fields containing at least 100 hemocytes were examined and the phago-
Fig. 3. (A) Bacterial presence in the hemolymph of Galleria mellonella primed with 0 (a) 1 × 103 (b) 1 × 104 (c) 1 × 105 (d) or 1 × 106 (e) cells of heat-killed Photorhabdus luminescens TT01 and followed by 50 cells of viable P. luminescens TT01 infection at 48 h after priming. The hemolymph was collected at 24 h after larval infection with viable P. luminescens TT01. Black arrows indicate bacterial cells. (B) Bacterial density in the hemolymph of G. mellonella primed with different doses of heat-killed P. luminescens TT01. Scale bar = 5 m. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
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Fig. 4. (A) Hemocytes phagocytosis images of Galleria mellonella larvae taken with a differential interference contrast microscope and a fluorescence microscope at 48 h after priming with 0 (a), 1 × 103 (b), 1 × 104 (c), 1 × 105 (d) or 1 × 106 (e) cells of heat-killed Photorhabdus luminescens TT01. (B) Changes in the phagocytic rate of hemocytes collected from G. mellonella larvae at 48 h after priming with different doses of heat-killed P. luminescens TT01. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
cytic rate was determined using the following formula: Phagocytic rate = [(phagocytic hemocytes)/(total hemocytes)] × 100%. 2.6. In vivo encapsulation assay An in vivo encapsulation assay was performed according to a modification of a method of Hu et al. [20]. Sephadex DEAE A-25 chromatography beads (Pharmacia) were used as encapsulation targets. To facilitate identification of the beads in vivo, they were stained in a 0.1% Congo red solution for 2 h. The beads were dried under UV light, and resuspended in PBS solution. Five groups of G. mellonella larvae were immune-primed by hemocoel injection of 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 of heat-killed P. luminescens TT01 cells per larva. At 48 h after the immune priming, 10 larvae were selected from each treatment, and approximately 30 beads were injected into each larval hemocoel using a microsyringe. The larvae were dissected 24 h post-injection, and the beads were examined under a microscope (as shown in Fig. 5A). For each bead, the radius (R) of the bead and the thickness (T) of the capsule were measured. To compare the extent of encapsulation of the injected beads, the bead capsules were classified into four grades according to their thickness as follows: 1, beads with adherent cells, but without a clear capsule were counted as unencapsulated; 2, beads with a clear capsule, but 0 < T/R < 0.5; 3, 0.5 < T/R < 1; and 4, T/R > 1 (as shown in Fig. 3A). The extent of encapsulation was estimated by weighted sum as follows: weighted sum = (P × A), where P represents the percent of encapsulated beads of every grade to the total beads, and A represents the grade number of encapsulated beads (1–4).
2.7. Real-time quantitative RT-PCR Total RNA from the hemocytes was isolated using a Trizol Extraction Kit (Invitrogen). Followed by DNase treatment, the firststrand cDNA was synthesized by iScriptTM cDNA Synthesis Kit (Bio-Rad). The qPCR was performed using a Bio-Rad IQ5 Real-Time PCR system with SYBR-Green detection (SYBR Premix, TIANGEN) according to the manufacturer’s instructions. The primers for S7e, gallerimycin, cecropin and galiomycin genes were chosen as described previously [21]. The primers used for PCR were as follow: S7e forward, 5 -ATG TGC CAA TGC CCA AGT TG-3 , S7e reverse, 5 GTG GCT AGG CTT GGG AAG AAT-3 ; gallerimycin forward, 5 -TAT CAT TGG CCT TCT TGG CTG-3 , gallerimycin reverse, 5 -GCA CTC GTA AAA TAC ACA TCC GG-3 ; cecropin forward, 5 -ATT TGC CTG CAT CGT AGC G-3 , cecropin reverse, 5 -CTT GTA CTG CTG GAC CAG CTT TT-3 ; galiomycin forward, 5 -TCG TAT CGT CAC CGC AAA ATG3 , galiomycin reverse, 5 -GCC GCA ATG ACC ACC TTT ATA-3 . All the products were of 131 bp length. The real-time qPCR conditions were: 95 ◦ C for 3 min, 44 × (95 ◦ C for 15 s, 60 ◦ C for 15 s, 72 ◦ C for 1 min). Each reaction was run in triplicate, after which the average threshold cycle (Ct) was calculated per sample. The amount of mRNA detected was normalized to ribosomal protein S7e mRNA values (a housekeeping gene, whose level was constant in all the conditions tested).
