Efficacy Of Transfluthrin Varies By Species And Placement In A Warm Temperate Mediterranean Environment¹

Transmission of pathogens by insect disease vectors, such as mosquitoes and sand flies, is a substantial threat to human health and may significantly affect military operations in the field (Lamattina et al. 2024). Unfortunately, consistent implementation of the US military integrated vector management (IVM) system, such as proper use of topical repellents, pesticide-treated uniforms, or bed nets and routine surveillance and control activity, is historically low among US and other military personnel in austere environments (Ribas and Carreno 2010, Schofield et al. 2012). Implementation of durable, long-acting spatial repellent sources in military scenarios, such as in and around shelter systems or tactical sentry positions where personnel congregate or linger, could form a passive solution to mitigate lack of IVM compliance (Achee et al. 2012; Govella et al. 2015; Norris and Coats 2017; Britch et al. 2020a, 2020b).

In previous work, our group has investigated the performance and longevity of a variety of passive transfluthrin (US Environmental Protection Agency 2018) spatial repellent applications in field scenarios in several environments targeting mosquitoes and biting flies (Britch et al. 2020a, 2020b,; 2021; McMillan et al. 2022; Bayer et al. 2023). One finding relevant to potential US military field applications of spatial repellents is that transfluthrin, though highly effective, may need to be applied and positioned with care when protecting some enclosed spaces such as small tents from mosquitoes in warm humid and warm arid environments because of an apparent disorienting effect that may increase the number of mosquitoes in a tent (Bayer et al. 2023). We also found that spatial repellents in warm humid environments may have a range of efficacy at preventing mosquito incursion into an area, depending on how enclosed the area is and which species are present (Dame et al. 2014, Britch et al. 2021). However, it is not known whether these phenomena would also be observed with mosquitoes and sand flies in a warm temperate Mediterranean environment.

We established 2 study sites at the US Department of Agriculture, Agricultural Research Service, European Biological Control Laboratory on the grounds of the American Farm School (AFS), Thessaloniki, Greece. We investigated the efficacy of transfluthrin-impregnated resin diffusers, designed for timed release of transfluthrin as a vapor, to protect small 2-person US military tents, as well as open-air locations from natural populations of Culex pipiens L. mosquitoes and Phlebotomus species sand flies. These 2 scenarios represented, respectively, a small combat outpost with personnel in a small tent encampment and a bivouac area where personnel were resting in place or conducting sentry duties in the open overnight. Prior entomological studies identified 3 sand fly species at AFS sites: Phlebotomus perfiliewi Parrot (>70%), followed by Phlebotomus simici Nitzulescu, and Phlebotomus tobbi Adler, Theodor, and Lourie (Chaskopoulou et al. 2016).

To establish the simulated combat outpost tent study site, we chose a wooded area with heavy underbrush along the contour of a hill within 100 m of livestock pastures (40.567392°N, 22.995639°E) that in preliminary surveys had high biting pressure of both Cx. pipiens and Phlebotomus spp. (data not shown). Other mosquito genera, such as Anopheles and Aedes, were present in collections but at low numbers and excluded from analyses. At this site, we pitched 8 US military COMBAT (Eureka, Binghamton, NY) 2-person tents in a line, grouped into 4 paired replicates. For each pair, tents were placed approximately 1 m apart, with door openings facing outward and flaps tied open, away from one another (Fig. 1A). Each pair of tents was separated from the next pair in the line by approximately 4 m. We suspended a Centers for Disease Control and Prevention (CDC) light trap (model 512, John W. Hock, Gainesville, FL), baited with incandescent light and CO₂ (approximately 2.5-kg dry ice pellets) inside each of the tents, for a total of 8 traps, to simulate the presence of humans. Each CDC trap was suspended from a loop sewn into the center of the ceiling, with the intake fan approximately 0.5 m from the ground (Fig. 1A, inset).

We conducted the first night of surveillance with no transfluthrin present to determine the tent in each pair with the highest collection numbers of mosquitoes and sand flies. Based on data from this collection, we suspended a resin transfluthrin diffuser (Dainihon Jochugiku Co., Ltd., Osaka, Japan) with a 250-day longevity inside the tent in each pair with the highest biting pressure, for a total of 4 diffusers, and left the diffusers in place for the remainder of the experiment. Each diffuser was hung from a loop sewn into the inside ceiling of the tent approximately halfway between the door opening and the CDC trap (Fig. 1A, inset). The 250-day diffuser was impregnated with 1,200-mg transfluthrin technical active ingredient, released at 5 mg per 24 h. The other tent in each pair was designated an untreated control with no diffuser present for the remainder of the experiment. We then conducted 5 overnight collections, at 5-, 13-, 18-, 27-, and 35-day postplacement of the transfluthrin.

