Introduction

The Formosan subterranean termite (FST, Coptotermes formosanus Shiraki) is a pest insect that causes significant expenditure in prevention and repair costs in the USA annually. Microbial species have been investigated as potential control measures to complement chemical control methods (Shimizu and Yamaji 2002; Su 2002). Isaria fumosorosea (Ifr) has been shown to control FST in the laboratory (Wright et al. 2003). Species of Paecilomyces sect. Isarioidea are synonymous with Isaria (Luangsa-Ard et al. 2005). Keratin hydrolysate was previously found to be a biocompatible surfactant with foaming properties that allows delivery of the fungus into the nest environment of FST in trees and other difficult-to-reach void spaces (Dunlap et al. 2007). With a foam carrier system available to deliver Ifr into the void spaces of termite nests, augmentation of the virulence of this and other entomopathogenic agents is especially worthy of further investigation. Combining Ifr with compounds such as cuticle-degrading enzymes can increase the pathogenic potential of the treatment in the field. A novel strain of Metarhizium anisopliae isolated from an FST alate has been shown to cause mortality of FST alates and worker termites in the lab and in the field (Wright et al. 2005). Beauveria species have been demonstrated as microbial control agents of insect pests (Rehner and Buckley 2005; Ghikas et al. 2010). In order for entomopathogens to kill termites, they must have the capability of bypassing termite pre-penetration defense mechanisms and also survive passage to nestmates through allogrooming (Chouvenc et al. 2011). Combining entomopathogens with enzyme or chemical applications may enhance the pathogenic effect (Bulmer et al. 2009)

Cuticle-degrading enzymes isolated from three bacteria were studied for their ability to increase effectiveness of Ifr against FST. The enzymes chitinase and lipase have been isolated from entomopathogenic fungi and have been correlated with enhanced virulence (Hegedus and Khachatourians 1988; Mendonsa et al. 1996; Hernandez-Torres et al. 2004; Nahar et al. 2004; Dahiya et al. 2006). Selection of an appropriate chitinase for use with fungal entomopathogens is critical because chitinases are thought to be expressed by plants to protect against pathogenic fungi (Taira et al. 2002) and are used to control plant pathogens in agricultural applications (Dahiya et al. 2006; Karasuda et al. 2003). Bacterial chitinases aid host nutrition and parasitism, and act as antibacterial agents and immunoenhancers (Wen et al. 2002; Dahiya et al. 2006).

Chitinases may increase penetration of pest insects by entomopathogens by degredation of chitin (St. Leger et al. 1996). This effect may lead to an increase in virulence (El-Sayed et al. 1989; Gupta et al. 1994). A mutant of the fungus I. fumosorosea that overexpresses chitinases caused a significant increase in mortality of whitefly compared to the parental strain (Hernandez-Torres et al. 2004). Conversely, a mutant strain of the fungus Nomuraea rileyi, with decreased chitinase expression, showed a comparative decrease in mortality of cabbage looper larvae (El-Sayed et al. 1989). Paecilomyces lilacinus is used as a biological control agent of plant parasitic nematodes and its chitinase activity correlates with that of Trichoderma spp. and M. anisopliae (Khan et al. 2003).

It has been proposed that chitinases will increase penetration of target insects by pathogenic fungi through degradation of structural chitin (St. Leger et al. 1996) with the potential to increase virulence (El-Sayed et al. 1989; Gupta et al. 1994). A stable mutant of I. fumosorosea that over-produces chitinases displayed twofold greater mortality of whitefly than the parental strain (Hernandez-Torres et al. 2004). By contrast, a mutant strain of the fungus N. rileyi with decreased chitinolytic activity compared to the parental strain also caused a decreased mortality rate among cabbage looper larvae (El-Sayed et al. 1989). P. lilacinus is a biological control agent for use against plant parasitic nematodes. Chitinase activity isolated from this species correlates with that found in Trichoderma spp. and M. anisopliae (Khan et al. 2003).