2.8. Bacterial infection of G. mellonella (in vivo bacterial growth) Five groups of G. mellonella larvae were immune-primed with 10 l of PBS containing 0 (control), 1 × 103 , 1 × 104 , 1 × 105 or
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Fig. 5. (A) Encapsulation of Sephadex A-25 beads by Galleria mellonella larvae hemocytes in vivo: (a) grade 1; (b) grade 2; (c) grade 3; (d) grade 4; (for details see Section 2.5). (B) Encapsulation index of hemocytes in G. mellonella larvae primed with different doses of heat-killed Photorhabdus luminescens TT01. Data were expressed as means ± SE. Values for different groups followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
1 × 106 of heat-killed P. luminescens TT01 cells per larva, respectively. At 48 h after the immune priming, 10 l of PBS containing approximately 50 cells of viable TT01 were injected into the hemocoel cavity of each G. mellonella larva. In all the experiments larvae were bled 24 h after the injection. Hemolymph was obtained from punctured prolegs of the larvae; whole hemolymph samples were then processed by two low speed centrifugations (700 g for 10 min, at 4 ◦ C) to remove hemocytes and tissue debris. The plasma was diluted in PBS solution. Aliquots of cell-free fractions of supernatants were placed on an NBTA plate. Petri dishes were incubated for 48 h at 28 ◦ C, and bacteria were quantified by Colony Forming Units (CFU) count. In addition, in order to examine the presence of growing bacteria in the host hemocoel cavity, five groups of
G. mellonella larvae were slected and treated as described above. Hemolymph samples in each group were diluted in five volumes of Grace’s insect medium and assayed visually by light microscope in microwells cultures.
2.9. Data processing and statistical analysis Differences between mean values were analyzed with one-way analysis of variance (ANOVA) by using the least significant difference (LSD) test and considered significant when P < 0.05. All experiments were replicated at least three times. Data were processed with DPS package.
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3. Results 3.1. The degree of immune protection of G. mellonella against viable P. luminescens TT01 infection depended on the doses of heat-killed P. luminescens TT01 with which they were inoculated As shown in Fig. 1, the larvae of the control group were killed significantly faster compared to larvae primed with 1 × 104 (P = 0.0001), 1 × 105 (P < 0.0001) or 1 × 106 (P < 0.0001) cells of heatkilled P. luminescens TT01 48 h prior to the viable P. luminescens TT01 infection. In addition, caterpillar killing speed depended on the dose of heat-killed P. luminescens TT01 with which they were inoculated. For example, larvae primed with 1 × 103 cells of heatkilled P. luminescens TT01 were killed significantly faster compared to larvae that received higher doses (1 × 105 or 1 × 106 cells/larva) of heat-killed P. luminescens TT01 priming when the larvae were challenged with viable P. luminescens TT01 48 h after priming (P < 0.0001). There was no significant difference in killing speed between the 1 × 103 cells of heat-killed P. luminescens TT01 priming group and the control group. 3.2. Administration of heat-killed P. luminescens TT01 leads to alterations in THC and bacterial density in the larval hemolymph In this work, the THC of larvae that had been inoculated with different doses of heat-killed P. luminescens TT01 was ascertained at 48 h after inoculation. The results (Fig. 2) indicated that after 48 h, THC of larvae that received a dose of 1 × 103 , 1 × 104 , 1 × 105 or 1 × 106 heat-killed P. luminescens TT01 per larva had increased to approximately 19.3 × 106 , 20.8 × 106 , 30.0 × 106 , 32.9 × 106 cells per larva, respectively and was significantly higher than the THC of the larvae inoculated with PBS (P < 0.05). The bacterial density in the larval hemolymph was also examined at 24 h after infection with viable P. luminescens TT01 in each primed group and the control group. Those larvae that had been primed with different doses (1 × 104 , 1 × 105 and 1 × 106 cells/larva) of heat-killedP. luminescens TT01 48 h prior to infection showed significantly lower bacterial densities (32.5 × 107 , 10.6 × 107 and 3.1 × 107 cfu/ml, respectively) at 24 h after viable P. luminescens TT01 infection than the control group (Fig. 3A, B). In contrast, the larvae that had been primed with the lower dose (1 × 103 cells/larva) of heat-killed P. luminescens TT01 showed significantly higher bacterial density than those larvae that had been primed with the higher doses of heat-killed P. luminescens TT01 (1 × 104 , 1 × 105 and 1 × 106 cells/larva) at 24 h after being challenged with viable P. luminescens TT01, and they displayed no significant difference from the control group. 