Mortality and repellent effects of microbial pathogens on Coptotermes formosanus (Isoptera: Rhinotermitidae)
© Wright and Cornelius; licensee BioMed Central Ltd. 2012
Received: 17 September 2012
Accepted: 22 November 2012
Published: 15 December 2012
Two entomopathogenic fungi, Isaria fumosorosea and Metarhizium anisopliae, and one bacterium, Bacillus thuringiensis, were tested for their ability to cause mortality of Formosan subterranean termites (FST), Coptotermes formosanus (Shiraki), after liquid exposure, and for their lack of propensity to repel FST.
The fungus Isaria fumosorosea at 108 spores/ml caused 72.5% mortality on day 7, significantly higher than the control and 106 spores/ml treatment. On day 14, the 106 and 108 concentrations caused 38.8% and 92.5% mortality, respectively, significantly higher than the control. On day 21, 82.5% and 100% of the termites were killed by the 106 and 108 treatments, respectively. I. fumosorosea did not repel termites at 106 nor 108 spores/g in sand, soil or sawdust. The fungus Metarhizium anisopliae at 108 spores/ml caused 57.5% mortality on day 7, 77.5% mortality on day 14 and 100% mortality on day 21.
On all three days the rate of mortality was significantly higher than that of the control and 106 spores/ml treatment with I. fumosorosea. Neither I. fumosorosea nor M. anisopliae caused repellency of FST in sand, soil or sawdust. The bacterium Bacillus thuringiensis did not cause significant mortality on days 7, 14 or 21. When termites were exposed to cells of B. thuringiensis in sawdust and when termites were exposed to a mixture of spores and cells in sand, a significantly higher number remained in the control tubes. Repellency was not seen with B. thuringiensis spores alone, nor with the above treatments in the other substrates.
Microbes have been considered as potential control agents for termites, as alternatives and adjuncts to chemical control measures. Termite behavior and grooming mechanisms present limitations to the effectiveness of termite microbial control , though it is suggested that combining pathogenic strains with other strains and with insecticides may improve efficacy . Behavior of mound building termites was found to limit spread of an isolate of Metarhizium anisopliae throughout the colony, with repellency being the primary inhibitory factor . A formulation of another strain with reduced repellency was shown to kill nests of Nasutitermes exitiosus termites by baiting in limited field trials. The microbes in this study were chosen because of evidence of their causing mortality to termites or other insects and are here screened for their degree of non-repellency.
M. anisopliae, when tested against the subterranean termite Reticulitermes flavipes, was found to cause alarm, aggregation and defensive reactions among termites that were untreated . Other fungi caused a lesser degree of alarm response which was followed by grooming and isolation of the infected termites. In addition, M. anisopliae was found to repel the Formosan subterranean termite (FST), Coptotermes formosanus, in tree-based mulches, however some of the repellency may have been attributable to substances from the mulches . Although, potential for M. anisopliae as a control agent for termites was demonstrated when, in a test of eight entomopathogenic strains against the subterranean termite C. gestroi, M. anisopliae was found to be the most virulent . A novel strain of M anisopliae was found to cause significantly greater mortality of FST alates and workers than a previously commercialized strain .
Isaria fumosorosea is an entomopathogenic fungus that has been previously shown to cause significant mortality to FST . I. fumosorosea is formulated in a wettable powder suitable for delivery with keratin foam. The keratin foam was developed as a biologically compatible delivery mechanism for termite microbial control agents [9, 10]. Species of Paecilomyces sect. Isarioidea are synonymous with Isaria.
Bacillus thuringienis is known to produce compounds toxic to some insects and to be pathogenic to others. Because Bacillus strains produce spores there is potential that this microbe will tolerate the nest environment of the termite, and produce infectious propagules in the soil and termite nest environment inhabited by termites. B. thuringiensis Berliner has caused mortality of the termite Nasutitermes ehrhardti. Bacillus isolates have been identified in the gut of C. formosanus, indicating the ability of the genus to survive, and potentially cause mortality of the termite .
