Psammaplin A and pentoxifylline, two chitinolytic enzyme inhibitors, were evaluated for their palatability, feeding deterrence, consumption, and subsequent mortality against the eastern subterranean termite, Reticulitermes flavipes (Kollar). Psammaplin A and pentoxifylline were incorporated into filter paper diets at 0.0375, 0.075, 0.15, and 0.3%, and 0.01, 0.02, 0.04, 0.08, and 0.21% active ingredient (w/w), respectively. The treated filter papers served as food source or bait for termite workers used in this study. No-choice and two-choice feeding bioassays assessed bait consumption, palatability, repellency, and biological activity over a period of 1 - 5 wk (no-choice assay) and 3 - 5 wk (two-choice assay) of feeding. Neither psammaplin A nor pentoxifylline treatment repelled eastern subterranean termite diet consumption (in both nochoice and two-choice feeding arenas) as feeding commenced on both diet types within 48 h. In no-choice feeding tests, diet consumption was significantly decreased in termites fed 0.3% (2 - 5 wk) and 0.15% (4 - 5 wk) psammaplin A treated diet. Pentoxifylline treatment decreased diet consumption at 0.21% (3 wk). Consumption ratio trends suggest increased diet consumption rates by survivors feeding on chitinase inhibitor treated diet. In two-choice tests, termites consumed almost equal amount of diet treated with psammaplin A or pentoxifylline and untreated diet (with the exception of diet treated with 0.3% psammaplin A). Under no-choice bioassays, termite mortality from all concentrations of both chitinase inhibitor treated diets was significantly greater than that from untreated diet. Biological activity of psammaplin A and pentoxifylline treated diets in 2-choice feeding arenas was reduced by greater than 50% in comparison with no-choice feeding arenas. These results indicate the novel potential of chitinase inhibitors as effective active ingredients for a termite baiting program.

Subterranean termites are important economic pests throughout many temperate regions. The control and repair costs due to subterranean termites are estimated globally to be US $32 billion annually (Rust and Su 2012). In the United States, it is estimated that prevention and control costs range from US $1 - $2 billion annually (Jones 2003, Peterson et al. 2006). Due to their cryptic and destructive nature, stopping, suppressing, or eliminating subterranean termites from invading a structure is the ultimate goal of any control practice. For nearly 40 years, soil treatment with termiticides has been the conventional technique for structural control of subterranean termites. Presently, both repellent and nonrepellent termiticide applications are being used to eliminate subterranean termites from a structure (Barile 2003, Remmen and Su 2005).

An alternative pest management strategy for subterranean termite control is the use of a monitors and baiting program (Su et al. 1995b, Grace et al. 2002). Baiting systems containing benzoylphenylurea (BPU) as insect growth regulators (IGRs) such as hexaflumuron, noviflumuron, and diflubenzuron have been used to control or eliminate invading colonies of subterranean termites (DeMark et al. 1995, Prabhakaran 2001, Su 2003, Getty et al. 2005). BPU IGRs function as chitin synthesis inhibitors, which disrupt normal molting and alimentary tract (peritrophic membrane) physiology. BPU IGRs are relatively nontoxic to mammals due to strong target site protein binding (mammals lack chitin) and relatively rapid catabolism to less toxic compounds (Bayoumi et al. 2003). Termite baiting program efficacy relies on a slow acting, highly palatable/nondeterring active ingredient that promotes trophalactic exchange between termites through grooming behavior and stomodeal and proctodeal feeding (Myles 1997). Efficacy of bait product varies across termite species due to palatability preference of certain active ingredients (Su and Scheffrahn 1993, Su and Scheffrahn 1996, Karr et al. 2004), the number of workers of a colony foraging on and distributing active ingredient throughout the colony (Grace et al. 1996), and most significantly due to effects of competing food sources (Perrott 2003). Due to these efficacy differences, Kubota et al. (2006) suggested that other BPU IGRs may be effective bait toxicants and should be examined.

