Behavioral response of insect larvae to bioluminescence produced by the bacterium Photorhabdus luminescens cultivated by in vitro and in vivo methods

Walter Patterson, Floyd Inman III, Devang Upadhyay and Leonard Holmes

Sartorius-stedim Biotechnology Laboratory, Biotechnology Research and Training Center

University of North Carolina at Pembroke, Pembroke, North Carolina 28372

The data obtained from the experiment displays a linear trend in the negative direction in regards to distance from the culture for both in vitro and in vivo. The data obtained from the experiment indicates the migration of G. mellonella to the culture of P. luminescens occurred. These data supporting the speculation that P. luminescens utilizes bioluminescence to attract host insect larvae. The reason for implementing this strategy is to attract a secondary host so that emerging IJs of H. bacteriophora can easily infect the host for survival. Bioluminescence would increase the transfer probability of both nematode and bacterial symbiont by decreasing the distance between a new host and the infected cadaver.

A possible explanation for the low correlation value in the trend is the fact that the bacterial luminosity of the in vitro culture aliquot is lower than its natural in vivo counterpart. In this experiment the maximum RLU value reached by any culture was 2.0 x 10⁶ when compared to H. bacteriophora infected larvae where RLU can be greater than 10⁷ RLU Another factor which may have impacted error is that the presented results are of behavioral data; which may yield low correlation values in any given population. There may also be some unknown mechanism by which G. mellonella may be able to detect a carcass of another G. mellonella . If such a mechanism did exist it may explain the differences in the intercepts and rates of the functions found.

Discussion

Conclusion

The results obtained from the experiment show, an inversely proportional linear relationship between intensity of bioluminescence and larval head distance. The data obtained from this experiment supports the speculation that Photorhabdus spp. utilizes bioluminescence to attract other insect hosts for survival of both symbiotically associated partners. Further study is required utilizing infected insect larvae to support this experiment; however, if the strategy of using bioluminescence for host attraction can be confirmed experimentally then this confirmation may be extrapolated across the entire genus of Photorhabdus.

Acknowledgements

Partial financial support was provided in part by the: North Carolina Biotechnology Center (grant # 2010-IDG-1008), UNC-Pembroke Department of Chemistry & Physics and Farm Bureau of Robeson County. Additionally, thanks is given to the UNCP Office of the Provost and Academic Affairs and to the following sponsors.

Results

The distance from the head of the G. mellonella to the culture was determined by comparing the number of pixels constituting a known distance within the system to the distance in pixels from the head to the culture as described in proposed mathematical model (EQUATION 1). The mathematical model was applied to each larval/bacterial system image as shown (FIGURE 3). Averages and standard deviations of 12 sets (FIGURES 4 and 5) resulted in single points that were graphed as a function of increasing intensities of bioluminescence..

Figure 3: Determination of distances between the larval head and the culture aliquot. Cm and D are units of distance (cm) and the Dy, Dx, Cy and Cx are

number of pixels.

Figure 4: Effects of in vitro luminosity on Galleria mellonella position relative to P. luminescens culture. The trend of in vitro culturing that depicts a linear relationship. The linear portion depicts an attraction rate of -7.5 mm/105 RLU.

Figure 5: Experimental correlation between distance and RLU determined during in vivo trials. A linear correlation is observed that depicts a decreasing rate of -0.75 mm/105 RLU which is similar to the trend obtained through in vitro trials.

Background

Galleria mellonella (the Greater Wax Moth) is a Lepidopteran insect commonly associated with the destruction of honey bee hives. G. mellonella is considered to be a model host used for infectivity, efficacy, pathogenicity studies of entomoparasitic nematodes (EPNs) and their symbiotically associated entomopathogenic bacteria. Larvae of G. mellonella are also used to rear EPNs in vivo in the laboratory setting.

Photorhabdus luminescens is a Gram-negative, insect toxin-producing, bioluminescent, terrestrial bacterium commonly found in a symbiotic relationship with the insect-parasitic nematode Heterorhabditis bacteriophora. P. luminescens is carried within the gut of infective juvenile (IJ) nematodes. As the IJs detect a susceptible host insect, they attack the host and penetrate through the cuticle of the host to gain access to the host’s hemolymph (i.e. “blood”). Upon entrance, IJs of H. bacteriophora will regurgitate P. luminescens into the insect as a response to unknown insect cues. As the bacterial symbiont proliferates, P. luminescens secrete a variety of insect toxins and digestive enzymes that kill and bioconvert the host into nutrition for both partners. Additionally, Photorhabdus spp. is the only currently known terrestrial bacterial genus capable of producing bioluminescence (FIGURE 1). However, the role of bioluminescence in Photorhabdus spp. is yet to be identified, but it has been speculated that bioluminescence is critical for both nematode and bacteria as this light may be involved with attracting other potential hosts.

Typical hosts of H. bacteriophora include different species of insect larvae belonging to the insect orders of Lepidoptera, Coleoptera and Diptera. Lepidopteron larvae such as G. mellonella have the ability to detect light using sensory organs called stemmata. In G. mellonella, such structures exist on both sides of the head (FIGURE 2). These stemmata contain rhodopsins inside them that are primarily used in light detection. Most species of Lepidoptera contain rhodopsins that have a λmax close to the λmax of bioluminescence produced by Photorhabdus spp. (490 nm). From understanding that Photorhabdus spp. are associated with EPNs of Heterorhabditis spp. and produce light at wavelengths close to the wavelengths of insect rhodopsins, one could hypothesize that the purpose of the bioluminescence in the Photorhabdus spp. is for the bioluminescent attraction of insect larval hosts.

Figure 1: Plated culture of P. luminescens in low light displaying bioluminescence.

Figure 2: Scanning electron micrograph of the head of G. mellonella.

Samples of H. bacteriophora were obtained through in-house mass production protocols. P. luminescens cultures were isolated from infected G. mellonella using the method of Inman III and Holmes [1]. Infected hemolymph was aseptically obtained by an incision of the foremost pro-leg and streaked for isolation onto 2X nutrient agar. Non-used infected Galleria from this step were used for in vivo luminosity studies. Though a series of two bacterial isolations colonies of P. luminescens were successfully obtained. P. luminescens was cultured in 2X concentrated nutrient broth containing 25mM dipotassium phosphate (K2HPO4), pH 7.3. Cultures were incubated at room temperature and agitated on an orbital shaker at 150 rpm overnight. Culture aliquots (1 mL) of P. luminescens were dispensed into microcentrifuge tubes. Luminosity of each aliquot was measured with a luminometer in relative luminosity units (RLU) and recorded.

Moist filter paper (9 cm Ø) was placed on the lids of gridded Petri dishes. Bacterial aliquots were placed onto each filter paper. To the Petri dishes containing the aliquots, larvae of G. mellonella larvae were added and placed into a low-light environment and overlaid with felt. After 10 minutes of luminous exposure, digital pictures were taken at approximately a 90° angle.

Methodology

Equation 1: Mathematical model used to calculate distances.

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