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Triatomines (Hemiptera: Reduviidae: Triatominae), commonly called "kissing bugs", are blood-sucking pests and vectors of the protozoan parasite Trypanosoma cruzi , the causative agent of Chagas disease (CD). Eleven species of kissing bugs occur throughout the southern half of the USA, four of which are well known to invade human dwellings. Certain kissing bugs in the USA are known to transmit T. cruzi to humans and other animals and their bites can also lead to serious allergic reactions, including anaphylaxis. In Florida, the kissing bug Triatoma sanguisuga frequently invades homes, bites residents, and has been found infected with T. cruzi , placing humans and companion animals at risk for CD. This review outlines integrated pest management (IPM) strategies for minimizing human exposure to T. sanguisuga and CD. A comprehensive IPM plan for kissing bugs includes detailed inspections, removal of vertebrate host nesting areas, and kissing bug harborage, home improvements to exclude kissing bugs from entering structures, pest removal, and judicious use of pesticides. This approach can limit or eliminate kissing bug entry into residential structures, thereby preventing kissing bug bites, and CD infections in humans and companion animals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three-host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin-resistant and three strains of fipronil-tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione-S-transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin-resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil-tolerant tick strains., (© 2017 The Royal Entomological Society.)

Determining the upper (CTmax) and lower (CTmin) critical thermal limits of invasive ants provides insight into how temperature could shape their distribution, seasonality, and daily activity. Understanding the potential distribution of invasive ants is imperative to improving quarantine and management efforts. Nylanderia fulva (Mayr) (tawny crazy ant) and Solenopsis invicta (Buren) (red imported fire ant) are invasive ants that are established throughout the southeastern United States. Recent studies have found that body size and thermal ramping rate can affect the estimation of critical thermal limits. However, the effects of both variables and their interactions on the thermal limits of N. fulva and S. invicta have not previously been described. Thus, we evaluated the impacts of body size and ramping rate on the critical thermal limits of N. fulva and S. invicta Overall, N. fulva had a narrower thermal breadth than S. invicta (Nf CTmin = 7.3°C and Nf CTmax = 41.3°C vs. Si CTmin = 4.1°C and Si CTmax = 45.3°C). For both species, slower ramping rates resulted in lower CTmax values and ants with smaller head capsules had a narrower thermal breadth than ants with larger head capsules. These data improve our understanding of the critical thermal limits of both species and could be useful for developing predictive models that estimate the future spread of these invasive ants in nonnative ranges., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Nylanderia fulva (Mayr), the tawny crazy ant, is an invasive pest established in Florida and several other Gulf Coast states. In their invasive ranges in the Southeastern USA, large N. fulva populations have reduced species abundance, even displacing another invasive ant, Solenopsis invicta (Buren). In North Florida, N. fulva populations survive winter temperatures that reach below freezing for extended periods. However, the shallow littoral debris used by N. fulva for nest construction offers little insulation to brood and reproductives when exposed to freezing temperatures. Field populations of N. fulva in North Florida were observed tunneling below ground, a previously undescribed behavior. Other invasive ants exhibit similar subterranean tunneling behavior as a means of thermoregulation. To test the hypothesis that N. fulva has the capacity to construct subterranean tunnels across a range of ecologically relevant temperatures, tunneling performance for N. fulva and S. invicta, another invasive ant that tunnels extensively, were compared at four temperatures (15.0, 18.0, 20.0, and 22.0 °C). Overall, N. fulva tunneled significantly less than S. invicta. Nylanderia fulva tunneled furthest at warmer temperatures whereas S. invicta tunneled furthest at cooler temperatures. However, N. fulva constructed subterranean tunnels at all temperatures evaluated. These data support the hypothesis that N. fulva is capable of tunneling in temperatures as low as 15.0 °C, confirming that this ant can also perform a behavior that is used by other ants for cold avoidance.