Just as BPU IGRs inhibit chitin synthesis, there exist several molecules that (effectively or can) inhibit chitin degradation (Kramer and Muthukrishnan 1997). In insects, chitin is degraded within the cuticle (during molting) and peritrophic membrane (during periods of molting and ingestion) by a binary enzyme system composed of chitinase and β-N-acetylglucosaminidase (Fukamizo and Kramer 1985, Filho et al. 2002). Chitinase and chitinase-like proteins are present in bacteria, fungi, plants, insects and other arthropods, and some marine and land animals (Watanabe and Kono 2002). The chitinase-like proteins found in higher animals, such as mice, chicken, and humans, function in defense against invading chitin-expressing pathogens (Nagano et al. 2002).

Several chitinase inhibitors with insecticidal biological activities have been identified through natural product chemistry (Spindler and Spindler-Barth 1999), such as allosamidin, the cyclic dipeptides cylco-(Proline-Tyrosine) and cyclo-(Histidine-Proline), and the modified fungal cyclic peptides argifin and argadin (Arai et al. 2000, Shiomi et al. 2000, Houston et al. 2002, Rao et al. 2003). These chemicals have been useful in elucidating the biochemistry of chitin catabolism, but have not been commercially formulated into insecticidal agents due to high production costs.

Two chitinase inhibitors with lower production costs are psammaplin A and pentoxifylline. Psammaplin A is a brominated tyrosine derivative isolated from the marine sponge, Aplysinella rhax. Psammaplin A (PA) inhibits chitinase by binding near and blocking the active site of the enzyme. Through initial screening (topical applications of 500 ppm), PA was shown to have insecticidal activity against diamondback moth larva with limited activity against other insects (Tabudravu et al. 2002). However, when incorporated into the diet of the peach-potato aphid, psammaplin A was shown to be an extremely toxic chitinase inhibitor (Saguez et al. 2006). Rao et al. (2005) screened a library of marketed drug molecules and identified the methylxanthine derivatives theophylline, caffeine, and pentoxifylline as chitinase inhibitors. Pentoxifylline provided the highest level of chitinase inhibition in fungal and human chitinase-like proteins (μM range of concentrations) by binding near the chitinase enzyme active site and mimicking a chitinase-chitin reaction intermediate. Because pentoxifylline is a molecule already being mass produced in the pharmaceutical industry, its production costs are far cheaper than all other chitinase inhibitors.

Delayed toxicities of psammaplin A and pentoxifylline against the eastern subterranean termite (via no-choice ingested diet assays) were assessed by Husen and Kamble (2013). LT50 values of psammaplin A treated diets (0.0375 - 0.3%) fed to eastern subterranean termite workers ranged from 21.3 - 38.5 d. LT50 values of pentoxifylline treated diets (0.01 - 0.21%) fed to eastern subterranean termite workers ranged from 32.2 - 44.6 d. Delayed toxicity (LT50 values) resulting from both chemicals were significantly lower than that of control treatments (at all concentrations tested). Concentration dependent mortality in pentoxifylline treated diet occurred only at 0.1 - 0.8% and was not observed in response to psammaplin A treatments. One hypothetical explanation of these results is that pentoxifylline is not palatable to/deters eastern subterranean termite feeding as treatment concentration increases whereas psammaplin A does not (Husen and Kamble 2013).

Active ingredients serving as bait toxicants must be slow acting (delayed toxicity), highly palatable, and not deter feeding. We conducted this study to assess the effects of chitinolytic inhibitors, psammaplin A, and pentoxifylline on diet consumption, palatability, feeding deterrence, and subsequent mortality in the eastern subterranean termites under no-choice and two-choice feeding conditions.

