Potential use of pinenes to improve localized insecticide injections targeting the western drywood termite (Blattodea: Kalotermitidae).

The western drywood termite, Incisitermes minor (Hagen), causes significant economic damage to wood structures in the United States of America, especially California. When infestation is not widespread, localized insecticide injections may be useful for remedial control. However, the extensive gallery structure of drywood termites and their tendency to aggregate at specific parts of the galleries can impact the efficacy of localized insecticide injection. Chemicals that attract termites from a distance may improve the localized insecticide injection by increasing the number of termites contacting the insecticide residues. Two volatile terpenes, α-pinene and ß-pinene, commonly found in many coniferous timber trees, were applied to artificial termite galleries to determine if termites were attracted from their original aggregation site. Furthermore, we examined if adding these pinenes would improve the overall efficacy of some insecticide products for drywood termite control. Behavioral assay results showed that the treatment with pinenes increased the likelihood that drywood termites would leave their original aggregation site and contact the treated part of the gallery. When tested with the pesticide products applied in a small area away from the termite aggregation, ß-pinene significantly accelerated the time course of mortality for the aqueous fipronil. The efficacy of disodium octaborate tetrahydrate dust was not influenced by the addition of pinenes. Implications for drywood termite management and future research directions are discussed.

Toxicity of isocycloseram, an isoxazoline insecticide, against laboratory and field-collected German cockroaches (Blattodea: Ectobiidae).

Isocycloseram is a new insecticide in the isoxazoline class that targets insect GABA-gated chloride channels. In this study, we evaluated a cockroach gel bait formulation containing 1% isocycloseram against a susceptible strain (UCR) and 5 field-collected strains (WM, RG386, Ryan, CDR, and SY) of the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), and compared it with several commercial insecticide baits in the laboratory. Using the Ebeling choice box method, we also tested a residual deposit of an SC formulation of isocycloseram against the UCR, RG386, and Ryan strains. The isocycloseram bait was among the fastest-performing treatments against adult males (mean survival time: 0.9-2.7 days) and mixed stages and sexes (mean survival time: 1.4-5.4 days) across all strains. Secondary transfer effects of the bait were demonstrated in the UCR strain by exposing new adult males to individuals killed by direct bait treatment. Physiological resistance was not detected in the WM, CDR, and RG386 strains with topical treatment of a diagnostic dose (3× LD95) of isocycloseram developed using the UCR strain. However, topical assays revealed resistance ratios (RR50) of 1.6 and 3.0× in the Ryan and SY strains, respectively. The performance of a 0.05% isocycloseram residual application against the Ryan strain was improved with the addition of piperonyl butoxide.

Overexpression of cytochrome P450 gene CYP6K1 is associated with pyrethroid resistance in German cockroaches (Blattodea: Ectobiidae) from California.

We investigated the expression of 4 cytochrome P450 genes (CYP4G19, CYP6J1, CYP6K1, and CYP4C21) in 4 field-collected strains (WM, RG386, CDR, and Ryan) of the German cockroach, Blattella germanica (L.), collected from California. The UCR susceptible strain was used as a comparison. Topical assays using a diagnostic dose (3× LD95) of deltamethrin revealed decreased sensitivity in all field-collected strains with mortality ranging from 0% to 58%, and the addition of PBO before deltamethrin treatment increased mortality to 52.5%-87.5%. Using qPCR to investigate the expression levels of CYP4G19, CYP4C21, CYP6J1, and CYP6K1, we found that only CYP6K1 was significantly overexpressed (2.1-5.8× higher) in all field-collected strains when compared to the UCR strain. Next, we investigated the role of the CYP6K1 gene by performing gene knockdown using RNAi. After dsCYP6K1 treatment, the expression levels of CYP6K1 in WM and Ryan strains were significantly reduced (P < 0.01) by 91%-94% vs. those treated with dsEGFP (control) on the third and sixth day posttreatment. RG386, CDR, and Ryan strains were more susceptible compared to their respective controls to topically applied deltamethrin 6 days after treatment with dsCYP6K1. This study provides evidence of the involvement of the P450 CYP6K1 gene in pyrethroid resistance in some populations of German cockroaches.

