Characterization of the oral secretion from Nicrophorus marginatus, N. carolinus, and N. tomentosis
Michiko Abe, Department of Biology, University of Nebraska at Kearney, Kearney, Nebraska 68849, USA.
Mentor: Dr. Julie Shaffer, Associate Professor, Department of Biology, University of Nebraska at Kearney, Nebraska 68849-1140, USA. Email: shafferjj@unk.edu)
To Cite This Paper:.
Abe, M. 2007. Characterization of the oral secretion from Nicrophorus marginatus, N. carolinus, and N. tomentosis. Research Thesis, Department of Biology, University of Nebraska at Kearney, Kearney, Nebraska 68849, USA.
Abstract: Burying beetles are known to produce oral secretions containing antimicrobial compounds. My research was to identify and characterize the proteinaceous compounds of the oral secretions from burying beetles. Oral secretions were collected June and July, 2007 from Nicrophorus marginatus, N. carolinus, and N. tomentosis. The oral secretions from the three collected species were compared for antimicrobial activity using a Microtox Model 500 Analyzer. Protein concentrations were identified using a BCA Assay. Dialysis and gel electrophoresis were used to identify the role peptides might play in antimicrobial activity. All three species produce antimicrobial oral secretion but N. marginatus produced the highest concentration of protein in its secretion. After the samples were diluted to equal protein concentrations, N. tomentosis exhibited the highest antimicrobial activity. These data also showed that N. carolinus exhibited the greatest variation in antimicrobial activity. It was also shown that peptides do not seem to be the antimicrobial agent. Antimicrobial activity is most likely due to enzymes present in the secretion, although this will need to be studied further.
All animals and plants coexist with bacteria and fungi, but bacteria or fungi have the ability to cause diseases due to virulence factors and their rapid growth in a host (Gabay, 1994). All living organisms, including microorganisms themselves, have various mechanisms to defend against microbial invaders. First line defenses are described as innate immunity. An innate immune system uses proteins to identify harmful substances in the body (Fearon and Locksley, 1996). An ancient and widespread defense strategy is the production of small antimicrobial peptides. Antimicrobial peptides can be synthesized in many ways and produced by a minimal input of energy in response to bacterial and fungal attacks (Broekaert et al., 1995; Cociancich et al., 1993). Insect immune systems include responses of hemocytes and synthesis of antimicrobial peptides and proteins (Gillespie et al., 1997). These three properties make a powerful and effective immune system for insects. More complex organisms have an expanded capacity to defend against infections (George and Karp, 1986). In mammals, the innate immune system is a rapidly induced first response to microbial attacks and also stimulates the adaptive immune system (Khush et al., 2002). Insect immunity is similar to an innate immune system in vertebrates.
Insects have remarkable and complex defensive immune systems. Insects have both cellular and humoral mechanisms. The cellular response is phagocytosis and encapsulation of invading foreign organisms by blood cells. The blood cells encapsulate invading materials by making nodules or forming an organized multicellular capsule around large invaders. The insect humoral response is primarily characterized by the synthesis of antimicrobial peptides (Lamberty et al., 1999). Systemic infection induces synthesis of antimicrobial peptides (Tzou et al., 2002).
The expression of gene-encoded antimicrobial peptides plays an important role in a first defense response against infections. The encoded proteins are used to identify potentially noxious organisms or substances by binding to bacterial or fungal polysaccharides (Gillespie et al., 1997). The different genes have different targets for specific microorganisms. The presence of several antimicrobial peptides can provide animals and plants with a more powerful defense against microbial infections. An abundant number of peptide structures have been reported, and fifty percent of them have been identified in invertebrates, which are predominantly insects (Bulet et al., 1991).
Insect antimicrobial peptides are synthesized by the fat body, a functional homologue of the vertebrate liver, and certain blood cell types. After synthesis, they are released rapidly into the hemolymph and directly kill invading pathogens. Antimicrobial peptides are found in the secretions of seminal fluid, lymph, and serum of insects (Gabay, 1994).
More than 200 antimicrobial peptides have been characterized from various species of insects (Lamberty et al., 2001). These molecules have the ability of phagocytes to kill ingested microorganisms (Gabay, 1994). Cecropins, defensins, drosocins, diptericins, and attacins are major antibacterial peptide families isolated from insects (Otvos Jr., 2000).