Termites. Termite infested logs were collected from Wilderness Park Recreation Area (Lincoln, NE) in July 2010, July and August 2011, and April 2012 and transported to the laboratory and stored in Roughneck trash cans (121 L, Rubbermaid, Huntersville, NC) under 85% relative humidity. Prior to experimental use, termites were extracted from the logs and maintained in Plexiglas containers (35 × 25 × 10 cm) with moistened substrate (sand, debris from log) and a corrugated cardboard food source. Termites were taxonomically identified to be R. flavipes using soldier morphology (Weesner 1965, Nutting 1990, Husen et al. 2006). No-choice chitinase inhibitor bioassays were conducted on 3 distinct termite colonies collected greater than 500 m apart (collected on July 2010, July 2011, August 2011). Chitinase inhibitor two-choice bioassays were conducted on 2 distinct termite colonies collected greater than 500 m apart (collected on August 2011 and April 2012). Colony distinction was also established by microsatellite genotyping (Vargo 2000). Undifferentiated 3rd to 5th instar termite workers were used for all bioassays. No-choice and two-choice chitinase inhibitor treated diet feeding bioassays were conducted on each termite colony within 1 month of their initial collection.

Chemicals. Psammaplin A (solid, C22H24Br2N4O5S2, molecular weight 664.39, Sigma Aldrich, St. Louis, MO, Prod. # P8749, Lot # 077K46101; solid, C22H24Br2N4O5S2, molecular weight 664.39, Santa Cruz Biotechnology, Santa Cruz, CA, Prod. # sc-258049A, Lot # A0512) and Pentoxifylline (solid, C13H18N4O3, molecular weight 278.31, Sigma Aldrich, St. Louis, MO, Prod. # P1784, Lot # 059K1682) were used for termite feeding bioassays. Multiple sources of psammaplin A were required due to manufacturer discontinuation after July 2010. Chitinase inhibitors were dissolved in HPLC grade acetone (Sigma Aldrich, St. Louis, MO, Prod. # 270,725, Lot # MKBD5310) for treatment of termite diet.

Treatment of filter paper as diet with chitinase inhibitors. Whatman grade 4 qualitative filter paper disks (55 mm diam., Whatman, Kent, UK) were treated with 3 ml of an acetone solution containing psammaplin A or pentoxifylline with both sides of the disk treated evenly (1.5 ml/side). Psammaplin A was incorporated into diet treatments at 0.0375, 0.075, 0.15, and 0.3% (w/w) and pentoxifylline was incorporated into diet treatments at 0.01, 0.02, 0.04, 0.08, and 0.21% (w/w). Untreated filter paper disks were prepared by applying 3 ml of acetone only as mentioned earlier. All treated filter paper disks were placed in a chemical hood and allowed to dry overnight at room temperature in RubbermaidTakealong plastic storage containers (669 ml, Rubbermaid, Huntersville, NC) until acetone had completely evaporated. Disks were then stored in plastic air-tight containers at 4°C until addition to experimental units.

Diet consumption and termite mortality in no-choice feeding test. No-choice feeding tests were used to assess bait consumption and mortality in response to ingestion of psammaplin A and pentoxifylline treated diets. Each experimental unit consisted of a plastic Petri dish (100 × 15 mm, BD Falcon, Becton, Dickenson, and Company, Franklin Lakes, NJ), 20 g of distilled water-washed and oven-dried sand moistened with 4 ml of deionized water, 40 worker termites (3rd - 5th instar) plus 1 - 2 soldier termites, and a treated or untreated filter paper disk as diet.

Each chitinase inhibitor concentration had 5 experimental unit replicates and was assayed against 3 termite colonies. Intercolony variation in diet consumption, palatability/deterrence, and subsequent mortality were determined to be minimal, thus bioassay data from independent colonies were combined leading to a total of 15 replicates of experimental unit for statistical analysis at each concentration and each time interval sampled.

At the onset of the feeding bioassays, experimental units were constructed and stored in 26.5 L Bella plastic storage units (Bella Contemporary Storage, Leominster, MA). High levels of humidity were maintained within the containers by placing moistened paper towels both above and below the experimental units. Paper towels were remoistened every 2 d. The experimental units were placed in a growth chamber at 23°C and complete darkness for 24 h to allow termites to acclimate to the Petriplate units. After the acclimation period, chitinase inhibitor treated or control diet was added to each unit.

