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. 2021 Jan 28;11(2):101. doi: 10.1007/s13205-021-02662-6

Toxicity of insecticidal proteins from entomopathogenic bacteria to Galleria mellonella larvae

Chunli Liao 1,2,3,✉, Yi Yang 1, Xingzhao Fan 1, Jiangnan Du 1, Jing Zhu 1, Mingbo Sang 1, Bingbing Li 1,2,3,✉
PMCID: PMC7843678  PMID: 33520586

Abstract

Entomopathogenic bacteria have great potential in insect control in the agricultural production because they produce a large variety of protein toxins that can kill their hosts by damaging the insect midgut. However, the mechanisms on how these toxins or specific insecticidal proteins act on insects are very diverse and elusive. Here we select Galleria mellonella larvae as the host to explore the effects of insecticidal proteins on the activities of three protective enzymes (SOD, POD, and CAT) and on the morphology of the midgut tissues. As a result, the activities of the three enzymes consistently increased and then decreased when the host was injected with the insecticidal proteins from the entomopathogenic bacterium Enterobacter cloacae. Moreover, the microscopy analysis showed that tissues, cells, and organelles of the host midgut are all diseased after uptake of the insecticidal proteins. Remarkably, the protein toxins contributed to the deformation of the midgut, blackening of the midgut surface, dissolution of cell membrane, shrinkage of cell nucleus, and chromatin condensation. Our findings will advance the explanation of G. mellonella pathogenesis caused by the insecticidal proteins.

Keywords: Galleria mellonella, Entomopathogenic bacteria, Insecticidal proteins, Toxicity, Midgut

Introduction

Entomopathogenic bacteria have caught considerable attention due to their utility for biocontrol of insect pests in agriculture and medicine (Azizoglu et al. 2020). These bacteria can produce a variety of protein toxins that can kill insects through disrupting the midgut epithelium cells (Cai et al. 2017; Skowronek et al. 2020). For one, Bacillus thuringiensis insecticidal proteins, including Cry, Vip, and Cyt, are able to kill different insect or nematodes through three major steps: (1) solubilizing and activating the insect midgut crystal, (2) binding the activated toxins to the midgut receptors, and (3) inserting the toxins into the midgut membrane to conduce to the formation of ion channels or pores (Bravo et al. 2015, 2017; Lee et al. 2003; Rajamohan et al. 1998). It is also reported that the key factors that determine the range of insect invaded by these entomopathogenic bacteria are the insecticidal proteins in the parasporal crystals (Chakroun et al. 2016; Syed et al. 2020). Other reports showed that Gram-positive spore-forming entomopathogenic bacteria are capable of producing protein toxins to help them invade, infect, and eventually kill their insect hosts (Liao et al. 2017, 2019; Malovichko et al. 2019). However, the detailed mechanisms on how these protein toxins kill Galleria mellonella via interfering with the immune system are still open questions.

In the long term, insects have evolved to form unique immune systems to prevent the invasion by foreign pathogens (Meunier 2015). Once infected, they can invoke their immune systems to generate excessive reactive oxygen species (ROS) that will help them kill foreign pathogenic bacteria or parasites (Hu et al. 2019). Normally, the level of ROS in cells is relatively low and, thus, does not cause any damage to organisms because there is a ROS-removing system that mainly consists of a variety of protective enzymes (Liao et al. 2016, 2015a, b), such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). For example, larvae of Ostrinia furnacalish have been reported to produce a large amount of ROS in hemolymph when they are invaded by Macrocentrus cingulum, along with a subsequent increase of the activity of SOD, CAT, and POD (Feng et al. 2002). Therefore, these protective enzymes play an important role in overcoming the adverse environmental conditions where an insect may encounter. Although there are many studies on the reaction between host insects and invaders, mechanisms on how these antioxidases respond to the insecticidal proteins from entomopathogenic bacteria are underexplored.

Many studies have reported that insects would exhibit a series of pathological changes in the midgut when feeding the cotton with a B. thuringiensis (Bt) insecticidal gene (González-Villarreal et al. 2020; Wang et al. 2019). However, the relevant mechanisms are mainly observed to be the disruption of the cellular structures of the midgut, especially for the destruction of microvillus, mitochondria, and cell membrane (Bravo et al. 2015, 2017; Prabu et al. 2020; Rajamohan et al. 1998). To expand the action mechanisms of insecticidal proteins, we employed a novel entomopathogenic bacterium Enterobacter cloacae NK isolated from the Heterorhabditis sp. PDSj2 (Liao et al. 2017), a parasite of G. mellonella. We expect to provide more information for the biocontrol of G. mellonella to advance the development of a sustainable agriculture.