Many insects produce antimicrobial peptides as part of their humoral responses in their innate immune systems. Burying beetles are one species that produce antimicrobial peptides. What is of particular interest is what these beetles may use the peptides for. They may not only be part of the humoral immune response, but they may also be secreted to protect their food sources against microorganisms in their natural habitats.
Carrion beetles (Coleoptera: Silphidae) have 13 genera and about 208 species all over the world (Ratcliffe, 1996). They consist of two subfamilies: the Silphinae and the Nicrophorinae (Hoback et al., 2004). The Nicrophorinae, the subfamily to which the burying beetles belong, have three genera and about 90 species. They are found in the Americas, Europe, and Asia. Most of the species are distributed in northern temperate regions (Ratcliffe, 1996).
The Nicrophorinae are known to produce oral secretions containing antimicrobial compounds (Hoback et al., 2004). The oral secretions of the Nicrophorinae are effective in protecting their food sources. The Nicrophorinae locate small vertebrate carcasses which they use to bury and prepare as food for their young. The beetles move the carcasses into a chamber which is a safe place of their own making, a few inches below the surface of the ground. This is why they are called burying beetles. They round the carcasses into a compact ball and remove the fur or feathers. Then, oral secretions are added to the carcasses to soften and preserve them (Milne and Milne, 1976). The larvae use the carcasses to obtain nutrients and develop after they hatch. The eggs are deposited on the carcasses or in the nearby soil. Eggs hatch within four days and larvae develop into pupae another six to eight days later. This species provide extensive biparental care for their offspring to protect the carcasses and the brood from conspecific competitors or microbial decomposition. These carcasses should be easily decomposed by microbial activity in the soil, so the carcasses must be protected to delay the decomposition in the chambers. This is why the beetles coat the carcasses with oral antimicrobial secretions (Hoback et al., 2004).
Antimicrobial compounds produced by burying beetles act as preservatives for the carcasses. The purpose of my research is to identify the characterization of oral secretions from Nicrophorus marginatus, Nicrophorus carolinus, and Nicrophorus tomentosis.
MATERIALS AND METHODS
Insect Collection. Nicrophorus marginatus, N. carolinus, and N. tomentosis were collected in Kearney County, Nebraska during the summer of 2007. They were collected on Tuesday through Thursday from 9:00 a.m. to 11:00 a.m. using baited pitfall traps. Rotted rats were used for bait and were replaced every week. Collected beetles were transported to the laboratory in BHS at the University of Nebraska at Kearney.
Secretion Collection. The secretions of beetles were collected by placing a sterile cotton swab in the mandibles (Hoback et al., 2004). The swab was placed in 0.1 M Tris at pH 7 to remove the oral secretion. Ten beetles’ oral secretion was collected per 1 mL of 0.1 M Tris. The samples were sterilized with a 0.22 µm filter and kept at -20oC until use.
Dialysis. The secretions from N. marginatus were dialyzed in 0.5M for 2 hours, 0.25M for 2 hours, and 0.1M Tris at pH 7 overnight in 7Kda and 10Kda molecular weight cutoffs Slide-a-lyzer dialysis membrane from Pierce (Rockford, IL).
SDS-PAGE. A 10% Acrylamide Tris-HCL gel was used to separate the proteins. Thirty μL of field sample, 7K dialyzed sample, and 10K dialyzed sample for N. marginatus were added to three different micro tubes and 100μL of reducing sample buffer was added to each tube. All tubes were put in near boiling water for 5 minutes to denature the proteins in the samples. Ten μL of each sample and 5μL of protein marker was loaded onto the gel. The gel was allowed to run for 45 minutes at 150 volts, stained with Coomassie Blue for a few hours, and destained with a decolorizer (175mL of distilled water, 15mL of methanol, and 10mL of acetic acid) overnight. Photographs were taken of the resulting gel using the Gel Doc System.
BCA Assay. A BCA kit from Pierce (Rockford, IL) was used to quantify protein. Stock solutions and samples were prepared in test tubes. A working reagent was added to each tube, and the tubes were incubated at 37°C for 30 minutes. Absorbance of each sample was measured with a Beckman DU 640 Spectrophotometer (Corona, CA) at 562nm twice, and compared to the standard curve of the stock solutions.
Toxicity Analysis. The antimicrobial activity of samples was tested with a Microtox Model 500 Analyzer (SDI, Newark, DE). Antimicrobial activity was quantified by measuring the decrease in bioluminescence of the bacteria Vibrio fisherii caused by cell death. All samples were tested three times using a 2% screening test, following the procedure in the MicrotoxOmniTM Software (AZUR Environmental, Newark, DE) with 0.1 M Tris at pH 7 as a control.