Prior to the addition of diet, each piece of filter paper was weighed (pretreatment diet weight) using an Ohaus GA110 digital scale (Ohaus Inc., Parsippany, NJ). After diet was added, experimental units were placed in the growth chamber at 23°C and complete darkness for the duration of the study. Experimental units were destructively sampled at 1, 2, 3, 4, and 5 wk intervals, and bait consumption and biological activity were recorded. At each of these sampling intervals, live termites were quantified within each unit to determine mortality rate over time. A termite was considered dead when it was turned on its back and did not move appendages when prodded (Mao et al. 2011). Over the course of the study, all experimental units were observed every 3 d, and dead termites were removed to reduce mortality resulting from cannibalism of dead termites and or microbial growth within the experimental unit. At the conclusion of all sampling intervals, each diet disk was cleaned with a soft brush, air-dried, and then reweighed to calculate diet consumption (posttreatment diet weight).

Mean amount of treated diet consumed and mean percent survival data from nochoice feeding test experimental units were used to create a consumption ratio (mean diet consumption divided by mean percent survival) in response to control, pentoxifylline, and psammaplin A diet treatments over 1 - 5 wk. The consumption ratio provides a general trend of diet consumption as it relates to survival. Therefore, a higher consumption ratio indicates a greater consumption rate by surviving termites.

Treated diet palatability, feeding deterrence and subsequent termite mortality in two-choice feeding test. Two-choice feeding tests were used to investigate the palatability and feeding deterrence of each chitinase inhibitor treated diet. Mortality response over time was also evaluated to examine mortality rate changes when a competitive food source is present. The experimental unit setup and concentrations of psammaplin A and pentoxifylline tested remained the same as in the no-choice feeding tests. Data from two independent termite colonies (5 replicates of experimental unit at each concentration per colony) was combined leading to a total of 10 replicates at each concentration. Each experimental unit was provisioned with half of a piece of chitinase inhibitor treated paper and half of a piece of control treated paper (denoted with 3 small markings with lead pencil). Untreated control replicates received 2 half pieces of acetone-treated filter paper. Prior to the addition of diet, each piece of filter paper was weighed (pretreatment diet weight) using an Ohaus GA110 digital scale (Ohaus Inc., Parsippany, NJ). After diet was added, experimental units were placed in the growth chamber at 23°C and complete darkness for the duration of the study. Five replicates of each experimental unit from each chitinase inhibitor treatment/control diet regimen were destructively sampled at 3, 4, and 5 wk intervals. Diet consumption and termite mortality were determined as in the no-choice feeding tests.

Statistical analyses. Statistical analyses were conducted at 1, 2, 3, 4, and 5 wk (no-choice feeding tests) and 3, 4, and 5 wk (two-choice feeding tests) intervals to compare termite diet consumption and subsequent mortality in response to ingesting chitinase inhibitor treated diet. Within both types of feeding tests, diet consumption was analyzed by ANOVA using the PROC MIXED procedure. Means were separated using Fisher least significant difference procedure with statistical differences tested by paired t-tests (all paired) (P ≤ 0.05) (SAS Institute 2003). Within both types of feeding test, percentage mortality was statistically analyzed using the arcsine of the square root transformation (percent mortality transformed to normal distribution) (Yamamura 2002). Transformed percent mortalities were analyzed by analysis of variance (ANOVA) using the PROC MIXED procedure to detect significant differences at α = 0.05. Differences between treatment means were evaluated by paired t-tests (all paired) (SAS Institute 2003).