Eicosanoids are biologically active, oxygenated C20 polyunsaturated fatty acid metabolites. They are involved in human immune responses in the form of prostaglandins and leukotrienes. In addition, they have been found to protect invertebrates from infection by bacteria (Miller et al. 1994, 1996; Stanley 1998). Eicosanoid biosynthesis inhibitors (EBI) were found to increase insect mortality when applied in combination with the bacterium Serratia marcesans to tobacco hornworms, Manduca sexta (L.) (Stanley-Samuelson et al. 1991). Insects form nodules in an active defensive response to bacterial infection. Nodule formation was inhibited when M. sexta, the tenebrionid beetle, Zophobas sp., and crickets, Gryllus assimilis (F.) were injected with both an EBI and bacteria (Howard et al. 1998; Miller et al. 1996, 1999).

Connick et al. (2001) exposed FST to combinations of the bacterium S. marcesans and the EBIs ibuprofen (IBU), ibuprofen sodium salt (IBUNA), and dexamethasone (DEX). Both IBU and DEX significantly increased mortality of FST workers versus mortality caused by S. marcesans alone, with DEX showing the greater effect on mortality. Based on other studies, it is believed that the increased mortality resulted from the suppression of the FST immune system by the EBIs (Stanley-Samuelson et al. 1991; Miller et al. 1994, 1999; Howard et al. 1998). IBUNA in combination with S. marcesans caused a lower mortality than the bacterium alone (Connick et al. 2001). IBUNA, unlike IBU, is water soluble and this factor may have affected the termite or the bacterium through dilution in the moist environment in which termites live and were tested. None of the EBIs, when used alone, caused significantly higher mortality than a peptone control. This was the first report of a pharmaceutical effect on insects exposed to EBIs through contact and normal feeding rather than injection. Cuticle-degrading enzymes and EBIs are here combined with entomopathogenic fungi to determine their potential to increase the rate of FST mortality.

I. fumosorosea (Ifr) has been developed as a biocontrol agent for whitefly control (Jackson et al. 1997). Methods to produce Ifr blastospores in a stable, wet-able powder formulation have been developed (Jackson 1999; Jackson et al. 2003). Screening of Ifr against the Formosan and native subterranean termite workers revealed that it was pathogenic to termites (Wright et al. 2003). Additional work with Ifr demonstrated that a 5 min exposure of termites to Ifr spores is sufficient to cause 100 % mortality in 4 to 7 days (Wright et al. 2003). EBIs are believed to suppress insect immune systems thereby aiding infection by microbial pathogens (Connick et al. 2001). A synergistic effect from the application of EBIs and the bacterium S. marcesans showed that DEX and IBU both enhanced the pathogenic effect of the bacterium (Connick et al. 2001).

Materials and methods

Microbial strains

Beauveria pseudobassiana strain ARSEF 8046 was obtained from the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) and was originally isolated from a deer tick. The culture was inoculated onto potato dextrose agar (PDA) plates and incubated at 30 °C for 7 days. After incubation, fungal spores were scraped from the plates into a sterile tube using 100 μL of sterile 0.01 % aqueous Triton X-100 and an inoculating loop. Spore concentrations were quantitated using a Levy hemacytometer (0.1 mm deep; VWR, West Chester, PA, USA). For initial mortality assays, the following concentrations were prepared: 2 × 103, 2 × 106, and 2 × 109 spores/mL. For assays in combination with enzymes, a concentration of 2 × 107 spores/mL was prepared.

I. fumosorosea (Ifr) strain 3581, isolated from Bemisia argentifolli in McAllen, Texas, was formulated in Hyflo Super-Cel (Celite Corp., Lompoc, CA). The formulation was produced by a previously reported method (Jackson et al. 1997) and provided by Dr. Mark Jackson of the USDA-ARS-NCAUR, Peoria, IL, USA. Stock cultures were grown on potato dextrose agar (PDA; DIFCO, Detroit, MI, USA) for 3 weeks at room temperature, cut into 1-mm2 agar plugs and stored in 10 % glycerol at −80 °C. Conidial plates were produced by inoculating PDA dishes with a conidial suspension from frozen stock cultures and incubating at 25 °C for 2–3 weeks. The spores were diluted in Hyflo to a final spore count of approximately 1.6 × 1010 spores/g for combination with chitinase from Streptomyces griseus, or to a final spore count of approximately 6 × 108 spores/g for combination with the chitinase from S. marcesans and lipase from Pseudomonas cepacia.

