[Bed Bug Supplement] Shedding Light on IGRs and Bed Bugs

Editor’s note: Portions of this article were adapted from a technical paper titled, “Effects of Juvenile Hormone Analog Formulations on Development and Reproduction in the Bed Bug,” in the entomology research journal Pest Management Science.

Insect growth regulators (IGRs) have a long history of effective use against urban pests, including fleas, cockroaches, termites, stored product invaders and mosquitoes. IGRs are attractive management tools because of their prolonged effect and low mammalian toxicity. They also have novel modes of action, making them useful when combatting resistant pest populations. Different types of IGRs have different modes of action. The class of compounds used to control termites disrupts molting by interfering with formation of chitin, a foundation of insect cuticle. IGRs the industry uses to fight fleas, cockroaches, mosquitoes (and bed bugs) on the other hand mimic insect juvenile hormone (JH). Naturally occurring levels of this hormone in insects regulate growth, development and reproduction. Juvenile hormone analogs (JHAs), such as hydroprene and methoprene, disrupt these processes by imitating abnormally high levels of JH in exposed individuals.

Gentrol (Central Life Sciences) with (S)-hydroprene as the active ingredient, is often employed in bed bug management programs. In fact, when companies were asked in a series of annual surveys which bed bug insecticides they most often used, Gentrol was the second most mentioned product in 2007 and 2010, and fourth most mentioned in 2011 (Potter et al. 2008, 2010, 2011). Nonetheless, published research on the effects of IGRs on bed bugs is sparse. The only previously reported work with hydroprene (Gentrol) found no adverse effect on nymphal development, although the previously exposed nymphs succumbed as adults (Todd 2006, Miller 2009). Bed bugs utilized in each of these studies were from long-maintained laboratory colonies (more than 23 and more than 32 years, respectively), whose vulnerability to insecticides may have influenced the outcome. In other published trials, substantial mortality resulted when bed bugs were exposed to methoprene — but only at application rates several times higher than on current product labels (Naylor et al. 2008, Shaarawi et al. 1981, 1982, Takahashi and Ohtaki 1975).

This article presents our findings on the impact of IGRs on bed bugs. Be forewarned that the experiments were fairly complex — but necessary to measure effects on reproduction and development as relates to management.

Evaluation Methods. Evaluating IGRs in bed bug-infested dwellings is problematic. Since the compounds are not intended to kill quickly, they’re usually applied in combination with other faster-acting materials. This makes it harder to segregate results attributable to the IGR alone. Consequently, experiments were designed to closely monitor effects under controlled conditions in the laboratory.

Three different bed bug populations (strains) were utilized: the Harlan (FD) population, which is highly susceptible to most insecticides, and two populations collected from Cincinnati (CIN-1) and New York (NY-1), which are pyrethroid-resistant. Colonies and test insects were fed weekly using an artificial feeding system. When evaluating delayed-action insecticides, repetitive blood meals are needed since the nymphs must feed between molts. (Recall that bed bugs normally molt five times en route to becoming adults, progressing through five nymphal stages or “instars.”) Adult females must also blood feed in order to lay eggs.

During experiments bed bugs were maintained on an artificial feeding system. Adults and nymphs were fed warmed blood through a thinly stretched membrane to simulate human skin.

Bed bugs of different life stages (eggs, nymphs and adults) were exposed to various concentrations of Gentrol IGR Concentrate [(S)-hydroprene] and Precor IGR Concentrate [(S)-methoprene] diluted in water. Bugs and eggs from the respective strains were exposed to the IGRs as a direct spray, and by confinement on previously treated surfaces. All experiments were replicated (repeated) four times.

Direct Spray Tests. Groups of 10 bed bug eggs from the NY-1 strain were sprayed directly with Precor at the labeled concentration (1x), or two times the label concentration (2x) for fleas; Gentrol at two times (2x) the labeled concentration for bed bugs (simulating two applications as mentioned on the label), three times (3x) the recommended concentration for bed bugs; or water alone. Each of the five treatments was applied to groups of eggs laid within the previous 24 hours (one-day old eggs) or 48 to 72 hours (two- to three-day old eggs). Immediately after spraying, the eggs were transferred to a clean container and monitored daily for effects on hatching.

The elevated (3x label) concentration of Gentrol compared to water alone was also sprayed directly onto groups of 20 third-instar NY-1 strain nymphs. The wetted third-instar nymphs were then transferred to new containers, fed weekly and observed for adverse effects. Those reaching adulthood were further examined for lethal or abnormal effects, re-fed and allowed to mate with an untreated adult of the opposite gender. Oviposition (egg production), hatching and mortality were then recorded for 20 days.