Bait consumption and termite mortality in no-choice feeding tests. When examining sampling interval specific psammaplin A mean diet consumption, there was a significant interaction between treatment type and treatment concentration (F = 3.01; df = 16, 225; P = 0.0001). At 1 wk, there were no significant differences in diet consumption between psammaplin A and untreated control (Table 1). At 2 wk, significantly more diet was consumed in control versus 0.3% psammaplin A treatments. However, at 3 - 5 wk, significant concentration-dependent differences in diet consumption were observed. At 3 wk, significantly less 0.3% psammaplin A treated diet was consumed when compared with that of the 0.0375% psammaplin A and untreated control. At 4 wk, significantly less 0.3% psammaplin A treated diet was consumed versus control and all other psammaplin A (0.0375, 0.075, and 0.15%) treated diets. Also, significantly less 0.15% psammaplin A treated diet was consumed compared with control and 0.0375% psammaplin A treatment. At 5 wk, significantly less 0.15% and 0.3% psammaplin A treated diet was consumed versus control and all other psammaplin A (0.0375% and 0.075%) treated diets (Table 1).

Table 1.

Mean psammaplin A (PA) treated diet consumption (mg) by R. flavipes workers in no-choice feeding tests.

Mean psammaplin A (PA) treated diet consumption (mg) by R. flavipes workers in no-choice feeding tests.
Mean psammaplin A (PA) treated diet consumption (mg) by R. flavipes workers in no-choice feeding tests.

When examining sampling interval specific pentoxifylline mean diet consumption, there was a significant interaction between treatment type and treatment concentration (F = 3.30; df = 16, 225; P = 0.0001). At 1 wk, significantly less 0.21% pentoxifylline treated diet was consumed than diet treated with 0.01% pentoxifylline (but did not differ from control and all other pentoxifylline treatments, Table 2). At 2 wk, diet consumption did not differ between control and pentoxifylline treated diets. At 3 wk, significantly less 0.21% pentoxifylline treated diet was consumed compared with untreated control and all other pentoxifylline (0.01%, 0.02%, 0.04%, and 0.08%) treated diets. At 4 and 5 wk, diet consumption did not differ between control and pentoxifylline treated diets (Table 2). At 3 wk, 0.21% pentoxifylline treatment was the only treatment to significantly reduce diet consumption when compared with other pentoxifylline treatments and the control (Table 2). Increases in diet consumption when compared with the control as a result of pentoxifylline treatment were observed at the 1, 2, 3, and 5 wk sampling intervals with the 0.01% pentoxifylline treatment significantly increasing diet consumption at 1 wk (Table 2).

Table 2.

Mean pentoxifylline (PX) treated diet consumption (mg) by R. flavipes workers in no-choice feeding tests.

Mean pentoxifylline (PX) treated diet consumption (mg) by R. flavipes workers in no-choice feeding tests.
Mean pentoxifylline (PX) treated diet consumption (mg) by R. flavipes workers in no-choice feeding tests.

When examining sampling interval specific psammaplin A and pentoxifylline treated diet and mean termite mortalities, there were significant interactions between treatment type and treatment concentration ((F = 3.68; df = 14, 335; P = < 0.0001) and (F = 2.00; df = 14, 335; P = 0.0326), respectively). Mean percentage mortality within the control treatment gradually increased over time with final control mortality of 5.43% at 5 wk. Mortality increased significantly in termite groups fed either psammaplin A or pentoxifylline treated diet (Tables 3, 4). All concentrations of both treated diet types resulted in significant mortality versus control treated diet at 1 - 5 wk. At 1 - 2 wk (psammaplin A treated diet) and 1 - 4 wk (pentoxifylline treated diet), no significant mortality differences between treatment concentrations. At 3 wk, mortality was significantly greater from feeding on 0.3% versus 0.0375% and 0.075% psammaplin A treated diet. At 4 wk, termite mortality was significantly lower at 0.0375% versus all other psammaplin A (0.075%, 0.15%, and 0.3%) treated diets. At 5 wk, resultant mortality was significantly lower at 0.0375% compared with 0.15% and 0.3% psammaplin A treated diets. Also, at 5 wk, termite mortality was significantly lower at 0.01% compared with 0.04%, 0.08%, and 0.21% pentoxifylline treated diet, and 0.3% psammaplin A treated diets.

Table 3.

Mortality of R. flavipes workers exposed to psammaplin A (PA) treated diet in no-choice feeding tests.