Materials and methods

Preparation and extraction of insecticidal proteins

E. cloacae NK was incubated in Luria–Bertani (LB) broth at 37 °C until the OD600nm was 0.35. Then, the culture was transferred into the fermentation medium with an inoculum of 6% (v/v) and the system was further cultivated at 37 °C for 28 h. The fermentation medium was composed of: 0.024 g/L (NH4)2SO4, 0.2 g/L peptone, 0.013 g/L MgSO4, 0.0072 g/L KH2PO4, 0.005 g/L K2HPO4, 0.015 g/L Na2SO4, and 0.9 g/L glucose, with a final pH of 7.0. Next, the fermentation broth was centrifuged and the cell pellets were obtained and disrupted with an ultrasonic processor for 10 min. After that, the sample was centrifuged again and 10 mL of the supernatant that contains intracellular proteins was collected and supplemented with the powder of (NH4)2SO4 in the ice-water bath until the degree of saturation reached 80%. This sample was placed at 4 °C for 6 h and then centrifuged for 20 min at 12000 r/min. Finally, the supernatant was discarded and the deposits were washed out with phosphate buffered saline (PBS) (pH = 7.4) to prepare the raw protein solution. This protein solution was collected with a dialysis bag (7 KDa) and stored at 4 °C for 24 h; and the dialysate was replaced every 4 h. After dialysis, the protein solution was freeze-dried and stored at − 80 °C.

Quantification and purification of raw proteins

For the measurement of protein activity, 0.1 mg/mL raw protein solution was made with distilled water and 1 mL of the protein solution was added into 5 mL of the coomassie brilliant blue (CBB, G-250) solution. After a 5 min reaction, the OD595nm of the sample was measured using the mixture of 1 mL of distilled water plus 5 mL of CBB as the blank. The content of protein was determined with the following formula:

Protein contentmg/mL=OD595nm-0.0188/0.9631.

For the purification of proteins, 5 mg/mL raw protein solution was made and purified via the gel chromatography (Sephadex G-100) with an injection volume of 3% of column volume. Next, the proteins that have a highly active peak were collected for freeze-drying and 2 mg/mL solution of such proteins was made for further purification through the ion-exchange column chromatography (DEAE-Sephrose FF) with an injection volume of 3% of column volume. After the second purification, the final proteins were tested through the native-PAGE electrophoresis to obtain the pure protein.

Effects of insecticidal proteins on activities of protective enzymes

G. mellonella larvae were cultured at 25 °C with a humidity of 75% under dark condition. Next, 5 μL of 10 g/L protein solution was injected from the middle of its first pair of pleopod. Each experimental group had 30 larvae. The control larvae were injected with PBS. Each treatment had three biological replicates. After that, 10 μL of hemolymph were taken into 1.5-mL tube every 6 h and 2% (v/v) phenylthiocarbamide was added into the tube to avoid oxidization. All these samples were then stored at 4 °C for further use. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in each sample were determined through the nitro blue tetrazolium photo-reduction method (Beauchamp and Fridovich 1971), the guaiacol (peroxidase) test (Jebara et al. 2005), and the ultraviolet absorption method (Jebara et al. 2005), respectively.

Midgut sampling and microscopy

Each experimental group had 30 larvae that were starved for 24 h in the same plate and then fed with the food containing 20 μg/g insecticidal protein. Larvae in the control group were fed with the normal food. Each group was carried out in three replicates. Next, the migut was individually sampled after 24 h, 48 h, and 72 h. All samples were immediately stabilized in 4% glutaraldehyde at 4 °C for further microscopy.

For the scanning electron microscopy observation, the midgut was cut into 1 mm × 1 mm pieces and then stabilized in 4% glutaraldehyde for 1 h. Then, the samples were washed three times with 0.1 mol/L PBS (pH = 7). The washing time was 15 min each time. After washing, samples were further stabilized in 1% osmic acid for 2 h and washed three times with 0.1 mol/L PBS (pH = 7). Next, all the samples were dehydrated with the gradient solutions of acetone: (1) one-time gradient dehydration with 50%, 70%, and 80% acetone for 10 min; (2) two-time gradient dehydration with 90% acetone for 10 min; and (3) two-time gradient dehydration with 100% acetone for 15 min. After dehydration, the samples were successively permeated using the mixture of acetone and embedding agent at a ratio of 2/1 (acetone/embedding agent in volume), 1/1, ½, and 0/1 for 1 h, repsectively. After that, these samples were first air-dried at room temperature for 2 h, then dried at 30 °C for 48 h, and finally baked at 60 °C for 48 h in an oven. Lastly, the samples were tailored and dyed for the scanning electron microscopy observation (JEM-1400, 100 kV).