RESULTS
Collection. Insects were late in emerging the summer of 2007, due to cooler temperatures and wet weather in Kearney. N. marginatus beetles were found in traps in significant numbers toward the end of June, and N. carolinus the second week of July. N. tomentosis was found in traps at the same time as N. marginatus, but they were gone by the first part of July.
Toxicity Analysis. The Microtox analysis was used to identify differences in the antimicrobial activity in the three species of beetles trapped the summer of 2007. After the secretions were collected, antimicrobial activity was compared (Figure 1). Controls did not contain any samples, and one was also used as the blank. The secretions from the three species were tested to determine which species had the greatest antimicrobial activity. There was no apparent difference in activity when comparing samples taken directly from the beetles. Due to small numbers of N. tomentosis collected, no statistical analysis could be completed. 
FIGURE 1. Average absorbance readings from Microtox Analyzer in control, N. marginatus sample, N. carolinus sample, and N. tomentosis sample. The absorbance of each sample was measured after 5 and 15 minutes.
Protein Quantification. A BCA assay was performed to compare the protein concentrations in the secretions from the three species collected. A standard curve was established (Figure 2), and the protein concentration from each sample was figured and averaged (Table 1). The secretion from N. marginatus contained the largest amount of protein and the smallest amount of protein from N. tomentosis. The protein concentration from N. carolinus was between the other two species. Once again due to an insufficient number of samples for N. tomentosis, no statistical comparisons could be made.
FIGURE 2. The BCA standard curve obtained by measuring absorbance in each 11 different protein concentrations.
Table 1. Average absorbance readings and calculated protein concentrations for the oral secretions of N.marginatus, N. carolinus, and N. tomentosis.
Toxicity Analysis. The antimicrobial activity was tested again after the secretions were diluted to the same protein concentration, 1300 µg/mL. Figure 3 shows that the antimicrobial activities in N. marginatus and N. tomentosis oral secretions exhibited a higher antimicrobial activity than antimicrobial activities in N. carolinus oral secretions of the same protein concentration. After 5 minutes of exposure to the secretion, N . carolinus was at least 20 Absorbance units higher than N. marginatus and N. tomentosis, meaning that N. carolinus did not exhibit as high of an antimicrobial activity. N. marginatus and N. tomentosis were only 6 Absorbance units different from each other with N. tomentosis having greater activity.
FIGURE. 3. Average absorbance readings from the Microtox Analyzer in the field samples of N. marginatus, N. carolinus, and N. tomentosis at the same protein concentration at 5 and 15 minutes.
Sample Fractionation. N. marginatus samples were dialyzed to remove peptides from the secretion. Gel electrophoresis was used to identify if the peptides were gone (Figure 4). Lane 3 contains the undialyzed sample. Peptide bands were obvious in that sample. In both dialyzed samples, no peptide bands were found. These data were in support of earlier work done by Megan Nelson. After removing the peptide, the samples were tested for antimicrobial activity using the Microtox analysis. The antimicrobial activity in the 10K dialyzed sample was higher than the 7K dialyzed and field samples (Figure 5). All undialyzed and dialyzed samples exhibited antimicrobial activity greater than the control. The protein concentration of these three samples was figured and averaged (Table 2). The field sample taken directly from the beetles contained the largest amount of protein (854.4μg/mL) and the smallest amount of protein (430.7μg/mL) from 10K dialyzed sample.
FIGURE. 4. Protein gel of dialyzed oral secretions from N. marginatus. Lane 1: Mass marker, Lane 3: Undialyzed sample, Lane 5: 7 kDa dialyzed sample, Lane 7: 10 kDa dialyzed sample. FIGURE 5. Average absorbance readings from the Microtox Analyzer in the field, 7K dialyzed, and 10K dialyzed samples from N. marginatus at 5 and 15 minutes.
Table 2. Average absorbance readings and calculated protein concentrations for the oral secretions of field, 7K dialyzed, and 10K dialyzed samples from N. marginatus. DISCUSSION
The beetles emerged one month later in 2007 than in 2006. This really set back the research that was completed this summer. The first experiment was to check the beetle samples for antimicrobial activity. In the toxicity analysis, all three species show antimicrobial activity (Figure 1). This experiment had to be completed because previous studies (Hoback et al., 2004) show that N. carolinus lacks antimicrobial activity in its oral secretion. In this study I found that it did exhibit antimicrobial activity but also had the highest standard error (Figure 1). This species exhibits the greatest variability in antimicrobial activity. Currently other members of the laboratory are looking at the factors that would influence the production of antimicrobial secretion and why N. carolinus sometimes lacks antimicrobial activity in its oral secretion.