Mortality of R. flavipes workers exposed to psammaplin A (PA) treated diet in no-choice feeding tests.
Mortality of R. flavipes workers exposed to psammaplin A (PA) treated diet in no-choice feeding tests.
Table 4.

Mortality of R. flavipes workers exposed to pentoxifylline (PX) treated diet in no-choice feeding tests.

Mortality of R. flavipes workers exposed to pentoxifylline (PX) treated diet in no-choice feeding tests.
Mortality of R. flavipes workers exposed to pentoxifylline (PX) treated diet in no-choice feeding tests.

Consumption ratios (mean diet consumption divided by mean percent survival) were calculated to display general trends of diet consumption as it relates to survival. A higher consumption ratio indicates a greater consumption rate by surviving termites. A gradual increase in consumption ratio was observed (Figs. 1, 2) at 1 - 5 wk of feeding on control diet. At 1 - 2 wk, the consumption ratios of both psammaplin A and pentoxifylline treated diets were consistent with that of the control diet. At 3 wk, consumption ratios increased at all psammpalin A treated diet concentrations and in 0.01% - 0.08% pentoxifylline treated diets. The consumption ratio in response to 0.21% pentoxifylline treated diet decreased (below the control treated diet ratio). At 4 wk, consumption ratios increased at 0.0375 and 0.075% psammaplin A and all pentoxifylline treated diet concentrations. Consumption ratios decreased in response to 0.15 and 0.3% psammaplin A diet treatment. At 5 wk, consumption ratios were increased (relative to control) at all concentrations of psammaplin A and pentoxifylline treated diet (Figs. 1, 2).

Fig. 1.

Consumption ratios (mean diet consumption (mg) divided by mean percent survival) of no-choice psammaplin A (PA) feeding bioassays over time.

Fig. 1.

Consumption ratios (mean diet consumption (mg) divided by mean percent survival) of no-choice psammaplin A (PA) feeding bioassays over time.

Close modal
Fig. 2.

Consumption ratios (mean diet consumption (mg) divided by mean percent survival) of no-choice pentoxifylline (PX) feeding bioassays over time.

Fig. 2.

Consumption ratios (mean diet consumption (mg) divided by mean percent survival) of no-choice pentoxifylline (PX) feeding bioassays over time.

Close modal

Diet palatability, feeding deterrence and subsequent mortality in two-choice feeding tests. In two-choice feeding tests, addition of half chitinase inhibitor treated/half control diet did not significantly deter consumption versus those fed only a control diet. Generally, a trend emerged of less consumption on chitinase inhibitor versus control treated diet at all psammaplin A concentrations and the highest pentoxifylline concentration (0.21%) (Tables 5, 6). However, significant feeding deterrence was only observed in response to 0.3% psammaplin A treated diet at 3 and 5 wk (Table 5). Pentoxifylline diet consumption (0.01 - 0.04%) was equal to or above that of control treatment at some sampling intervals though this increase was not statistically significant (Table 6).

Table 5.

Mean psammaplin A (PA) treated diet consumption (mg) by R. flavipes workers in two-choice feeding tests.

Mean psammaplin A (PA) treated diet consumption (mg) by R. flavipes workers in two-choice feeding tests.
Mean psammaplin A (PA) treated diet consumption (mg) by R. flavipes workers in two-choice feeding tests.
Table 6.

Mean pentoxifylline (PX) treated diet consumption (mg) by R. flavipes workers in two-choice feeding tests.

Mean pentoxifylline (PX) treated diet consumption (mg) by R. flavipes workers in two-choice feeding tests.
Mean pentoxifylline (PX) treated diet consumption (mg) by R. flavipes workers in two-choice feeding tests.

Termite mortality from two-choice feeding tests with psammaplin A and pentoxifylline treated diet did not significantly differ from those fed on untreated control. There were no significant differences in percent mortality within treatment type or versus control among termites exposed to psammaplin A or pentoxifylline treated diets within the two-choice feeding tests. Percentage mortality levels were greatly reduced from those of the no-choice feeding tests. Resultant percent mortalities from psammaplin A and pentoxifylline treated diets ranged from 10.5 (0.0375%) - 28.8% (0.3%) and 8.5 (0.01%) - 18.8% (0.08%) (Tables 7, 8), respectively.