Statistical analysis

All experiments were performed in triplicate. One-way ANOVA test was conducted using Microsoft Origin 8.5 to test for differences between the treated and control groups. Subsequently, Tukey’s honest significant difference was determined at a discrimination level of P < 0.05 or P < 0.01. Results of variance analysis were shown as error bars in each figure.

Results

Effects of insecticidal proteins on SOD, POD, and CAT

To evaluate the influences of insecticidal proteins of E. cloacae NK on the immune system of G. mellonella larvae, we determined the activities of SOD, POD and CAT at different intervals (Fig. 1). Generally, the activities of the three enzymes had not changed significantly in the control group (P > 0.05). However, the activities of these enzymes in the treated groups went up first, peaked at the middle of experiment, and significantly dropped eventually (P < 0.01). Specifically, the activity of POD in the experimental group showed a significant increase only between 12 and 18 h (P < 0.05), though there was an insignificant increase at 6 h (P > 0.05), compared to that in the control group (Fig. 1a). After 18 h, the activity in the experimental group decreased sharply to almost 30% of the normal level at 36 h. Similarly, within 24 h the activity of SOD in the treated group was higher than that in the untreated group, but it only increased significantly between 12 and 18 h (P < 0.05), peaking at 18 h. After 24 h, the activity of SOD also dropped significantly (P < 0.05), with the final activity halving. Differentially, the activity of CAT in the treated group increased initially, then peaked at 12 h, and dropped dramatically thereafter, with the final activity being a quarter of the normal level. Compared with the normal larvae, these changes in the activities of the three enzymes indicated that insecticidal proteins have largely disrupted the immune system of the treated G. mellonella larvae.

Fig. 1.

Fig. 1

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Comparison of the activities of three protective enzymes between the treated and untreated groups. Data were shown as mean and error bars were the standard deviations

Effects of insecticidal proteins on the morphology of midgut

To investigate the impact of insecticidal proteins on the midgut of G. mellonella larvae, we first observed the entire morphology of midgut. Interestingly, the midgut morphology of the larva fed with insecticidal proteins changed obviously, compared to that of the normal larvae (Fig. 2). The thickness of the normal midgut was quite even and the midgut was intact with a clear internal spine. However, the thickness of the treated larvae was uneven and smaller than that of the normal ones. Moreover, the treated midgut got black and lost the internal spine; and this influence was more obvious over time. These findings suggested that insecticidal proteins might damage the midgut of G. mellonella larvae.

Fig. 2.

Fig. 2

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Comparison of the morphology of midguts between the treated and untreated groups on a microscope under a magnification of 4 × 10

Changes of midgut goblet cells

To further determine the changes in the midgut structure of G. mellonella larvae, we employed the scanning electron microscope. As a result, changes of the structure of the goblet cells were mainly the disruption of cellular nucleus and loss of internal spines of mitochondria. Specifically, cellular nucleus was normally elliptical before the larvae being fed with insecticidal proteins. However, cellular nucleus was deformed at 24 h, shrunk at 48 h, and disrupted completely at 72 h (Fig. 3). Moreover, chromatin was evenly distributed before the treatment, whereas it started to condense after 24 h and was marginalized till the nucleus disruption. It is well known that cellular nucleus is the center of cell metabolisms and genetics. Therefore, it indicated that insecticidal proteins could kill G. mellonella larvae through destroying the cellular nucleus of the midgut goblet cells. On the other hand, the amount of internal spine in mitochondria was found to decrease over time (Fig. 4), indicating that insecticidal proteins might result in the damage of mitochondria and kill the larvae through interfering the intercellular energy metabolism.

Fig. 3.

Fig. 3

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Comparison of the changes of cellular nucleus of the midgut goblet cells between the treated and untreated groups under a scanning electron microscope. a Normal cellular nucleus of larvae untreated with insecticidal proteins, b–d cellular nucleus of larvae treated with insecticidal proteins at 24 h, 48, and 72 h, respectively

Fig. 4.