We established the simulated bivouac site in an AFS olive grove approximately 0.5 km from the tent site. We hung a CDC trap baited with light and CO₂ (approximately 1-kg dry ice) from a limb on 4 pairs of olive trees, with intake fans approximately 1 m from the ground (Fig. 1B). Each of these 8 trees was selected to provide at least 15 m between treated and untreated areas and between each CDC trap. One tree in each pair was treated with a single 250-day effect transfluthrin diffuser suspended within 0.25 m of the CDC trap (Fig. 1B), for a total of 4 diffusers left in place for the remainder of the experiment. The other tree in each pair was designated an untreated control with no diffuser present for the remainder of the experiment. We then conducted collections at 5-, 13-, 18-, 27-, and 35-day postplacement of the transfluthrin.

Mosquito and sand fly collection data from previous preliminary work with transfluthrin at AFS suggested that transfluthrin may migrate into surrounding materials, potentially confounding results. To reduce opportunity for collection bias, we assigned a specific CDC trap, CO₂ reservoir, battery, and collection bag to each position across both study sites. We measured the relative efficacy of transfluthrin by calculating the percentage difference in mosquito and sand fly collections in treated compared with untreated replicates. We also conducted a t-test using Wizard2 (version 2.0.16 (267), Evan Miller, New York, NY, wizardmac.com) for each collection date at each site to determine whether mean collections at treated versus untreated control replicates were significantly different. All collection data were log transformed as ln(x + 1) prior to analysis per Bidlingmayer (1969). We used the benchmark for spatial repellent efficacy as ≥61% reduction in collections per the Miller et al. (2022) review that established expected field efficacy of this intervention.

The outcome of the 5 collections at each site is shown in Fig. 2. We observed that transfluthrin affected collections of mosquitoes and sand flies differently within and between both scenarios (Fig. 2A–2D). In tents, Cx. pipiens mosquito collections were higher (mean 89.3% increase; not significant) in tents where transfluthrin was present (Fig. 2A). The percentage difference generally increased over the collection periods but became a significant increase only at 27 days (P = 0.028, t(6) = 2.875) compared with the untreated control. In contrast, collections of Phlebotomus spp. sand flies in tents with transfluthrin diffusers were consistently, though not significantly, lower (mean 33.3% reduction; Fig. 2B). In bivouacs, where transfluthrin was placed in the open without a shelter, Cx. pipiens collections were overall slightly reduced (mean 13.1%; not significant) in locations where transfluthrin was present, despite an initial increase of this species in treated locations in the first collection (Fig. 2C). In contrast, Phlebotomus species sand fly collections in bivouacs were consistently and significantly overall lower (mean 74.1% reduction; P < 0.001, t(38) = 4.234) in locations where transfluthrin was present, compared with untreated controls (Fig. 2D). Sand fly reductions in the treated bivouac locations were significant at day 5 (64.3%; P = 0.038, t(6) = 2.644), day 13 (78.2%; P = 0.008, t(6) = 3.937), and day 27 (77.5%; P = 0.017, t(6) = 3.294).

Military personnel in austere field environments are potentially exposed to vector insects such as mosquitoes and sand flies for extended periods, equating to higher risk of transmission of pathogens and persistent nuisance and painful biting, altogether degrading operational capabilities. We found that a spatial repellent such as transfluthrin may, as demonstrated here with significant reductions in Phlebotomus species sand flies in the bivouac scenario, provide a substantial layer of passive protection to personnel in the field in such conditions. However, some patterns in our results also indicate that spatial repellents need to be used with care. For example, we found that efficacy of transfluthrin may not be consistent across all high-threat biting Diptera present in an area. The devices performed very well against sand flies in an open area, reducing Phlebotomus species sand flies on average by 74.1%, which was greater than the expected minimum benchmark of 61% reduction (Miller et al. 2022). However, transfluthrin was less effective at reducing mosquitoes in that same area. This phenomenon of species-dependent efficacy echoes previous spatial repellent investigations with transfluthrin and other active ingredients targeting mosquitoes and biting flies in a variety of environments and scenarios (Britch et al. 2020a, 2020b, 2021; McMillan et al. 2022; Bayer et al. 2023). We hypothesize that a suite of spatial repellent active ingredients, or a variety of delivery systems, may be required to protect personnel in the field from the full community of vectors at a given location.