Termite antennae play a significant role in grooming . Termites without antennae did not remove conidia of I. fumosorosea and M. anisopliae as efficiently as did termites with antennae. Also, termites reared individually were more susceptible to microbial infection than were termites reared in groups and subject to grooming by nestmates [15, 16]. To effectively control termites using microbes it will be critical to select pathogens that are capable of not only causing mortality but also withstanding detection and removal. Microbial strains that are both virulent and non-repellent have a greater likelihood of being spread within a termite nest and controlling termites in the field. Results are described here for virulence and non-repellency of potential microbial control strains.
Results and Discussion
A concern when applying microbial control agents is whether they will repel the target insect rather than infect and kill them. Studies with termites in the laboratory show the ability of microbial agents to kill termites, however few of these experiments have been translated to the field [1, 3, 17]. FST are known to remove infected nestmates from the nest and to partition infected areas of the nest and this has the potential to limit availability of inoculum [1, 15]. By selecting strains of fungi and bacteria that are pathogenic and also not repellent to termites, the probability of applying a microbial agent that functions successfully in the field is increased.
Each of the microbial agents was evaluated for the degree of non-repellency toward termites. Non-repellent agents are less likely to be detected and avoided by termites, thereby increasing the probability of causing a pathogenic effect . Termites were tested by exposure to the three microbes in sand, soil and sawdust. The number of FST remaining in tubes containing an entomopathogen was compared to the number of termites remaining in control tubes following 24 hrs in a paired choice test. Repellency was evident by termite foraging behavior in treated arenas differing significantly from termite behavior in untreated controls. Non-repellency was reported as no statistical difference between the numbers of termites in tubes.
Mean (±SEM) number of C. formosanus in a paired choice test where tubes were filled with substrate treated with fungal spores at the indicated concentrations, after 24 h exposure
Number of termite in tubes
I. fumosorosea 10 6 spores/g
36.3 ± 13.5a*
60.2 ± 17.3a
96.1 ± 11.1a
77.4 ± 10.6a
92.5 ± 9.6a
72.8 ± 10.2a
I. fumosorosea 10 8 spores/g
46.0 ± 6.5a
50.8 ± 4.5a
71.3 ± 16.0a
82.7 ± 17.1a
49.3 ± 9.8a
56.1 ± 9.7a
M. anisopliae 10 6 spores/g
23.9 ± 5.5a
45.0 ± 13.0a
82.3 ± 7.4a
76.0 ± 7.0a
93.4 ± 9.2a
62.7 ± 9.3a
M. anisopliae 10 8 spores/g
12.3 ± 2.0a
23.0 ± 5.9a
78.3 ± 12.6a
77.6 ± 12.8a
31.0 ± 3.9a
36.5 ± 4.5a
Mean (±SEM) number of C. formosanus in a paired choice test where tubes were filled with substrate treated with Bacillus thuringiensis strain ATCC 33679 at a concentration of 10 9 propagules/g, after 24 h exposure
Number of termites in tubes
43.5 ± 15.0a*
66.5 ± 17.1a
51.6 ± 8.9a
82.0 ± 10.9a
29.3 ± 6.6a
130.8 ± 9.6b
26.1 ± 6.7a
70.2 ± 10.6a
77.1 ± 12.2a
65.8 ± 7.3a
70.5 ± 13.8a
50% Cells + 50% Spores
31.5 ± 4.4a
88.3 ± 12.3b
41.1 ± 8.4a
60.3 ± 12.6a
66.3 ± 11.9a
66.8 ± 12.0a
Of the microbes tested, I. fumosorosea demonstrated the highest rate of mortality when termites were exposed to the spores in liquid. This is consistent with previous mortality studies that showed a significant pathogenic effect of this fungus against FST [8, 18]. In this study I. fumosorosea was also found to not repel termites in a paired choice test in sand, soil or sawdust. For any microbial agent to be effective as a termite control agent the cells or spores must not be repellent, as repellency will result in detection and avoidance by the members of the colony . I. fumosorosea has the added advantage of being produced as a stable powder . This fungus has also been formulated in a biologically-compatible foam suitable for application to termite nest environments . The foam has the potential to be used with M. anisopliae and other microbial agents.
Of the microbes tested, B. thuringiensis cells were found to repel termites only when in sawdust, and in the combination of cells and spores in sand. The remaining treatments, cells in sand and soil; spores in sand, soil and sawdust; and a combination of cells and spores in soil and sawdust, were not repellent to FST. However, when termites were exposed in liquid to the bacterium it was found to not be significantly pathogenic.