Termites used for mortality bioassays

FST workers, third instar or older based upon size, and soldiers were provided from bucket traps (Su and Scheffrahn 1986) located in New Orleans, LA, USA. Termites from four different colonies were selected to prevent colony vitality biasing of data. Each colony represented one replicate. Each treatment was applied independently to 18 workers and 2 soldiers from each replicate. Only one half of the termites in each experimental sample were directly exposed to the fungus to confirm that the previously demonstrated transfer of fungal spores from termites to their nestmates occurred. Varying mortality rates of controls are attributed to seasonal changes in termite viability. All termites for each individual experiment were collected at the same time.

Cuticle-degrading enzymes

Proteinase A (ProA) was isolated from Aspergillus melleus and proteinase B (ProB) is from a bacterial source. One unit of each will hydrolyze casein to produce color equivalent to 1.0 μmol (181 μg) of tyrosine/min at pH 7.5 at 37 °C. Lipase A (LipA) was isolated from Thermomyces lanuginosus and lipase B (LipB) was isolated from P. cepacia. One unit of each liberates 1 μmol oleic acid/min at pH 8.0 and 40 °C. Chitinase A (Chi A) was isolated from S. griseus, and chitinase B (Chi B) was isolated from S. marcesans. One unit of each will liberate 1.0 mg of N-acetyl-d-glucosamine from chitin/h at pH 6.0 at 25 °C in a 2-h assay. All enzymes were obtained from Sigma (St. Louis, MO, USA). Each was prepared at a concentration of 2 units of enzyme/mL in water. A 500 μL aliquot (1 unit) of the enzyme was used to wet the filter paper, with an equal volume of water applied to filter paper in the control replicates.

B. pseudobassiana and cuticle-degrading enzymes

Twenty FST from each colony were placed into a 2 mL conical microcentrifuge tube containing 0.5 mL of a suspension containing the stated concentration of spores in sterile, deionized water for 2 min, 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 sterile water only. These termites were then transferred to a 55 mm Whatman filter in the lid of a 60 × 15 mm Petri dish with an equal number of untreated nestmates. The paper disc served as a moisture and nutrient source. For control and fungus-only assays, the filter paper was wetted with water. For enzyme-only or fungus with enzyme assays, the filter paper was wetted with the appropriate enzyme solution at a concentration of 1 unit per filter disc in water. Bioassays were incubated at 25 °C and 85 % humidity, optimized for fungal and termite viability, while mortality was monitored. For initial mortality assays mortality was monitored on days 7, 14, and 21. For assays with combined fungal and enzyme treatments, mortality was monitored on day 7.

I. fumosorosea and cuticle-degrading enzymes

Chi A and Chi B were diluted to working concentrations of 0.06 and/or 0.6 mg/mL in water. For enzyme-only treatments, 20 termites from each colony were placed on a 55-mm disc of filter paper, wetted with 0.5 mL of the appropriate enzyme, in the lid of a 60 × 15 mm Petri dish as enzyme controls. For fungus-only treatments, 10 termites from each colony were placed in a 100 × 15 mm Petri dish containing 2 g of diluted Ifr spores and were allowed to walk on the spores for 5 min, independently of termites from other colonies. The termites were then transferred to a 55 mm disc of filter paper, wetted with 0.5 mL of water, in the lid of a 60 × 15 mm Petri dish along with an equal number of nestmates not exposed to the fungus. For the control, 20 termites from each colony were placed on a 55 mm disc of filter paper, wetted with 0.5 mL of water, in the lid of a 60×15 mm Petri dish. Combined treatments exposing 20 termites from each colony to both Ifr and the respective enzyme concentration were prepared as described in the following steps: a 55 mm disc of filter paper, in the lid of a 60 × 15 mm Petri dish, was wetted with 0.5 mL of one of the chitinase solutions. For fungus-only treatments, 10 termites from each colony were exposed to Ifr spores as described. The termites were then transferred to a filter paper disc, wetted with 0.5 mL of the appropriate concentration of chitinase, along with an equal number of nestmates not directly exposed to the fungus.