Permethrin is a commonly used acaricide for tick control on domestic animals and in residential environments, while fipronil use is restricted to on-animal treatment. Following widespread reports of permethrin and fipronil application failures to control indoor infestations of Rhipicephalus sanguineus (Latreille), the brown dog tick, 31 tick populations were obtained from Florida and Texas for acaricide resistance screening. These field-collected ticks from kennels and residential facilities were challenged with technical grade permethrin and fipronil to create dose response curves that were compared with an acaricide-susceptible strain. Permethrin resistance was successfully screened in nine populations, all of which were resistant or highly resistant. Tick susceptibility to fipronil was conducted on four populations, which were found to be tolerant, with resistance ratios below 10. This is the first documentation of R. sanguineus permethrin resistance and fipronil tolerance in the United States. Potential causes of resistance development and recommendations on future brown dog tick control management plans are discussed., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

RNA interference (RNAi) represents a breakthrough technology for conducting functional genomics research in non-model organisms and for the highly targeted control of insect pests. This study investigated RNAi via voluntary feeding in the economically important pest termite, Reticulitermes flavipes. We used a high-dose double-stranded (ds) RNA feeding approach to silence two termite genes: one encoding an endogenous digestive cellulase enzyme and the other a caste-regulatory hexamerin storage protein. Contrary to results from previous low-dose studies that examined injection-based RNAi, high-dose silencing of either gene through dsRNA feeding led to significantly reduced group fitness and mortality. Hexamerin silencing in combination with ectopic juvenile hormone treatments additionally led to lethal molting impacts and increased differentiation of presoldier caste phenotypes (a phenotype that is not capable of feeding). These results provide the first examples of insecticidal effects from dsRNA feeding in a termite. Additionally, these results validate a high-throughput bioassay approach for use in (i) termite functional genomics research, and (ii) characterizing target sites of conventional and novel RNAi-based termiticides.

Lower termites rely on cellulolytic protozoa to aid in the digestion of their wood-based diet. However, despite the major contribution of protozoa to the lower termite digestive system, few techniques have been developed to monitor shifts in protozoan populations. This study investigated whether quantitative real-time PCR (qRT-PCR) and/or cellulase enzyme assays can be used to monitor changes of cellulolytic protozoan populations in the lower termite, Reticulitermes flavipes (Kollar). Previously developed cellulase primer sets were used to test for changes in cellulase gene expression, while three different cellulase enzyme assays were used to assess changes in cellulase enzyme activity. The results from this study indicate that qRT-PCR is a reliable method to monitor shifts in cellulolytic protozoan populations. Specifically, qRT-PCR can serve as a useful monitoring technique during high-throughput screening of novel termite control agents such as cellulase inhibitors, and help to answer questions relating to whether or not such control agents impact cellulolytic protozoan populations.

Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it (e.g., wood and humus). Lower termites, which are equipped with both endogenous (i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut+salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.

In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression.

Efficacy and speed of action of fire ant (Hymenoptera: Formicidae) baits that claim fast control of colonies were compared with a standard bait. More than 85% of red imported fire ant, Solenopsis invicta Buren, laboratory colonies provided bait containing the active ingredient indoxacarb died within 3 d, and all colonies were dead in 6 d. Standard bait containing hydramethylnon resulted in death of 60% of the colonies in 9 d. Bait containing spinosad did not cause colony death. Under field conditions, one-half of the areas treated with the indoxacarb bait did not have any active fire ant nests within 3 d, whereas 11 d was needed to reach the same level of control with the hydramethylnon bait. Spinosad had a maximum of 17% of the treated areas without nests after 3 d. The delay in death of S. invicta adults treated in the laboratory with the indoxacarb and spinosad baits was shorter than the standard hydramethylnon bait, which had mortality similar to the traditional delayed toxicity criterion of < 15% mortality after 24 h and > 89% mortality over the test period. Indoxacarb caused mortality of 57% at 24 h and 100% at 48 h; however, visual symptoms of toxicity were not readily observed for at least 8 h before the abrupt increase in death. Spinosad caused 96% mortality by 24 h, and initial mortality became apparent at 4 h. Time required for death of 15% of a treated population (LT15) of spinosad, indoxacarb, and hydramethylnon was 3, 9, and 16 h, respectively. Delayed toxicity characteristics of the fast-acting indoxacarb bait may be useful for the development of other fast-acting ant baits.

