Evaluation of Hydrogen Peroxide Fumigation and Heat Treatment for Standard Emergency Arthropod Inactivation in BSL-3 Insectaries

Abstract

Climate change and global movements of people and goods have accelerated the spread of invasive species, including insects that vector infectious diseases, which threaten the health of more than half of the world's population. Increasing research efforts to control these diseases include the study of vector - pathogen interactions, involving the handling of pathogen-infected vector insects under biosafety level (BSL) 2 and 3 conditions. Like microbiology BSL-3 laboratories, BSL-3 insectaries are usually subjected to fixed-term or emergency room decontamination using recognized methods such as hydrogen peroxide (H₂O₂) or formaldehyde fumigation. While these procedures have been standardized and approved for the inactivation of diverse pathogens on surfaces, to date, there are no current standards for effective room-wide inactivation of insects in BSL-3 facilities in case of an emergency such as the accidental release of a large number of infected vectors. As H₂O₂ is often used for standard room decontamination in BSL-3 facilities, we evaluated H₂O₂ fumigation as a potential standard method for the safe, room-wide deactivation of insects in BSL-3 insectaries in comparison to heat treatment. To account for physiological diversity in vector insect species, six species from three different orders were tested. For the H₂O₂ fumigation we observed a strong but also varying resilience across all species. Lethal exposure time for the tested dipterans ranged from nine to more than 24 h. Furthermore, the coleopteran, Tribolium castaneum, did not respond to continuous H₂O₂ exposure for 48 h under standard room decontamination conditions. In contrast, temperatures of 50°C effectively killed all the tested species within 2 to 10 min. The response to lower temperatures (40-48°C) again showed a strong variation between species. In summary, results suggest that H₂O₂ fumigation, especially in cases where a gas generator is part of the laboratory equipment, may be used for the inactivation of selected species but is not suitable as a general emergency insect inactivation method under normal room decontamination conditions. In contrast, heat treatment at 48 to 50°C has the potential to be developed as an approved standard procedure for the effective inactivation of insects in BSL-3 facilities.

Keywords: BSL-3 insectary; containment; gene drive; heat susceptibility; hydrogen peroxide; inactivation procedures; infectious disease; vector insects.

FIGURE 1

Equipment used for the H₂O₂ fumigation experiments at Ortner Reinraum GmbH. (A) airlock; (B) table within the airlock showing the setup of experimental cages and instruments [H₂O₂ gas sensor (1), see also (D) and data logger for temperature and humidity (2), see also (E)]; (C) Steris Steraffirm chemical H₂O₂ indicator strips; (D) ATI Portasens II portable gas detector used inside the airlock; (E) Testo 176H1 data logger for temperature and humidity monitoring inside and outside (control cohorts) of the airlock.

FIGURE 2

Mosquito treatments at 400 ppm H₂O₂ and post-treatment survival monitoring. Adult mosquitoes were subjected to 400 ppm H₂O₂ treatments for 7 h (A,B) or 14 h (C) and dead individuals counted after exposure termination. Survivors were further monitored until the time of death. The Kaplan-Meier estimator was used to estimate survival. Survival numbers from the two biological replicates per species were combined for the analysis. The curves show the probability of survival per time interval. The dashed vertical line indicates the end of H₂O₂ exposure. The black arrow indicates the time point where the remaining survivors of the two vials were combined into one for further monitoring, v, treatment cohort; c, control cohort.

FIGURE 3

Determination of the maximum lethal exposure time for mosquitoes, medfly, and D. melanogaster. Ae. aegypti (A), An. stephensi (B), C. capitata (C), and D. melanogaster in the absence of food (D) were continuously exposed to 400 ppm hydrogen peroxide and viability assessed at regular intervals until the last individual was dead. The Kaplan-Meier curves show the probability of survival per time interval for the treatment (v) and the control (c). The dashed vertical line in the graphs indicates the end of the H₂O₂ exposure. Survival numbers from all biological replicates per species were combined for the analysis. Data shown are based on three biological replicates for the mosquitoes (v and c) and the medfly control, two for the Drosophila and medfly treatment, and four for the Drosophila control.

FIGURE 4

Effects of long-term H₂O₂ exposure on the milkweed bug and Drosophila. Sp. pandurus nymphs and adults (A), and D. melanogaster adults (B) were exposed to 400 ppm H₂O₂ for up to 47 h and death assessed at intervals. The Kaplan-Meier curves show the combined probability of survival per time interval for the treatment and post-treatment monitoring time, as described in Figure 2. The dashed vertical lines indicate the end of H₂O₂ exposure. Data shown here is based on two biological replicates for Sp. pandurus and five biol. replicates for D. melanogaster; v, treatment; c, control.

FIGURE 5

Lethality induced by short-term exposure to 1000 ppm H₂O₂. Insects were continuously exposed to 1000 ppm hydrogen peroxide for up to 5 h and death assessed in intervals. Survivors were monitored for another 5–8 days after treatment end and death assessed in intervals. Data analysis shown here was performed as described in Figure 2 and is based on one large experimental cohort for An. stephensi (A), C. capitata (B), and Sp. pandurus (C), and two biological replicates for D. melanogaster (D). The dashed vertical lines indicate the end of the H₂O₂ exposure; v, treatment; c, control.

FIGURE 6

Exposure of Ae. aegypti eggs to hydrogen peroxide. (A) Pictures of Ae. aegypti eggs displaying different degrees of bleaching after exposure to 400 ppm H₂O₂ for 7, 14, or 21 h, and to 1000 ppm for 5 h. The control batch was kept next to the airlock for the duration of the exposure. (B) Eggs from (A) were submerged in water to assess the influence of the H₂O₂ treatment on egg viability. Shown is the pupation rate (number pupae/number eggs*100) for the different treatments. Data are based on one large experimental cohort of more than 1000 eggs for each treatment.

FIGURE 7

Comparison of insect heat tolerance at five different temperatures. Cohorts of 20 insects were exposed to 40°C (A), 42°C (B), 45°C (C), 48°C (D), and 50°C (E) for increasing time periods. For every combination of temperature and time a fresh cohort was used, and death was monitored for 24 h post exposure. Box and whisker charts are showing the inclusive median of survival numbers of the insects depending on the treatment temperature and exposure time. The means are indicated by an x, outliers are displayed as dots. Except for T. castaneum, the survival numbers shown here for every combination of temperature and treatment time are based on two to five repetitions with 20 individuals each. The survival numbers displayed for the controls are based on three to 12 repetitions with 20 individuals each.

FIGURE 8

Comparison of the heat tolerance of Ae. aegypti female mosquitoes at different age. Influence of age on heat sensitivity was investigated at two moderately increased temperatures, 40 and 42°C (A,B), and at a near-lethal temperature, 48°C (C). “Young” females were between 1 and 10 days old, “old” females between 10 and 29 days. Lethality for each exposure temperature and time was assessed in two to four biological replicates (i.e., each replicate was performed with an experimental cohort from a different cage). Each cohort consisted of 20 individuals. The box and whisker charts display the inclusive median of the replicate values for each exposure time. The means are indicated by an x, outliers are shown as dots. The survival numbers displayed for the controls are based on two to eight repetitions with 20 individuals each.