Lip A and Lip B were diluted to working concentrations of 2 and 10 mg/mL. For both enzyme control and combined enzyme/Ifr treatments, 20 FST from each colony were placed into a 2-mL microcentrifuge tube containing 0.5 mL of the appropriate lipase solution for 2 min independent of termites from the other colonies. The termites were then transferred to a 90-mm disc of Whatman filter paper and allowed to air dry. Termites exposed to an enzyme only were transferred to a 55-mm disc of filter paper, wetted with 0.5 mL of water. Water control termites were exposed as described as above, but the microcentrifuge tube contained water rather than an enzyme. For combined treatments, half of the termites were subsequently exposed to Ifr spores as described above. Ten termites exposed to both the lipase and the fungus were placed onto a filter paper, wetted with 0.5 mL of water, along with 10 nestmates which were exposed to the lipase only. All Petri dishes containing termites were incubated at 25 °C and 90 % humidity while mortality was monitored for up to 21 days.

I. fumosorosea and eicosanoid biosynthesis inhibitors

IBU and DEX (Sigma Chemical Co., USA) solutions were made in ethanol to a final concentration of 50 μg/mL and a 50 μg/mL solution of IBUNA (Sigma Chemical Co., USA) was made in sterile water. All solutions were used immediately. To prevent the ethanol from affecting the termites, 1 mL of the DEX and IBU solutions were pipetted onto Whatman #4 filter paper discs and allowed to dry. The filter paper was then wetted with 1 mL of sterile water. FST were exposed to the EBI when they walked on and consumed the filter paper.

Ten FST workers from each of four colonies were allowed to walk on a sporulated fungal culture on a PDA plate for 2.5 min. The exposed FST workers were then transferred to Petri dishes (Falcon, Franklin Lakes, NJ, USA) which contained 10 of their nestmates and a Whatman #4 filter paper disc. The filter paper was dampened with either 1 mL of sterile water (Solution 2000 Water Purification System, Solution Consultant Inc., Jasper, GA, USA), or 1 mL of a 50 μg/mL solution of one of the EBIs. The Petri dishes were then incubated at 25 oC and ∼99 % humidity for the duration of the experiment. Controls included exposure of termites allowed to walk on Ifr alone, exposed to one of the EBIs alone, or exposed to 1 mL of sterile water only.

Data analysis

All data were analyzed using analysis of variance (ANOVA) and least significant difference (LSD) at p ≤ 0.05 (Cody and Smith 1997). One hundred percent mortality represents an absolute value of 80 termites. Treatments with the same letter on the same day are not significantly different. All analyses were run using the SAS System Software (Cody and Smith 1997).

Human and animal rights

No humans or other animals were subjects in this study.

Results

B. pseudobassiana and cuticle-degrading enzymes

On day 7, the mortality rate for termites exposed to the control treatment which contained only water was 3.8 %. On the same day, termites exposed to both 103 and 106 spores/mL had mortality rates of 2.5 %. Termites exposed to 109 spores/mL reached a mortality rate of 100 % by day 7 (Fig. 1). By day 14, 7.5 % of the control termites had died, while the mortality rate for the 103 and 106 treatment levels was 3.8 and 11.3 % respectively. By day 21, the 106 spore concentration treatment mortality rate equaled that of the control at 12.5 %, while the 103 treatment was 10.0 % on the same day.

Fig. 1
figure 1

Mortality (mean %) of FST by B. pseudobassiana strain ARSEF 8046 at concentrations of 103, 106, and 109 spores per mL on 7, 14, and 21 days after exposure; termites were exposed to the stated concentration of the fungal strain in liquid, and incubated on moistened filter paper; mortality was determined weekly

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Once it was determined that B. pseudobassiana caused mortality of FST, the potential for increased mortality with enzymes capable of degrading termite cuticle was investigated. Termites were exposed to 1 × 107 spores/mL of B. pseudobassiana, and 1 unit each of two proteinases, one lipase and one chitinase. These concentrations were chosen because they did not cause significant mortality individually, and would allow identification of an increase in mortality caused by synergism. Each enzyme was then combined with the fungus for determination of potential increased virulence on day 7. None of the individual treatments were significantly different from one another. The combined treatments of the fungus and the lipase were not significantly different from the individual treatments. Bpb + Lip A caused a mortality rate of 10.0 %, while exposure of termites to Bpb + Lip B resulted in a mortality rate of 13.8 % (Fig. 2). The combination of Beauveria with Pro A caused a 21.3 % mortality, while the combination of the fungus with Pro B resulted in a mortality rate of 22.5 %. Pro A is from the fungus Aspergillus and Pro B is from a bacterium. Both caused mortality significantly higher than that of the control, the Beauveria strain alone, or either Pro A or Pro B alone. The combination of Beauveria with chitinase resulted in a mortality rate of 32.5 %, the highest observed in this study.