Previous molecular studies on the termite Reticulitermes flavipes have revealed that two hexamerin proteins serve an important status quo role in the regulation of juvenile hormone (JH)-dependent caste differentiation. Here, we report sequence data and other experimental evidence suggesting how these two hexamerins function in achieving caste regulation. The two hexamerin genes, named Hex-1 and Hex-2, encode highly unique sequence features relative to the 100+ other known insect hexamerins. These features include a long hydrophobic tail and prenylation motif in Hex-1, and a long hydrophilic insertion plus several putative protease cleavage sites in Hex-2. Both hexamerin genes are primarily expressed in fat body tissue, but only Hex-2 expression is substantially induced by JH. SDS-PAGE showed that the hexamerin proteins constitute a major proportion of total soluble termite protein. Also, although each protein occurs in both the membrane and soluble protein fractions, Hex-2 has stronger membrane affinity. Anti-JH antiserum specifically recognizes hemolymph-soluble Hex-1 protein, supporting that the unique prenylation site in Hex-1 facilitates covalent JH binding to the primary amino acid chain. Finally, increased ratios of Hex-2 to Hex-1 transcription occur in caste phenotypes and developmental stages that differentiate in response to rising JH titers. Two main conclusions can be taken from these studies. First, elevated ratios of Hex-2 to Hex-1 expression are associated with caste phenotypes that differentiate in response to rising JH titers (i.e., workers, presoldiers and soldiers). Second, due to their unique structural features and other observed characteristics, our findings support the hypothesis that the two hexamerins participate in the regulation of caste-differentiation by modulating JH availability.

Lower termites express a unique form of eusocial polyphenism in that totipotent workers can differentiate into either soldier or reproductive caste phenotypes. In this initial effort using RNA interference in termites, we found that two hexamerin genes, Hex-1 and Hex-2, participate in the regulation of caste polyphenism. Our methodology involved a dual gene-silencing approach that used a single short-interfering RNA fragment to silence the two homologous hexamerin genes. We performed validation studies that evaluated effects on nontarget housekeeping genes, silencing of a nonhousekeeping control gene, and effects at the protein level. We found that the two hexamerin proteins, which are inducible by the morphogenetic juvenile hormone and which constitute a significant proportion of total termite protein, suppress juvenile-hormone-dependent worker differentiation to the soldier caste phenotype. This mechanism allows termite colonies to retain high proportions of altruistic worker caste members, thus apparently enhancing colony-inclusive fitness. These findings demonstrate a unique status quo regulatory mechanism for termite worker caste retention and provide an example of previously undescribed preadult developmental/caste-regulatory genes from any social insect.

Eastern subterranean termites, Reticulitermes flavipes (Kollar), workers were introduced into arenas containing low, moderate, and high compaction builder's sand (1.05 g/cm3, 1.18 g/cm3 or 1.35 g/cm3 bulk densities, respectively), and they immediately began tunneling. Termites built the tunnel network significantly fastest in soil of low compaction compared with moderately or highly compacted soil. In soil of low compaction, 221.67 +/- 4.73 cm of total tunnel distance was constructed in 1 d compared with only 96 cm of tunneling in highly compacted soil. At 14 d, total tunnel distance averaged 216.83 +/- 4.56 cm in soil of low compaction compared with 169.70 +/- 4.10 and 181.18 +/- 6.13 cm in moderately and highly compacted soil, respectively. Decreases in total tunnel distance between 1 and 14 d were caused by backfilling of seldom-used tunnels. Termites did the majority of tunneling during the first day of introduction into arenas. In soil of low and moderate compaction, termites essentially constructed the entire tunnel network within the first day, only modifying it by backfilling or maintaining tunnels. In highly compacted soil, 53% of the final tunnel network was constructed during the first day, 87% was constructed by the third day, and 97% was constructed by the seventh day. Soil compaction did not affect the number of primary tunnels or the number and diameter of secondary tunnels. The angle between the secondary tunnel and primary tunnel also was not significantly affected by soil compaction. However, the number of secondary tunnels in soil of low compaction (5.89 +/- 0.51) was significantly greater than in moderately (2.74 +/- 0.36) and highly (3.58 +/- 0.59) compacted soils.