Another example from our data that suggests the need for care in implementing spatial repellents in IVM is that transfluthrin showed varying efficacy, depending on placement. When the transfluthrin source was placed inside a partially enclosed area (tent), it was less effective at reducing sand fly collections compared with outdoor placement in the bivouac scenario. Transfluthrin was also associated with increased collections of mosquitoes when dispersed inside the tents. We and others have observed and discussed this phenomenon of apparent attraction of mosquitoes when a spatial repellent is present (Choi et al. 2016, Bibbs et al. 2020, Britch et al. 2021, McMillan et al. 2022, Bayer et al. 2023). Briefly, accumulation of mosquitoes in a spatial repellent-treated area could indicate a disorienting effect (reduces ability to escape the area), an excitatory effect (increases likelihood of flight through the suction zone of the CDC trap), or a naivete effect (curiosity draws mosquitoes to the active ingredient to investigate). Note that the effect of accumulation of Cx. pipiens mosquitoes in treated tents increased over the course of sampling to the point of statistical significance later in the collection periods (Fig. 2A). Transfluthrin is known to migrate into surrounding materials (Govella et al. 2015) and could have accumulated in the tent material instead of dispersing away in the air, therefore creating more treated surfaces and increasing the realized diffusion rate above the expected 5 mg per 24 h. Data from a semifield study of efficacy of transfluthrin in a novel controlled release passive device suggest that placement of the device at the tent entrance, instead of inside the tent, could reduce entry of colony-reared Aedes, Anopheles, and Culex species in a north Florida subtropical and warm humid environment (Rajagopal et al. 2023). Also, apparent accumulation of mosquitoes in areas treated with transfluthrin could be underestimating protective efficacy: it has been shown that presence of mosquitoes in spatial repellent-treated areas may not equate to host seeking, probing, or biting (Bibbs et al. 2020, Swai et al. 2023).

One consideration to explain the difference in efficacy of transfluthrin between mosquitoes and sand flies in this study is pyrethroid susceptibility. In a recent study conducted in the Thessaloniki region, Cx. pipiens populations showed moderate levels of resistance to deltamethrin (Fotakis et al. 2017), while pyrethroid resistance mutations at the L1014 site of the para-type sodium gene were detected at allele frequency of 71.1% (L1014C and L1014F mutations). In contrast, no phenotypic insecticide resistance or associated mutations were reported from sand fly populations from the AFS study site (Fotakis et al. 2018). A recent laboratory study with Anopheles gambiae Giles found significant cross-resistance of a deltamethrin-resistant mosquito line to transfluthrin and vice versa (Zoh at al. 2023). Perhaps, reduced deltamethrin susceptibility and increased pyrethroid resistance allele frequencies observed in Cx. pipiens from Thessaloniki may partially explain the reduced efficacy of transfluthrin against Culex species mosquitoes in this study. Additional investigations are warranted toward improving our understanding on the link between pyrethroid-resistant field populations and transfluthrin efficacy under different operational scenarios.

Although impacts of pyrethroid resistance and optimal placement and dispersal of spatial repellents such as transfluthrin should be investigated further, the transfluthrin diffusers were highly effective at reducing sand fly incursion into small outdoor protected areas in this investigation. Therefore, a transfluthrin diffuser could protect personnel situated in hasty positions outdoors (e.g., during sentry duty, reconnaissance, or resting) and should be investigated further in additional environments and scenarios and target additional vector and nuisance Diptera. These use case scenarios are not unique to military stakeholders, and parallel considerations should be included in development of spatial repellents for civilians. In addition to tuning spatial repellents to placement and target species, future work should continue to investigate survival, blood feeding behavior, and fecundity of mosquitoes and sand flies postexposure under field conditions to estimate risk of evolution of resistance to these compounds before they are fielded in force (Achee et al. 2012, Bibbs and Kaufman 2017, Norris and Coats 2017).

We thank F. V. Golden and R. L. Aldridge for logistic support throughout the study. Comments from 2 anonymous reviewers improved this work. Funding was provided by the US Department of Agriculture, Agricultural Research Service, and the US Department of Defense, Deployed Warfighter Protection Program.