Oral toxicity of an artificial sweetener sucralose on the German cockroach (Blattodea: Ectobiidae) and its impact on water balance and gut microbiome.

Artificial or non-nutritive sweeteners are indigestible by most animals. Some sweeteners are orally toxic to insects and have received recent interest as potential safe insecticides due to their low mammalian toxicity. In this study, we investigated the oral toxicity of sucralose on insecticide-susceptible and resistant German cockroaches, Blattella germanica (L.). In a nonchoice test, we evaluated 5, 10, and 20% sucralose solutions. Depending on the cockroach strains, mean mortality ranged from 62.5 to 92.5%, 15 to 55%, and 2.5 to 27.5% for 20, 10, and 5% sucralose, respectively. Next, we measured the impact of a 20% sucralose treatment on water loss rates in the cockroach strains. All strains lost 23.0-30.29% of body water by 6 d. Dehydrated cockroaches were more prone to be killed by sucralose than nondehydrated ones. Lastly, we evaluated the effect of 20% sucralose treatment on gut bacterial composition and found the diversity of gut bacteria in treated cockroaches was significantly reduced after 3 days, implicating a rapid change in the alimentary environment.

Histamine excretion in common indoor and hematophagous arthropods.

Histamine is a biogenic amine that regulates multiple physiological functions in diverse organisms, specifically playing a central role in the mammalian immune response. The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), excretes histamine in large amounts in its feces as a component of its aggregation pheromone, which contaminates homes. The potential health risks associated with the presence of indoor histamine are unclear, but to predict future exposure risks, it is critical that we understand if other arthropods excrete histamine in any discernible phylogenetic pattern. In the present study, we evaluated histamine excretion by various arthropods; specifically those commonly found in large numbers indoors, other hematophagous species, and other species in the order Hemiptera. To evaluate arthropods for histamine excretion, rearing containers for each arthropod were swabbed and/or the harborage substrates were collected. Samples were then analyzed for the presence of histamine using gas chromatography-mass spectrometry. For those arthropods where histamine was present above the method detection limit, total histamine excretion was quantified over a period of 2 wk. Our results indicate that histamine excretion is limited to hematophagous hemipterans (bed bugs, bat bugs, tropical bed bugs, and kissing bugs), suggesting that indoor histamine contamination in the United States can be primarily linked to bed bugs.

The impact of selenium on insects.

Selenium, a naturally occurring metalloid, is an essential trace element for many higher organisms, including humans. Humans primarily become exposed to selenium by ingesting food products containing trace amounts of selenium compounds. Although essential in these small amounts, selenium exhibits toxic effects at higher doses. Previous studies investigating the effects on insects of order Blattodea, Coleoptera, Diptera, Ephemeroptera, Hemiptera, Hymenoptera, Lepidoptera, Odonata, and Orthoptera revealed impacts on mortality, growth, development, and behavior. Nearly every study examining selenium toxicity has shown that insects are negatively affected by exposure to selenium in their food. However, there were no clear patterns of toxicity between insect orders or similarities between insect species within families. At this time, the potential for control will need to be determined on a species-by-species basis. We suspect that the multiple modes of action, including mutation-inducing modification of important amino acids as well as impacts on microbiome composition, influence this variability. There are relatively few studies that have examined the potential effects of selenium on beneficial insects, and the results have ranged from increased predation (a strong positive effect) to toxicity resulting in reduced population growth or even the effective elimination of the natural enemies (more common negative effects). As a result, in those pest systems where selenium use is contemplated, additional research may be necessary to ascertain if selenium use is compatible with key biological control agents. This review explores selenium as a potential insecticide and possible future directions for research.

The Potential of Fluralaner as a Bait Toxicant to Control Pest Yellowjackets in California.

The western yellowjacket, Vespula pensylvanica (Saussure), is an important seasonal pest of recreational and outdoor venues in the western United States. Its propensity to scavenge food increases the likelihood of stinging incidences. Control measures are limited to intensive trapping and treating subterranean nests. The only toxicant registered for baiting in the US is esfenvalerate, which is ineffective. The objective of this study was to determine the potential of the isoxazoline fluralaner as a bait toxicant. With microsatellite genotyping, a minimum of 27 different colonies were shown to forage at a single monitoring site. Some colonies disappeared after baiting, and new colonies were detected. The implications for baiting and monitoring are discussed. Minced chicken and hydrogel baits containing 0.022% and 0.045% fluralaner significantly reduced foraging yellowjackets. Several bait applications covering large areas will be necessary to provide long-term control.