The second step of the experiment was to check the protein concentration of the beetle secretions. N. marginatus exhibited the greatest protein concentration and N. carolinus the lowest protein concentration from a BCA assay (Table 1). However, only one sample of N. tomentosis was obtained this summer. This species emerges early (Ratcliff, 1996), and I did not decide to compare it until the beetle was nearing the end of its cycle. Only ten beetles could be captured, creating problems when wanting to do statistical comparisons of the data. Next summer, more beetles will have to be sampled to complete this comparison among all three species.
All oral secretions from three species were diluted to equal protein concentrations based on the BCA assay in the second step to compare antimicrobial activity of these three species. These results showed that N. marginatus and N. tomentosis exhibit the greater antimicrobial activity in their oral secretion than N. carolinus (Figure 4). The fact that N. carolinus has the least active secretion may account for the lack of antimicrobial activity seen by Hoback et al. (2004). N. carolinus may not need as high of an activity because they are larger beetles (Ratcliff, 1996). They may be able to produce more secretion total than the smaller beetles. They may also not need to produce as much secretion because they are raising their broods in sandier, drier soils. Lack of water will limit the growth of microorganisms, so perhaps they do not have as great of contamination on the brood balls. Further studies would have to identify differences in brood ball decay under different conditions.
In order to decide if peptides were causing the antimicrobial action, samples were dialyzed in 7K and 10 K cartridges. In dialysis, molecules that are smaller than the pores of the membrane can move through the membrane. The 7K dialysis cassette removes molecules smaller than 7,000 kDa and 10K dialysis cassette removes molecules smaller than 10,000 kDa. As can be seen in Figure 5, peptides bands are apparent in the undialyzed sample. In both dialyzed samples, all peptide have been removed. This removal did not affect antimicrobial activity. The dialyzed samples exhibited greater antimicrobial activity than the field samples (Figure 5).
After all oral secretions from three species were diluted to equal protein concentrations based on the BCA assay, the Microtox analysis shows that N. marginatus and N. tomentosis exhibit the greatest antimicrobial activity in their oral secretion (Figure 3). The proteinaceous compounds in the secretions from the burying beetles have the antimicrobial activity, because after the peptide in the secretions was removed, the samples still showed the antimicrobial activity (Figure 5).
To be sure that the proteins were not diluted too much by the dialysis, protein was quantified in the dialyzed versus field samples. Dialysis diluted the protein by half (Table 2), but did not reduce antimicrobial activity (Figure5). In fact at 15 minutes, samples were slightly more active than the field sample. These data suggest that the peptides may actually have been interfering with the antimicrobial action.
Antimicrobial compounds have potential to be applied in clinical therapies. There are many antibiotics to kill microorganisms that cause infectious diseases, but they are losing their effectiveness due to increasing antibiotic resistance. A few years after the first antibiotic, penicillin, was used, penicillin resistant infections caused by the bacterium Staphylococcus aureus emerged (Bren, 2002). Bacteria have begun to gain resistance against many different antibiotics. Antimicrobial peptides are not affected by antibiotic resistance mutations and have the potential to replace antibiotics. They have the ability to kill microorganisms rapidly and have a broad activity on gram-positive and gram-negative bacteria and fungi. The novel antimicrobial peptide MBI-226 (Micrologix Biotech, Vancouver) is being used to treat infections caused by skin bacteria (Hancock and Scott, 2000). The identification of the antimicrobial compounds secreted by the burying beetles has potential use in the field of pharmacy.
Acknowledgements: I thank Dr. Julie Shaffer for her expertise and assistance, Mr. Darby Carlson for his cooperation in collecting the insects and secretions, Mrs. Judy Kuebler who supplied the place for trapping, and Shusaku Akahane for his help with my project. I would also like to thank the INBRE grant for funding this work.
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Abe, M. 2007. Characterization of the oral secretion from Nicrophorus marginatus, N. carolinus, and N. tomentosis. Research Thesis, Department of Biology, University of Nebraska at Kearney, Kearney, Nebraska 68849, USA. |