3.3. Priming G. mellonella with heat-killed P. luminescens TT01 caused dose-dependent elevation in phagocytosis and encapsulation abilities of the hemocytes The phagocytic images (Fig. 4A) generated by merging images taken with a differential interference contrast microscope and a fluorescence microscope clearly reveal changes in phagocytic activities 48 h after priming the larvae with different doses of heat-killed P. luminescens TT01. Phagocytic activities of hemocyte collected from larvae primed with 1 × 104 , 1 × 105 or 1 × 106 cells of heatkilled P. luminescens TT01 increased to 50.5%, 56.4% and 61.8%, respectively and were significantly higher than that of the control group (Fig. 4B). In contrast, there was no significant difference between the 1 × 103 cells of heat-killed P. luminescens TT01 per larva priming group and the control group at 48 h after-priming. We also observed similarly changed patterns in the encapsulation index. Compared with the control, the larvae primed with the higher doses (1 × 105 or 1 × 106 cells/larva) of heat-killed P. lumi-
Fig. 6. Quantitative RT-PCR analysis of cecropin, galiomycin, and gallerimycin transcripts in total RNA isolated from the hemocytes of Galleria mellonella larvae at 48 h after priming with different doses of heat-killed Photorhabdus luminescens TT01. Expression of each gene was normalized to the expression of S7e ±SD. Values for the same antimicrobial peptide gene followed by different letters are significantly different (P < 0.05) according to ANOVA and the least significant difference (LSD) test.
nescens TT01 significantly enhanced the encapsulation index of the hemocyte, while no significant differences could be observed in the other treatment groups (Fig. 5B). 3.4. Administration of heat-killed P. luminescens TT01 results in increased gene expression levels of cecropin and gallerimycin and galiomycin in the hemocytes of G. mellonella larvae We studied the appearance of transcripts for AMPs in the hemocytes of the larvae primed with different doses of heat-killed P. luminescens TT01 cells and noticed a gradual increase in the amount of transcripts for cecropin and gallerimycin and galiomycin as the priming dose of heat-killed P. luminescens TT01 increased. In other words, at 48 h after immune-priming, the expression of each tested gene was significantly higher in the hemocytes of the larvae preexposed to the higher doses of heat-killed P. luminescens TT01 (1 × 104 , 1 × 105 or 1 × 106 cells/larva) in comparison to the nonprimed (PBS control) larvae (Fig. 6). 4. Discussion Here we showed that priming G. mellonella larvae with different doses of heat-killed P. luminescens TT01 protects the insects from a challenge 48 h later with a lethal dose of viable P. luminescens TT01. As a response to the priming with heat-killed P. luminescens TT01 the larvae also showed elevated levels of humoral (expression of genes coding for AMPs) and cellular (hemocyte number, phagocytosis and encapsulation abilities of hemocytes) immune parameters 48 h after priming, offering a potential explanation for the immune protection. Previous work has demonstrated that sub-lethal doses of Aspergillus fumigatus, Candida albicans or Saccharomyces cerevisiae have a similar immune priming effect on inoculated G. mellonella larvae [12,22]. Although our previous work revealed that the heat-killed P. luminescens TT01 cells can elicit immune priming in G. mellonella larvae [18], the work presented here demonstrated for the first time a correlation between the dose of heat-killed P. luminescens TT01 inoculated and the levels of change in cellular and humoral immune parameters evident in G. mellonella larvae. Priming the larvae with doses of 1 × 104 , 1 × 105 or 1 × 106 heat-killed P. luminescens TT01 cells per larva was sufficient to render the larvae resistant to viable P. luminescens