Laboratory evaluations of biodegradable boric acid hydrogel baits for the control of Argentine ant (Hymenoptera: Formicidae).

Due to their mutualistic relationship with plant pests, the Argentine ant is considered a major pest in subtropical fruit orchards and vineyards. Besides insecticide sprays, liquid baiting has been demonstrated as an effective method to suppress the Argentine ant populations. To improve the economic feasibility of liquid baiting, hydrogel materials have been recently tested as a carrier for liquid baits containing various insecticidal active ingredients. Here, we tested boric acid as a toxicant in the aqueous sugar bait delivered in a biodegradable calcium alginate hydrogel. Laboratory tests demonstrated that boric acid (1%) liquid bait incorporated in the calcium alginate hydrogel effectively killed Argentine ant workers. Potassium sorbate (0.25%) added to the liquid bait as a preservative did not impact the efficacy of boric acid even though it significantly reduced the degree of swelling of the hydrogel beads in the bait solution. Testing with 2-month-old bait suggested that long-term storage might impact bait efficacy even with potassium sorbate preservative.

Effect of Bed Bug (Hemiptera: Cimicidae) Aldehydes on Efficacy of Fungal Biopesticides.

The use of the entomopathogenic fungus Beauveria bassiana (Bals. - Criv.) Vuill. (Hypocreales: Cordycipitaceae) has been recently incorporated in the management of bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae). Bed bugs produce a set of aldehydes that are known to affect the growth of some fungi. Considering that bed bugs or their exuviae release these aldehydes, it was suspected that the bed bugs' aggregation sites would contain an increased level of the bed bug aldehydes. The current study examined if elevated levels of the bed bug aldehydes in the microhabitats would impact the efficacy of B. bassiana. Following a brief exposure to the residues of commercial products containing B. bassiana, the treated bed bugs were kept in a vial with or without a natural or artificial blend of bed bug aldehydes (i.e., exuviae or synthetic compounds). For a B. bassiana product that is not currently registered for bed bugs control, the presence of aldehydes significantly reduced 15-d mortality (61-62%) compared to the no aldehydes control (97.7%). However, when tested with a B. bassiana formulation designed for bed bug control, the aldehydes only caused delayed mortality for the treated bed bugs. When tested in culture, the growth rate of B. bassiana on a medium was significantly reduced when the bed bug aldehydes were provided in the headspace. Implications on practical bed bug management using fungal biopesticides are discussed.

Genetic Evidence for Multiple Invasions of Coptotermes formosanus (Blattodea: Rhinotermitidae) in California.

New infestations of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae), were discovered in southern California, namely in Rancho Santa Fe and La Mesa (San Diego County) and Highland Park (Los Angeles County) in 2021. We investigated whether these new infestations were related to the previous infestations in La Mesa (2018) and Canyon Lake, Riverside County (2020). We used two mitochondrial genes (COI and COII) and seven polymorphic microsatellite markers to infer the genetic relationship between southern California colonies and their breeding systems. The samples collected from seven localities belonged to five colonies (inter-colony distances ranged from ~160 m to 185 km, with an average of 97 km). Of these five colonies, two were simple families, and three were extended families. Structure analyses of microsatellite genotypes grouped the termite samples into three distinct genetic clusters, suggesting at least three independent introduction events in southern California.

Wash-off potential of pyrethroids after use of total release fogger products.