Fig. 2
figure 2

Mortality (mean %) of FST by B. pseudobassiana strain ARSEF 8046 (Bpb) at a concentration of 107 spores/mL on day 7 in combination with proteinase (Pro), lipase (Lip), and chitinase (Chi) enzymes; termites were exposed to the fungal strain and enzymes in liquid, then incubated on moistened filter paper; mortality was determined weekly

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I. fumosorosea and eicosanoid biosynthesis inhibitors

To measure potential synergism between EBIs and a fungal biocontrol agent, FST workers were exposed to Ifr for 2.5 min in this experiment, half the time previously found to be effective. Through day 5 of the experiment, none of the chemical treatments enhanced the pathogenicity of Ifr (Figs. 3, 4, and 5). From day 6 through day 9, the Ifr + Dex (Fig. 3) and Ifr + IBUNA (Fig. 4) treatments showed increased mortality over Ifr alone. Both Ifr alone and Ifr with the Dex supplement reached 100 % mortality by day 12 (Fig. 3). Mortality in excess of 50 % with termites exposed to Ifr alone confirms previous studies that the fungus was transferred among nestmates. Ifr with the IBUNA supplement reached 100 % mortality by day 16 (Fig. 4). IBU, which was shown to enhance pathogenicity of S. marcesans (Connick et al. 2001), did not have a positive effect on the pathogenicity of Ifr. The bacterium and the IBU were delivered as liquid solutions while the termites were exposed to the fungus and IBU separately. This may have changed any solubility effects between the chemical and the microbes. At no point did the mortality rate of the Ifr + IBU combination exceed that of Ifr alone (Fig. 5). On day 6, when the DEX and IBUNA treatments exceeded the Ifr treatment, Ifr + IBU had killed only 26 % of the termites compared to 48 % killed by Ifr alone. On day 16, the Ifr, Ifr + IBU, and IBU treatments had reached 100, 99, and 6 % mortality, respectively (Fig. 5).

Fig. 3
figure 3

Comparative mortality of FST workers exposed to dexamethasone (Dex) and/or I. fumosorosea (3581), only half the termites were directly exposed to the fungus

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Fig. 4
figure 4

Comparative mortality of FST workers exposed to ibuprofen sodium salt (IBUNA) and/or I. fumosorosea (3581), only half the termites were directly exposed to the fungus

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Fig. 5
figure 5

Comparative mortality of FST workers exposed to ibuprofen (IBU) and/or I. fumosorosea (3581), only half the termites were directly exposed to the fungus

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I. fumosorosea and cuticle-degrading enzymes

On day 7, the mortality rate for termites exposed to the control treatment which contained only water was 2.5 %. On the same day, termites exposed to Ifr alone had a mortality rate of 22.5 % and reached a maximum of 63.8 % mortality on day 21.

Ifr spores in combination with Chi A at 0.06 mg/mL caused 43.8 % mortality at day 7, 70.0 % at day 14 and 88.8 % at day 21 (Fig. 6). The same enzyme at a concentration of 0.6 mg/mL when combined with Ifr spores caused 26.3 % mortality at day 7, 50.0 % at day 14 and 68.8 % at day 21. Ifr alone caused 41.3, 80.0, and 85.0 % mortality on days 7, 14, and 21, respectively. The mortality rates of the fungus in combination with the enzyme were not significantly different from those of the fungus alone. In fact, the values for the 0.6 mg/mL chitinase are somewhat, although not statistically, lower than those of the Ifr only treatment. On days 7 and 14, these treatments were also statistically not different from the untreated and enzyme-only controls. On day 21, all Ifr treatments were significantly different from the water control which showed only 21.3 % mortality. The combined Ifr + 0.06 mg/mL chitinase treatment, with 85.0 % mortality, was significantly different from the chitinase only controls, which at 0.06 mg/mL caused 20.0 % mortality, and at 0.6 mg/mL caused 15.0 % mortality.