Fig. 4

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Comparison of the changes of mitochondria of the midgut goblet cells between the treated and untreated groups under a scanning electron microscope. a Normal mitochondria of larvae untreated with insecticidal proteins, b–d mitochondria of larvae treated with insecticidal proteins at 24 h, 48, and 72 h, respectively

Changes of midgut columnar cells

To further investigate the change in the midgut of larvae, both microvillus and cell membrane of columnar cells were scanned under a scanning electron microscope (SEM). Gut cell membrane has important roles in both nutrient absorption and discharge of metabolic waste. However, the SEM analysis showed that the treated larvae exhibited a dissolution of cell membrane after being fed with insecticidal proteins and even the structure of cell membrane completely disappeared at 72 h (Fig. 5). A large amount of wrinkles and linear microvilli were observed on the internal surface of the normal midgut (Fig. 6). However, it seems that these microvilli started to be broken at 24 h when treated with insecticidal proteins. They were almost completely disrupted at 48 h and finally dissolved after 72 h. Therefore, these findings indicated that insecticidal proteins could kill G. mellonella larvae through destroying the midgut microvilli and membrane of columnar cells, leading to the disruption of the gut digestion system.

Fig. 5.

Fig. 5

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Comparison of the changes of cellular membrane of midgut columnar cells between the treated and untreated groups under a scanning electron microscope. a Normal cellular membrane of larvae untreated with insecticidal proteins, b–d cellular membrane of larvae treated with insecticidal proteins at 24 h, 48, and 72 h, respectively

Fig. 6.

Fig. 6

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Comparison of the changes of microvilli of midgut columnar cells between the treated and untreated groups under a scanning electron microscope. a Normal microvilli of larvae untreated with insecticidal proteins, b–d microvilli of larvae treated with insecticidal proteins at 24 h, 48, and 72 h, respectively

Discussion

In organisms, SOD, POD, and CAT are three typical enzymes that protect cells and tissues from the external damage of oxidation (Huang et al. 2019; Liao et al. 2015a, b; Liu et al. 2014). Insecticidal proteins can induce the immune response of G. mellonella larvae and result in the increase of reactive oxygen species (ROS), leading to the growth of SOD, POD, and CAT activities (Hu et al. 2019; Rizwan et al. 2018). This pattern is also consistent with our observation that insecticidal proteins of the entomopathogenic bacterium E. cloacae NK have significantly contributed to an enhanced activity of all the three enzymes to detoxify the induced ROS when infecting the larvae at the earlier and middle stages. However, the excessive production of ROS induced by insecticidal proteins would further inhibit the activity of the protective enzymes (Huang et al. 2019), which agrees with the decrease of the activities of SOD, POD and CAT at the end of the infection by insecticidal proteins in this study. The excessive ROS would largely damage the tissue of G. mellonella larvae and, thus, leads to the death of the larvae eventually. These findings add more information on the immune response of G. mellonella to insecticidal proteins of entomopathogenic bacteria and provide further evidence for elucidating the insecticidal mechanisms of entomopathogenic bacteria.

It is reported that lepidopterous larvae that are fed with the protein Cry1Ac or the engineered plants with a cry1Ac gene have exhibited serious pathological changes, such as fall-off of microvilli, disruption of nucleic and plasma membranes, unclear internal spines of mitochondria, and destruction of endoplasmic reticulum (Liu et al. 2020; Naik et al. 2018; Zhu et al. 2020). Similarly, we observed the pathological changes in the midgut of G. mellonella larvae when fed with insecticidal proteins of E. cloacae NK, which will greatly support the other mechanism that insecticidal proteins can also kill G. mellonella larvae through disrupting their gut digestion systems (Bowling et al. 2019; González-Villarreal et al. 2020).

In summary, the present study elucidates the toxicological mechanisms of insecticidal proteins from entomopathogenic bacteria against G. mellonella larvae. These findings will greatly advance the biocontrol of G. mellonella using entomopathogenic bacteria. Further studies should focus on molecular mechanisms of these insecticidal proteins in the control of G. mellonella, which would help develop more biological insecticides for green, sustainable agriculture and environment (Liao et al. 2014; Liu 2020; Zhou et al. 2020) or open up the possibility to engineer a crop that can resist the damage of G. mellonella (Liao et al. 2015c).

Acknowledgements

We gratefully acknowledge the financial support by the young teachers’ project of the Henan University of Urban Construction (Grant No. G2016009).

Author contributions

Conceptualization: CL and BL; methodology: CL, XF and JZ; data curation: CL and MS; writing: CL, BL, XF, JD, YY and JZ; supervision: CL and BL. All authors have read and agreed to the published version of the manuscript.

Funding

This work is funded by the young teachers’ project of the Henan University of Urban Construction (Grant No. G2016009).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Contributor Information

Chunli Liao, Email: liao20130427@163.com.

Bingbing Li, Email: libingbing@hncj.edu.cn.

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