Pyrethroids are frequently detected in urban wastewater. Even though treatment facilities remove most pyrethroids (> 90 %) in wastewater, residual concentrations can exceed thresholds that are acutely toxic to sensitive aquatic species. Total release foggers (also known as "bug bombs") are widely used by the general public for insect control. It was hypothesized that these products serve as a source of pyrethroids entering the urban wastewater through the deposition of the active ingredients on various surfaces and subsequent transfer from the contaminated surfaces to the waste stream through cleaning activities. Based on experiments conducted in an enclosure, we found that substantial amounts of a pyrethroid (i.e., cypermethrin) were deposited on various surfaces after a total release fogger use. A series of experiments simulating scenarios that would be representative of common residential cleaning activities indicated that the pyrethroid could be transferred from the contaminated surfaces to other adsorptive materials via physical contact (with or without water as a solvent). The pyrethroid was readily extracted from the adsorptive materials (cotton fabric and filter paper) when water was used as a solvent. Adding a small amount of detergent to the water significantly increased the extraction efficiency compared to water alone. These results indicate that insecticides used in total release foggers can contribute to insecticide loading into the wastewater treatment system via several possible routes, such as contact with or cleaning of exposed surfaces and washing contaminated clothing after their use within a structure.

Combined metabolic and target-site resistance mechanisms confer fipronil and deltamethrin resistance in field-collected German cockroaches (Blattodea: Ectobiidae).

Despite insecticide resistance issues, pyrethroids and fipronil have continued to be used extensively to control the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae) for more than two decades. We evaluated the physiological insecticide resistance in five German cockroach populations collected from 2018 to 2020 and measured the extent of metabolic detoxification and target-site insensitivity resistance mechanisms. Topically applied doses of the 3 x LD95 of deltamethrin, fipronil, DDT, or dieldrin of a susceptible strain (UCR, Diagnostic Dose) failed to cause >23% mortality, and the 10 x LD95 of deltamethrin or fipronil failed to cause >53% mortality. All field-collected strains possessed a combination of metabolic and target-site insensitivity mechanisms that cause reduced susceptibility. Elevated activities of esterase and glutathione S-transferase were measured, and the synergists piperonyl butoxide or S,S,S-tributyl phosphorotrithioate increased topical mortality up to 100% for deltamethrin and 93% for fipronil 10 x LD95. The target-site mutations L993F of the para-homologous sodium channel and A302S of the GABA-gated chloride channel associated with pyrethroid and fipronil resistance, respectively, were found at ~80-100% frequency in field populations. Pyrethroid and fipronil spray formulations also were ineffective in a choice box assay against field-collected strains suggesting that these treatments would fail to control cockroaches under field conditions.

Reduced Susceptibility Towards Commercial Bait Insecticides in Field German Cockroach (Blattodea: Ectobiidae) Populations From California.

Gel bait insecticides have been extensively used to manage the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), but issues with reduced effectiveness of such formulations are becoming increasingly common. We collected five field strains of German cockroaches in California and evaluated them against five commercial bait products [Maxforce FC Magnum (0.05% fipronil), Maxforce Impact (1% clothianidin), Advion Evolution (0.6% indoxacarb), Optigard (0.1% emamectin benzoate) and Siege (2% hydramethylnon)]. Increased survivorship and incomplete mortality towards all baits were recorded in the field strains. We assessed susceptibility to the active ingredients fipronil, clothianidin, indoxacarb, abamectin, hydramethylnon, and deltamethrin using topical bioassays with diagnostic doses (3 × LD95 and 10 × LD95) developed from the UCR susceptible strain. Low mortality was registered when tested with the 3 × LD95's of deltamethrin (0%), fipronil (0-3%), and clothianidin (13-27%); low to moderate mortality when treated with the 3 × LD95 of indoxacarb (13-63%), and moderate to high mortality after treatment with the 3 × LD95 of abamectin (80-100%) and hydramethylnon (70-83%). The mortality of all strains remained low after treatment with the 10 × LD99 of deltamethrin (0-20%) and low to moderate with fipronil (20-70%). We found negative correlations (P < 0.05) between Advion Evolution mean survival time and indoxacarb 10 × LD95 mortality and between Maxforce Impact and clothianidin 10 × LD95 mortality. These findings demonstrate multiple resistance towards all tested commercial bait insecticides except Optigard, suggesting the effectiveness of avermectin products in resistance management programs.

Identification of Chemicals Associated Gambusia affinis (Cyprinodontiformes: Poeciliidae), and Their Effect on Oviposition Behavior of Culex tarsalis (Diptera: Culicidae) in the Laboratory.