Fig. 6
figure 6

Comparative mortality of FST workers exposed to chitinase from S. griseus and/or I. fumosorosea (3581), only half the termites were directly exposed to the fungus

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Ifr spores when combined with 0.6 mg/mL of Chi B caused significantly greater mortality, 17.5 %, than the fungus alone, 3.8 %, as early as day 4 post-exposure (Fig. 7). By day 16, the combined treatment had caused 100 % mortality. The maximum mortality on day 16 was 56.3, 47.5 and 15.0 % for the fungus alone, enzyme alone and water control, respectively. Each value was significantly different from the combined treatment.

Fig. 7
figure 7

Comparative mortality of FST workers exposed to chitinase from Serratia marcesans and/or I. fumosorosea (3581), only half the termites were directly exposed to the fungus

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Ifr spores when combined with lipase at a concentration of 2 mg/mL caused significantly greater mortality than the fungus alone as early as day 2 post-exposure, 38.8 % mortality vs. 1.3 % mortality (Fig. 8). A rate of 100 % mortality in the combined treatment was reached at day 14. The 10 mg/mL treatment in combination with the fungus caused significantly greater mortality by day 4 and reached a maximal mortality of 97.5 % at the end of the experiment on day 16. The water control, 2 mg/mL lipase and 10 mg/mL lipase controls reached maximal mortality rates of 28.8, 56.3, and 51.3 % on day 16. All were significantly lower than the mortality rates of the combined treatments.

Fig. 8
figure 8

Comparative mortality of FST workers exposed to lipase and/or I. fumosorosea (3581), only half the termites were directly exposed to the fungus

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Discussion

Use of cuticle-degrading enzymes to enhance virulence of entomopathogenic fungi must be balanced with selection of enzymes that do not inhibit the biological control agent. Fungal inhibition would be evident by decreased activity when compared to the activity of the pathogen alone. Appropriate combinations can result in synergism, utilizing lower concentrations of each component than if they were applied individually. These results demonstrate an increased mortality rate as early as day 7 between B. pseudobassiana strain 8046 and both proteinases, as well as the chitinase. The lipase tested here did not produce an effect that was significantly different from the control. These data suggest that the proteinase and chitinase enzymes have potential use with this fungus. Further investigation of interactions between entomopathogenic fungi and cuticle-degrading enzymes may yield increased FST control conditions.

The desire to utilize cuticle-degrading enzymes to enhance virulence of entomopathogenic fungi must be tempered with selection of enzymes that do not negatively impact the biological control agent. Proper selection has the potential to generate enzyme/fungus combination treatments that control the target insect utilizing less of each component than if they were applied individually. These results demonstrate effect on mortality by chitinase from S. griseus and suggest a negative influence on the fungus’ pathogenic properties by this enzyme. The other two enzymes tested, chitinase from S. marcesans and lipase from P. cepacia, did increase the mortality rate of the FST when applied with Ifr when compared to application of the fungus or enzyme alone. These data suggest that these two enzymes have potential for use with Ifr in particular, and that further investigation of interactions between other entomopathogenic fungi and cuticle-degrading enzymes may yield compatible pairings. Further research is needed to determine if other treatments can improve control, and to optimize the ratio of enzymes to fungal spores.

Termites are cryptic pests that are difficult to detect and treat. Control methods will ideally affect colony suppression to prevent re-infestation. Termites will remove dead termites from the main body of the colony and can detect and avoid some chemical and biological control agents. Biological and chemical control agents have the potential to enhance the effect of each other. Of particular interest are chemicals that inhibit the immune system of termites making them more susceptible to microbial infection. As with successful chemicals, the selected microbe must not be repellent to termites or it will be avoided before a sufficient inoculum can be delivered. The microbe must also be delivered to nest members that do not forage to the delivery site. Inoculated foraging termites must also live long enough to travel back to the nest and transfer the microbe to nestmates for the colony to be suppressed. The EBIs DEX and IBUNA are shown here to enhance pathogenicity of Ifr. These EBIs can potentially be delivered to termites in the field in a foam or gel by injection into an infested tree or wood structure, or they could be impregnated into a solid substrate such as paper or wood in a bait station. Pathogenicity of fungal spores delivered simultaneously would then be enhanced. These studies and previous work with EBI and a bacterium indicate that further study in this area is warranted to yield ideal integrations of biological and chemical control agents.