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TT01 infection and provoked the increases in humoral and cellular immune parameters found at 48 h. In contrast larvae primed with heat-killed P. luminescens TT01 at a dose of 1 × 103 cells per larva showed no significant difference in killing speed relative to that observed in PBS inoculated (control) larvae and no significant increase in humoral and cellular immune parameters or decrease in the bacterial density. These results indicated that G. mellonella mount an immune response proportionate to the size of the inoculums to reduce the immunological cost to the larva. Previous work demonstrated that the hemocyte number of insects is altered by the presence of pathogenic stimuli or by microbial cell wall components [13,23]. Our results showed that the hemocyte number of the larvae increases as priming dose of heatkilled P. luminescens TT01 cells increases. A Previous study showed that the elevated levels of hemocytes were possibly due to the release of hemocytes attached to the internal organs within the insect [24]. In another study, the higher number of hemocytes correlated positively to a greater ability to fight invaders [25]. In this study we found that the higher level of hemocyte density in larvae primed with 1 × 106 heat-killed P. luminescens TT01 cells correlated positively with increased resistance to subsequent infection. Therefore, we can conclude that the higher level of hemocyte density in the larvae benefits to phagocytosing and killing invaded pathogens. We also observed a high correlation between the protection level and the increase in both phagocytosis and encapsulation abilities of the hemocytes in the heat-killed P. luminescens TT01 primed larvae. The larvae primed with the highest dose (1 × 106 cells/larva) of heat-killed P. luminescens TT01 showed significantly higher phagocytosis rates and encapsulation indices than the larvae that received the lower doses (1 × 103 , 1 × 104 cells/larva) of heat-killed P. luminescens TT01. These result is in line with a previous study showing that pre-exposure of juvenile shrimp to a formalin-inactivated pathogenic vibrio results in an increased phagocytic uptake of this bacterium by the hemocytes [26]. Phagocytosis and encapsulation represent the main elements of insect cellular responses that play key roles against invading pathogens. The up-regulation activity of phagocytosis and encapsulation following immune priming is of great benefit to resisting a subsequent lethal P. luminescens TT01 infection—but it does impose a cost on the insect and can even be fatal [27]. Therefore it is reasonable that entry of a low level of pathogen or pathogen associated material would appear to activate a response designed to eliminate the threat. The work presented here demonstrates that administration of heat-killed P. luminescens TT01 induces immune priming, and it is clear that the amount of heat-killed P. luminescens TT01 administered to the insect determines the degree of the immune response. Analysis of the expression of genes coding for antimicrobial peptides in the hemocytes of the larvae after immune-priming and examination of bacterial density in the hemolymph of immuneprimed larvae after viable P. luminescens TT01 infection were performed to establish whether there were correlations with decreased larval mortality following priming with heat-killed P. luminescens TT01 bacteria. Our results showed that when larvae are immune-primed with appropriate doses of heat-killed P. luminescens TT01 there is an increase in the expression of genes coding for antimicrobial peptides, such as gallerimycin, cecropin, and galiomycin. We also observed that the level of transcripts increased in a dose-dependent manner. This is consistent with the changes of bacterial density in the larval hemolymph; and the larvae primed with higher dose of heat-killed P. luminescens TT01 showed significant lower bacterial density when infected with viable P. luminescens TT01. Consequently, we can conclude that activation of defence mechanisms after immune priming is also noticeable at the molecular level. The elevated mRNA levels of genes coding for antimicrobial peptides have previously