Dry Residue Tests. In a second series of experiments, Masonite wood disks were treated with 1x or 2x label concentrations of Precor, 2x or 3x concentrations of Gentrol, or water alone, and allowed to air-dry for 24 hours. Groups of previously fed, unmated adult bed bugs (5 males, 10 females; NY-1 strain) were held continuously on the treated surfaces and mortality, oviposition and egg hatch were recorded daily for 20 days. Groups of 20 third-instar nymphs from insecticide susceptible (FD) and resistant (NY-1) bed bug colonies were also confined on disks treated in the aforementioned manner — plus a very high (10x) rate of Gentrol, i.e., 10 times the labeled concentration. Each group of continuously exposed nymphs was fed weekly and observed daily for molting and mortality. Upon reaching adulthood, each individual was paired with an untreated adult bed bug of the opposite gender to monitor any subsequent effects on oviposition, hatching and mortality. Each bug was also examined for deformities and females were dissected to count any eggs remaining in the abdomen.

A final experiment involved confining bed bugs on IGR-treated surfaces for their entire development time. Masonite disks were treated with 3x the label concentration of Gentrol or water alone, allowed to air-dry for 24 hours, and placed in individual containers. Groups of 30 fed and mated adult females from three different colonies (FD, NY-1 and CIN-1) were placed on each treatment and allowed to lay eggs for 24 hours, after which the adults were removed. Upon hatching, 20 newly-emerged (first-instar) nymphs from each treatment were fed and returned to their respective containers (additional nymphs were discarded). The nymphs were then followed through development to adults, with weekly feedings and observations on molting and mortality. Upon reaching adulthood, they were again fed, paired and mated with untreated adult bed bugs and monitored for effects on oviposition, hatching, etc.
 

Figure 1. Average number of eggs laid by adult bed bugs of two different strains (FD, NY-1) reared from third-instar nymphs on surfaces treated with 2x, 3x or 10x label concentrations of Gentrol. Average number of eggs laid by adult bed bugs of three different strains (FD, CIN-1 and NY-1) reared from first-instar nymphs on surfaces treated with 3x label concentration of Gentrol.

Results. What follows are the results for the different types of treatments.

Direct Spray of Eggs and Nymphs. No adverse effects were observed when bed bug eggs were sprayed directly with Precor or Gentrol, regardless of concentration applied or whether eggs were treated at one or three days of development. Hatching rates for eggs ranged from 95 to 100 percent and were statistically no different from spraying only water. Similarly, no adverse effects were noted when third-instar nymphs were sprayed directly with an elevated (3x) concentration of Gentrol. The mean percentage of nymphs developing to adulthood was not significantly reduced compared to wetting the bugs with water alone (86.3 vs. 93.8 percent). There also were no significant differences in subsequent numbers of eggs laid per female after a blood meal (7.1 with water vs. 6.5 with Gentrol), nor hatching (97 vs. 94 percent). Furthermore, no structural abnormalities were noted in any of the adults when examined under a microscope.

Adults Continuously Exposed to Dry Residues. No mortality or effects on egg production or hatching occurred from confining adult bugs on any IGR treatments. Average eggs laid per female (after one feeding) ranged from 5.6 to 6.5 for the various concentrations of Precor and Gentrol, vs. 6.1 eggs for water alone. Differences in hatch rate (89 to 94 percent for IGR treatments, 90 percent for water) were also non-significant.

Nymphs Continuously Exposed to Dry Residues. Neither IGR adversely affected nymphal development despite prolonged exposure to treated surfaces. Nymphs continued to grow, molt and feed normally even when confined from hatch-ing to adulthood on Gentrol deposits 10 times higher than labeled concentration. The lack of effect on developing nymphs was consistent for each of the three bed bug strains we tested.

Dissected bed bug female showing eggs retained in abdomen after being reared on elevated residues of Gentrol. The retained eggs did not hatch, but the embryos inside were partly developed. Tiny red eyespots can be seen on the two embryos to right of center, while the dark structure toward the bottom is the gut.