Table 7.

Mortality of R. flavipes workers exposed to psammaplin A (PA) treated diet in two-choice feeding tests.

Mortality of R. flavipes workers exposed to psammaplin A (PA) treated diet in two-choice feeding tests.
Mortality of R. flavipes workers exposed to psammaplin A (PA) treated diet in two-choice feeding tests.
Table 8.

Mortality of R. flavipes workers exposed to pentoxifylline (PX) treated diet in two-choice feeding tests.

Mortality of R. flavipes workers exposed to pentoxifylline (PX) treated diet in two-choice feeding tests.
Mortality of R. flavipes workers exposed to pentoxifylline (PX) treated diet in two-choice feeding tests.

Numerous insecticide baits are currently in use to control subterranean termite infestations. Benzoylphenyl urea compounds (primarily thought to function as chitin synthesis inhibitors) are the primary active ingredients in these baits (Kubota et al. 2006). Termite baits, and, thus, their active ingredients, must be slow acting, highly palatable, and not deter feeding. This study evaluated the effects of diet (filter paper) treated with the chitinolytic enzyme inhibitors, pentoxifylline and psammaplin A, on diet consumption/deterrence and subsequent mortality in the eastern subterranean termite. A range of concentrations of each chitinase inhibitor was tested to examine concentration-dependent trends in diet consumption/feeding deterrence and resultant termite mortality in no-choice and two-choice feeding arenas.

The active ingredient in bait must be nonrepellent, highly palatable, and not deter feeding (Su and Scheffrahn 1998). Chitin synthesis inhibiting active ingredients were tested at many concentrations for palatability, feeding deterrence, and repellency prior to the formulated concentrations being used commercially today (Su and Scheffrahn 1993, Su 2003). In terms of repellency, neither psammaplin A nor pentoxifylline repelled eastern subterranean termites when incorporated into the termite diet (in both no-choice and two-choice feeding arenas) as feeding commenced on all chitinase inhibitor treated or control diets within 0 - 48 h after addition to experimental units. In no-choice feeding tests, diet consumption was significantly decreased in termites fed 0.3% (2 - 5 wk) and 0.15% (4 - 5 wk) psammaplin A treated diet. Pentoxifylline treatment decreased diet consumption at 0.21% (3 wk).

Consumption ratios show increased rates of diet consumption (with the exception of 0.15 and 0.3% psammaplin A treated diet at 4 wk and 0.21% pentoxifylline treated diet at 3 wk) when compare with untreated control. Thus, surviving termites in chitinase inhibitor treated diet arenas consumed diet at a greater rate than survivors eating untreated diet. One hypothetical explanation of this trend could be that chitinase inhibitor treated diets are adversely affecting microorganisms (protozoa, bacteria and fungi) in the termite mid and hindgut, hampering the acquisition of nutrients necessary to maintain efficient metabolic processes, and thus causing surviving termites to consume more diet. Microbial metabolism produces the majority of termite nutritional and citric acid cycle sources (Odelson and Breznak 1983). Chitin synthesis inhibiting active ingredients such as noviflumuron, lufenuron, diflubenzuron, and hexaflumuron were all shown to cause significant reduction in termite gut protozoan populations with potential impacts on gut homeostasis (Perrott 2003, Lewis and Forschler 2010). In two-choice tests, termites consumed almost equal amount of diets in psammaplin A, pentoxifylline and untreated control (with the exception of 0.3% psammaplin A treated diet). Thus, under two-choice conditions, psammaplin A (0.0375 - 0.15%) and pentoxifylline (0.01 - 0.21%) treated diets did not deter feeding.