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been recorded in larvae challenged with bacteria. For example, gallerimycin and cecropin have been shown to increase in expression when larvae were inoculated with Bacillus thuringiensis [28]. Galiomycin has been shown to be induced in larvae infected with Escherichia coli and to have antifungal as well as antibacterial activities [29]. The production of AMPs, a key component of the humoral response, serves to kill pathogens that have escaped or withstood the cellular immune response. This work indicated that prior exposure to a heat-killed P. luminescens TT01 primes the G. mellonella immune system and allows the larvae to withstand a subsequent viable P. luminescens TT01 infection. This effect is also mediated by the production of elevated levels of AMPs which protect the insect from the second exposure to the pathogen. A dose-dependent immune response in G. mellonella larvae to heat-killed P. luminescens TT01 inoculation indicated that insects can modulate their immune responses depending upon the nature and size of the microbial challenge. Upregulation of immune responses in bumble bees following exposure to low levels of pathogen, but in the absence of compensatory feeding can be fatal [27], thus indicating that immune protection does have a cost for the insect. Elevated immunity following such immune priming offers protection to G. mellonella larvae and would have an obvious survival advantage but does have a cost in terms of utilization of resources [16,30]. Therefore, we can conclude that G. mellonella larvae mount an immune response proportionate to the amount of the priming agent to ensure survival but minimize the use of resources. The present study will increase our knowledge of invertebrate immunity and can be of benefit for understanding the evolution, ecology, and epidemiology of economic invertebrates diseases. Acknowledgements This work was supported by the National Science of Foundation of China (No. 21301034) and the National Science of Foundation of Guangdong Province (S2013040014083). References [1] S.A. Armitage, R. Peuss, J. Kurtz, Dscam and pancrustacean immune memory−a review of the evidence, Dev. Comp. Immunol. 48 (2015) 315–323. [2] C.J. Coates, T. Whalley, J. Nairn, Phagocytic activity of Limulus polyphemus amebocytes in vitro, J. Invertebr. Pathol. 111 (2012) 205–210. [3] C. Anagnostou, E.A. LeGrand, M. Rohlfs, Friendly food for fitter flies?−Influence of dietary microbial species on food choice and parasitoid resistance in Drosophila, Oikos 119 (2010) 533–541. [4] A. Tokura, G.S. Fu, M. Sakamoto, H. Endo, S. Tanaka, S. Kikuta, et al., Factors functioning in nodule melanization of insects and their mechanisms of accumulation in nodules, J. Insect Physiol. 60 (2014) 40–49. [5] A.L. Flores-Villegas, P.M. Salazar-Schettino, A. Cordoba-Aguilar, A.E. Gutierrez-Cabrera, G.E. Rojas-Wastavino, M.I. Bucio-Torres, et al., Immune defence mechanisms of triatomines against bacteria, viruses, fungi and parasites, Bull. Entomol. Res. 105 (2015) 523–532. [6] C.J. Tsai, J.M. Loh, T. Proft, Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing, Virulence (2016) 1–16. [7] J.C. Junqueira, Galleria mellonella as a model host for human pathogens, Virulence 3 (2012) 474–476. [8] N. Browne, M. Heelan, K. Kavanagh, An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes, Virulence 4 (2013) 597–603. [9] J. Renwick, E.P. Reeves, F.B. Wientjes, K. Kavanagh, Translocation of proteins homologous to human neutrophil p47(phox) and p67(phox) to the cell membrane in activated hemocytes of Galleria mellonella, Dev. Comp. Immunol. 31 (2007) 347–359. [10] R.S. Khush, F. Leulier, B. Lemaitre, Drosophila immunity: two paths to NF-kappa B, Trends Immunol. 22 (2001) 260–264. [11] B.M. Sadd, P. Schmid-Hempel, Insect immunity shows specificity in protection upon secondary pathogen exposure, Curr. Biol. 16 (2006) 1206–1210. [12] J.P. Fallon, N. Troy, K. Kavanagh, Pre-exposure of Galleria mellonella larvae to different doses of Aspergillus fumigatus conidia causes differential activation of cellular and humoral immune responses, Virulence 2 (2011) 413–421. [13] P. Mowlds, C. Coates, J. Renwick, K. Kavanagh, Dose-dependent cellular and humoral responses in Galleria mellonella larvae following beta-glucan inoculation, Microbes Infect. 12 (2010) 146–153.
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