No decline in production or viability of eggs resulted from adults exposed as nymphs to 1x or 2x concentrations of Precor, or 2x concentrations of Gentrol. In three related experiments, however, statistically fewer eggs were laid by adults previously exposed as nymphs to higher concentrations of Gentrol. (See Figure 1 above.) In experiments 1 and 2, adult (NY-1 strain) bed bugs confined since they were third-instar nymphs on 3x label concentrations of Gentrol laid 77 percent and 49 percent fewer eggs, respectively, than bugs exposed to water alone. Oviposition was 100 percent curtailed (zero eggs laid) when NY-1 strain bugs were reared on 10x label concentrations in experiment 2. Oddly, no decline in egg production occurred when third-instar nymphs of the long-maintained laboratory (FD) strain were confined on 3x Gentrol concentrations — although 38 percent fewer eggs were laid when FD strain bugs were reared on 10x label concentrations. In a third experiment, where insects from all three strains were confined from hatching to adulthood on 3x Gentrol deposits, NY-1, CIN-1 and FD bugs laid 40, 36 and 6 percent fewer eggs, respectively. The physiological basis for the greater inhibitory effect on the two field strains versus the laboratory (FD) strain is unclear.

Subsequent dissections revealed that many non-laying females exposed to elevated concentrations of Gentrol had in fact formed eggs — but the eggs were retained in the abdomen. The retained eggs did not hatch, but showed partial development of the embryo, with eyes, legs and segmentation visible. (See photo above) Other abnormalities were also noted at elevated Gentrol levels, especially the 10x concentration. Irregularities in formation of cuticle, malformed wing pads, and in extreme cases, bursting of the gut through weakened areas of cuticle on the dorsal surface of the abdomen were observed, especially in the NY-1 strain. (See photos below.) Similar effects were reported previously by other investigators (Todd 2006, Miller 2009).

The Bottom Line. Based on these studies, hydroprene and methoprene seemingly should have minimal effect on bed bug reproduction and development when used at label concentrations. Methoprene (the active ingredient in Precor) had no effect on bed bugs at 1x and 2x the label concentration for fleas. These results are in agreement with Naylor et al. (2008), who reported significant effects on bed bugs at about 8.4 times (but not 4.2 times) the Precor recommended concentration. It should be noted that Precor IGR Concentrate is not currently registered for bed bugs although industry surveys indicate use by some companies.

Structural deformities were common in bugs reared on high (10x label) concentrations of Gentrol. Left: thin/weakened cuticle and bursting through of gut contents. Right: Inability to shed former skin (darkened area is ruptured gut contents).

Adverse effects on bed bugs were seen with hydroprene (the IGR in Gentrol), although it’s questionable whether they would occur in commercial practice. Applications of Gentrol repeatedly had no acute (immediate) effect on eggs, nymphs or adults, even when the respective life stages were sprayed directly. Gentrol also did not affect nymphal development — nymphs continued to grow, molt and blood feed normally even when confined from hatching to adulthood on Gentrol deposits 10 times higher than label concentration. Moreover, the lack of effect on development was consistent for each of the three bed bug strains tested.

Elevated (3x and 10x) label concentrations of Gentrol did reduce the number of eggs laid by two different bed bug field strains. Egg retention within IGR-treated females was unusual, and to our knowledge, has never been reported. Up to 36 partially developed eggs were found within a single treated female, whereas those treated only with water had two or fewer eggs. However, these effects occurred only from prolonged exposure to higher than labeled concentrations. Throughout the extended period, nymphs and adults also continued to blood feed normally.

In commercial practice, Gentrol is often applied repeatedly in combination with other insecticides. While such usage might enhance the impact of this IGR, insecticides tend to perform their best in the confines of the laboratory. In infested dwellings, many more variables can impact performance, including non-continuous coverage, insect mobility and intermittent exposure to insecticide deposits. Nonetheless, given the scarcity of bed bug management tools with novel modes of action, further evaluation and review of label concentrations may be warranted.

Mark H. Goodman, Michael F. Potter and Kenneth F. Haynes are a Ph.D. student, extension professor, and professor, respectively, at the University of Kentucky. Funding for the study was provided by Central Life Sciences.

Photos © M. H.Goodman and M.F. Potter

References
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Shaarawi, FAI, WAM Radwan and SA Soliman. 1982. Effects of juvenoids on some biological aspects of the bed bug, Cimex lectularius L. II. Effect on egg viability and embryogenesis. Bull Fac Earth Sci (King Abdulaziz Univ.) 6: 35-47.

Takahashi, M., and T. Ohtaki. 1975. Ovicidal effects of two juvenile hormone analogs, methoprene and hydroprene, on the human body louse and the bed bug. Jpn J Sanit Zool 26: 237-239.

Todd, R.G. 2006. Efficacy of bed bug control products in lab bioassays: Do they make it past the starting gate? Am Entomol 52: 113–116.