Termiticide bait active ingredients must be slow acting allowing time to exchange between exposed foraging termites and those remaining within the nest or foraging elsewhere. Metabolic inhibitors such as sulfluramid were originally proposed as bait active ingredients due to their slow acting nature (Su et al. 1995a); however, they were not successful termite baits because they had short lethal time which did not allow active ingredient exchange with other colony members (Su and Lees 2009). Currently used chitin synthesis inhibiting active ingredients such as noviflumuron, diflubenzuron, hexaflumuron, and bistrifluron all exert lethality in a dose-independent manner. Doseindependent lethal time is defined as a fixed time span before the onset of death regardless of the amount of toxicant ingested by a termite (Rust and Su 2012). Dose independence within BPU insecticides is thought to be due to their main activity occurring only during molting.

When examining termite mortality from no-choice feeding arenas, both chitinase inhibitors tested in this study are slow acting chemicals. These results confirm previous findings of delayed toxicity of chitinase inhibitor treated diets against eastern subterranean termites (Husen and Kamble 2013). Under no-choice conditions, termite mortality from all concentrations of both chitinase inhibitor treated diets was significantly greater than that from control treated diet. In the no-choice tests, 0.0375 - 0.15% psammaplin A did not elicit an immediate spike in mortality (23 - 40% mortality, respectively, at 3 wk of feeding). Pentoxifylline was also slow acting with mortality response not reaching 50% in any of the tested concentrations until the 5 wk sampling interval. In instances where diet consumption was significantly decreased (or the consumption ratio decreased below control ratio), percent mortality remained high (71 % at 5 wk with 0.3% psammaplin A treated diet). These results are likely due to the overall amount of chitinase inhibitor consumed as treatment concentration increased even although treated filter paper diet consumption decreased (Kubota et al. 2006). Biological activity of psammaplin A and pentoxifylline treated diets in two choice feeding arenas was reduced by greater than 50% in comparison with no-choice feeding areanas. Further research into the biological activity of both of these compounds under more realistic field conditions (multiple competing food sources, longer feeding duration) should be undertaken.

When examining data on the biological efficacy of any active ingredient, it is important to question the legitimacy of mortality data. Groups (replicate sets from each colony tested) of experimental units used in these studies were only used for analysis when control mortality was under 10%. This insured the health of the collected colony and legitimized the resultant mortality effects of psammaplin A and pentoxifylline on R. flavipes workers and the corresponding effect on diet consumption over time. Whereas the mode of action responsible for the slow acting mortality of 3rd - 5th instar termite workers fed chitinolytic inhibitors is not completely clear at this time, mortality data reported here were not due to molting disruption, as no dead termites were found attempting to molt and a molt was never induced. Disruption of chitinous structures within the peritrophic matrix and other gene-enzyme systems within the termite (and its intestinal microbes) are possible modes of action.

These results indicate that chitinolytic enzyme inhibitors possess termiticidal biological activity with a slow rate of action against R. flavipes. Psammaplin A (0.0375 - 0.075%) and pentoxifylline (0.01 - 0.08%) did not decrease consumption or deter feeding on treated diet under no-choice and two-choice feeding conditions. These results display that chitinase inhibiting chemicals have potential to be used as termite bait active ingredients whether as stand-alone active ingredients or as additions to currently existing commercial termiticides. Further investigation of optimal concentrations (slow acting biological efficacy combined with lack of feeding deterrence) especially under two-choice conditions, trophallaxis-induced mortality, and kinetics of uptake, clearance, and metabolism of psammaplin A and pentoxifylline should be undertaken.

We are grateful to Julie Stone, Department of Biochemistry, University of Nebraska-Lincoln and Blair Siegfried, Department of Entomology, University of Nebraska-Lincoln for providing valuable technical guidance and revision of this manuscript. We greatly appreciate the support and assistance of Ralph Narain during this study. This research was supported by the Nebraska Agricultural Research Division and Department of Entomology, Institute of Agriculture and Natural Resources, University of Nebraska, Lincoln, NE.

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Author notes

2Waltham Services, Waltham, MA 02453, USA.