Based on the data reported here the fungi tested were found to not be repellent to FST. Both strains are pathogenic to this species of termite and have potential to control it in the field. The Bacillus strain had the lowest rate of mortality and, when exposed as cells in sawdust or as a combination of cells and spores in sand, was repellent to FST. Of the three microbes tested it would be the least likely to be selected for further development. The method reported here can be used to screen other Bacillus strains, and other potential bacterial entomopathogens, for mortality of FST in liquid. Using this method more closely approximates the liquid-based application which will ultimately be used in the field. The fact that the I. fumosorosea and M. anisopliae strains tested were pathogenic to FST and were here found to not repel termites makes them viable candidates for control of FST.
Isaria fumosorosea strain ARSEF 3581 was provided as blastospores in a wettable powder formulation with kaolin clay as the inert carrier by Dr. Mark Jackson (NCAUR, Peoria, IL) . Metarhizium anisopliae strain NRRL 30905 (ARS Patent Culture Collection, Peoria, IL, USA) was isolated in this laboratory from dead FST alates . It was inoculated onto potato dextrose agar (PDA) plates and incubated at 25°C for 7 d. Spores were harvested from the plates by scraping with a sterile loop. Bacillus thuringiensis Berliner strain ATCC 33679, isolated from diseased insect larvae, was obtained from the American Type Culture Collection (Manassas, VA, USA). A 100 μl aliquot of cells was removed from a tube stored at −80°C and used to inoculate 10 ml of LB. The culture was incubated at 28°C and 225 rpm for approx 6 hr, then used to inoculate 100 ml of LB which was incubated at 28°C and 225 rpm overnight. To encourage spore formation, a 10 ml culture of B. thuringiensis in LB was used to inoculate 100 ml of LB prepared at 25% (w/v) of the manufacturer’s standard recipe. The bacterial mass was harvested by centrifugation at 13 krpm for 20 min at 4°C in an angle rotor. The pellet was resuspended in water. Fungal spores, and bacterial cells and spores were enumerated using a Levy hemacytometer (0.1 mm deep; VWR, West Chester, PA, USA). B. thuringiensis cultures were determined to have reached 50% cells + 50% spores, and 100% spores by enumeration using the hemacytometer.
Termites were collected from City Park, New Orleans, LA from bucket traps . Four colonies were used for each treatment to prevent colony vitality biasing of data. Twenty FST from each colony were placed into a 2 ml conical microcentrifuge tube containing 0.5 ml of the spore/cell solution for 2 minutes, independent of termites from the other colonies. Tubes were agitated by hand during the incubation time to ensure that the termites were submerged in the liquid. The termites were then transferred to a 90 mm disc of filter paper (Whatman, Maidstone, England) in the lid of a 100 × 15 mm Petri dish where they were allowed to air dry. Control termites were exposed as described above, but the microcentrifuge tube contained water only without the addition of spores or cells. The termites were then transferred to a 55 mm Whatman filter paper disc moistened with water, which served as a moisture and nutrient source, and placed in the lid of a 60 × 15 mm Petri dish. Termites were incubated at 25°C and 85% humidity while mortality was monitored.
Termites were kept in the lab in 5.6-L covered plastic boxes containing moist sand and blocks of spruce Picea sp. until they were used in experiments. Treated substrates (sand, soil, or red oak sawdust) were inoculated with the stated concentration of microbe (w/w) and placed in a ½ gallon plastic bottle (Nalgene, Rochester, NY, USA). The bottle was rotated at 2 rpm (80% motor speed) for 6 hrs on a Wheaton Roller Apparatus (Millville, NJ, USA) at room temperature to ensure even distribution of cells and/or spores prior to transfer to the test containers. Control substrates did not contain any of the microbes.
For mortality bioassays, data were analyzed using analysis of variance (ANOVA) and least significant difference (LSD) at P≤ 0.05 . All analyses were run using SAS Software. For repellency bioassays, differences in the number of termites in treated or control tubes were compared using a paired choice t-test.
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
This study was funded by the United States Department of Agriculture, Agricultural Research Service. The authors wish to thank Bridgette Duplantis, Erin Lathrop and Christopher Florane for technical assistance.
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