Dogs were trained to detect Eastern subterranean termites, Reticulitermes flavipes (Kollar), using the United States Customs method of scent detection dog training modified with a food reward. Dogs were tested with various numbers of Eastern subterranean termites placed in vented PVC containers. Trained dogs were 95.93% accurate in finding > or = 40 Eastern subterranean termite workers (positive indications) and incorrectly indicated the presence of termites in 2.69% of the containers without termites. Multiple species of termites [dark southern subterranean, R. virginicus (Banks); Formosan subterranean, Coptotermes formosanus Shiraki; powderpost, Cryptotermes cavifrons Banks; and southeastern drywood termites, Incisitermes snyderi (Light)], were similarly evaluated. Dogs trained to locate Eastern subterranean termites were also 100% accurate in finding dark southern subterranean termites, 98.89% accurate in finding Formosan subterranean termites, 97.33% accurate in finding powderpost termites, and 88.89% accurate in finding southeastern drywood termites. Dogs were able to discriminate live termites from non-termite material. Trained dogs' false response rate was 25.33% to Eastern subterranean termite-damaged wood, 6.67% to American cockroaches, Periplaneta americana (L.), and 2.67% to Florida carpenter ants, Camponotus floridanus Buckley.

A portable, low-frequency acoustic system was used to detect termite infestations in urban trees. The likelihood of infestation was rated independently by a computer program and an experienced listener that distinguished insect sounds from background noises. Because soil is a good insulator, termite sounds could be detected easily underneath infested trees, despite the presence of high urban background noise. Termite sounds could be detected also in trunks, but background noise often made it difficult to identify termite signals unambiguously. High likelihoods of termite infestation were predicted at four live oak (Quercus virginiana Mill, Fagacae), two loblolly pine (Pinus taeda L., Pinacae), and two baldcypress (Taxodium distichum Rich. Pinacae) trees that wood-baited traps had identified as infested with Coptotermes formosanus Shiraki. Infestations also were predicted at two pine trees with confirmed recoveries of Reticulitermes flavipes (Kollar). Low likelihoods of infestation were predicted in four oak trees where no termites were found. Additional tests were conducted in anechoic environments to determine the range of acoustic detectability and the feasibility of acoustically estimating termite population levels. There was a significant regression between the activity rate and the number of termites present in a wood trap block, with a minimum detectable number of approximately 50 workers per liter of wood. The success of these field tests suggests that currently available acoustic systems have considerable potential to detect and monitor hidden infestations of termites in urban trees and around building perimeters in addition to their present uses to detect and monitor termite infestations in buildings.

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.

Toxicity and metabolism of t-permethrin were evaluated in two colonies (UF and ARS) of the eastern subterranean termite, Reticulitermes flavipes (Kollar), collected in Gainesville, FL. The UF colony (LC50 = 1.86 micrograms per vial) was approximately twofold more tolerant of t-permethrin than the ARS colony (LC50 = 0.89 microgram per vial) at the LC50. The synergists piperonyl butoxide and S,S,S-tributylphosphorotrithioate increased t-permethrin toxicity four- and threefold (at the LC50) in the UF and ARS colonies, respectively. Despite these differences in t-permethrin susceptibility, microsomal oxidase activities toward surrogate substrate (aldrin epoxidase, and methoxyresorufin O-demethylase), cytochrome P450 content, and microsomal esterase activity toward alpha-naphthyl acetate did not differ significantly between the colonies. Moreover, no significant differences in qualitative and quantitative metabolism of [14C]t-permethrin were observed between the UF and ARS colonies for three enzyme sources (microsomal oxidase, microsomal esterase, and cytosolic esterase). Based on in vitro metabolism assays, the major detoxification route of t-permethrin in the UF and ARS termite colonies appears to be hydrolysis catalyzed by microsomal esterases.