The western mosquitofish, Gambusia affinis (Baird & Girard), has been used worldwide for the control of larval mosquitoes for more than 100 yr. We found that the western encephalitis mosquito, Culex tarsalis Coquillett (Diptera: Culicidae), can detect the presence of G. affinis in oviposition sites based on associated chemicals, leading to a decrease in the number of egg rafts laid. Three volatile chemical compounds were identified in the headspace above the water where G. affinis had been held for 24 h. Oviposition bioassays conducted using standards of the volatile compounds identified (dimethyl disulfide [DMDS], dimethyl trisulfide [DMTS], and S-methyl methanethiosulphonate) found that females reduced oviposition only when low concentrations of DMTS were present, but this response was not consistent across all trials and concentrations tested. DMDS, DMTS, and S-methyl methanethiosulphonate are known bacterial metabolic waste products and may be the source of the compounds. Two nonvolatile compounds of interest were found to be present in the Gambusia-exudate water. After tasting Cx. tarsalis were deterred from ovipositing onto Gambusia-treated water from which the bacteria had been removed by filtration, indicating that the kairomone may consist of nonvolatile compound(s). One of the nonvolatile compounds isolated from the Gambusia-treated water has a benzene ring structure similar to that of cholesterol but the structure of the two nonvolatile deterrents remains to be fully characterized. Our research shows that three volatile compounds and two nonvolatile compounds are present in water associated with G. affinis (Poeciliidae: Gambusia) and affect the oviposition behavior of Cx. tarsalis in laboratory bioassays.

Development and Demonstration of Low-Impact IPM Strategy to Control Argentine Ants (Hymenoptera: Formicidae) in Urban Residential Settings.

Argentine ants are one of the most common nuisance pest ants treated by pest management professionals (PMPs) in southern and western urban residential areas of the United States. Two new technologies (spraying with a pheromone adjuvant and using a biodegradable hydrogel bait delivery method) were used to develop a unique low-impact integrated pest management (IPM) protocol for Argentine ants in urban residential settings. The IPM protocol included a one-time perimeter spray treatment with 0.03% fipronil (mixed with a pheromone adjuvant) at the beginning of the ant season to achieve a quick knockdown. The initial spray application was followed by a biodegradable hydrogel baiting with 1% boric acid as a maintenance treatment. This low-impact IPM protocol was compared with two other conventional methods: (1) one initial fipronil application and one pyrethroid spray application for maintenance, or (2) one initial fipronil application and one essential oil insecticide spray application for maintenance. Based on Argentine ant foraging activity, the protocols were compared for their control efficacy. Insecticide use information and treatment time were also recorded and compared among different treatment protocols. Our results provided empirical data to support the effectiveness and economic feasibility of the low-impact IPM protocol for managing Argentine ants in urban residential settings.

Genetic Analysis of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Populations in California.

A new infestation of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae), was discovered in Canyon Lake, Riverside County, California. We used three mitochondrial DNA (COI, COII, and 16S) and seven polymorphic microsatellite markers to characterize the genetic relationship of the colony with two other colonies that were collected in 1992 and 2018 in La Mesa, San Diego County. Maximum likelihood phylogeny of C. formosanus based on concatenated COI and COII sequences revealed that the two La Mesa populations (CA01 and CA02) and the Canyon Lake population (CA03) were from different maternal lineages. Based on the 14 COII haplotypes of C. formosanus found world-wide, CA01 and CA02 belonged to a haplotype widely distributed across the United States, while CA03 was grouped under a haplotype predominantly found in Asia. Microsatellite allele frequencies across all loci for both La Mesa populations were relatively similar, but significant genetic differences were found between CA02 and CA03 colonies (FST = 0.24; Dest = 0.30; G″ ST = 0.55; P < 0.01).

The Impact of Artificial Sweeteners on Insects.

Artificial sweeteners are sweet-tasting additives found in consumable products as substitutes for naturally occurring sugars. They are derived from plant extracts or manufactured by chemical synthesis. Ingestion of sweeteners by insects can lead to significant physiological effects, such as mortality, decreased fecundity, and behavioral change. Due to their low toxicity toward humans and the issues associated with conventional insecticide usage, artificial sweeteners have recently gained attention for their potential use as biorational insecticides. Here, we review their impact on insects and potential as novel insecticides.

Ontogenesis of Aldehyde Pheromones in Two Synanthropic Bed Bug Species (Heteroptera: Cimicidae).

Bed bugs produce volatile aldehydes that have alarm and aggregation functions. Using two synanthropic bed bug species, Cimex lectularius L. and C. hemipterus (Fabricius), developmental changes were examined for (E)-2-hexenal, 4-oxo-(E)-2-hexenal, (E)-2-octenal, and 4-oxo-(E)-2-octenal, the four most abundant aldehydes shared between the two species. Quantitative analyses of the aldehydes in the nymphal exuviae indicated that the aldehydes' ratio remained similar throughout nymphal development. In general, (E)-2-octenal was most abundant, and (E)-2-hexenal and 4-oxo-(E)-2-octenal were least abundant. The fourth aldehyde, 4-oxo-(E)-2-hexenal, was present in intermediate quantities. The quantities and percent abundances of the aldehydes in nymphal exuviae and the adults were significantly different between C. lectularius and C. hemipterus. The ratio between (E)-2-hexenal and (E)-2-octenal was determined in adult male and female bed bugs of each species. Adult C. hemipterus had a higher proportion of (E)-2-hexenal than C. lectularius, while no sex differences were found. This work provides the first systematic quantification of four aldehydes [(E)-2-hexenal, 4-oxo-(E)-2-hexenal, (E)-2-octenal and 4-oxo-(E)-2-octenal] for all five of the nymphal stages for both C. lectularius and C. hemipterus.

Volatile Essential Oils Can Be Used to Improve the Efficacy of Heat Treatments Targeting the Western Drywood Termite: Evidence from Simulated Whole House Heat Treatment Trials.

Colonies of western drywood termites, Incisitermes minor (Hagen) (Blattodea: Kalotermitidae), are difficult to detect and treat due to their cryptic nature. The use of heated air to create lethal temperatures within infested wood serves as a nonchemical treatment option targeting whole structure or large portions of the structure. However, the presence of hard-to-heat areas and potential risk of damage for heat-sensitive items are recognized as important challenges. Here, we tested if a localized injection of volatile essential oil could be utilized to address the heat sink issue, potentially increasing the overall efficiency of heat treatments against drywood termites. Artificially infested wooden blocks were placed in several locations of the test building, and heat treatments were conducted. For the treatment group, a small amount of essential oil (methyl salicylate) was added in the blocks prior to the heat treatment. All blocks placed in uninsulated wall voids had 92-100% termite mortality by day 7. However, the presence of a large concrete wall in the subarea hindered heating of blocks therein, resulting 36-44% mortality by day 7 when there was no essential oil treatment. Incorporation of the essential oil substantially increased the control efficacy for the subarea, resulting in more than 90% mortality. This approach might also be helpful in reducing the risk of potential heat damage during heat treatment without compromising its control efficacy.

Development of a lateral flow test for bed bug detection.

Lateral flow strip tests are a cost-effective method for detecting specific proteins in biological samples, which can be performed in the field without specialized expertise. While most recognizable in the pregnancy tests, there are many other applications for lateral flow strip technology. The pest control industry has increasingly emphasized the importance of pest monitoring to reduce unnecessary applications, focus interventions into locations with active pest infestations, and to develop records of pest infestation. Due to their cryptic behavior, the detection of bed bugs often necessitates labor-intensive, time-consuming and invasive visual inspections. A lateral flow strip test for the detection of bed bugs would represent a novel use for a well-established technology, which can enable pest control operators to rapidly confirm the presence or absence of bed bugs in a room. In the current report, we present an effort to develop and calibrate the lateral flow test devices for the detection of a bed bug specific protein. A variety of bed bug residue samples were prepared by varying several parameters: bed bug infestation level (1 bed bug/3 bed bugs), surface type (wood/fabric), feeding status (fed/unfed), and bed bug time-on-surface (1 d/7 d). Using a prototype sensor and test strip, we examined how these variables influenced the detection of the bed bug specific proteins in the sample and to what degree. We discuss how this lateral flow test device can be an effective tool to determine the presence or absence of bed bug proteins on a surface, providing highly credible evidence on bed bug infestations.