Insect Sources
Insect colonies of R. dominica and T. castaneum maintained continuously at the USDA-ARS Center for Grain and Animal Health Research were used. This included T. castaneum collected in Eastern KS (USA) from 2012, and R. dominica collected from Eastern KS in 2019. Tribolium castaneum and had been reared on a mixture of 95% unbleached, organic flour and 5% brewer’s yeast, while R. dominica was reared on tempered organic whole wheat. Adults that were 4–6-week-old were used for experiments. Colonies were maintained at 27.5°C, 65% RH, and 14:10 (L:D) h photoperiod.
Treatments
The following netting treatments were used: negative control (e.g., no netting), positive control (netting identical to LLIN but without insecticide; Item#1721-9668, Casa Mesh White, Casa Solid, Joann’s Fabrics, Hudson, OH, USA), 0.34% w/w alpha-cypermethrin LLIN (Carifend, BASF Corps, Ludwigshafen, Germany), and a 0.4% w/w deltamethrin LLIN (D-Terrence, Vestergaard Inc., Lausanne, Switzerland).
Laboratory food dust assay
To evaluate the effect of food dust on the efficacy of LLIN, there were two food dust regimes. Netting was either used as is or fully dipped into organic flour (Heartland Mills, Marienthal, KS, USA) that filled a 9 × 9 cm square Petri dish. After exposure to food dust, the netting was used to line a new, clean 9 × 9 cm Petri dish. Rhyzopertha dominica and T. castaneum adults were tested in cohorts of 20 and exposed on the netting for 10 min continuously in the Petri dishes, then their conditions were checked at 1, 24, 48 h, and 168 h after exposure. Insects were held in an environmental chamber set to 27.5°C, 65% RH, and 14:10 L:D. Conditions were classified as the percentage that were alive (normally moving around unimpeded), affected (showing abnormal or sluggish movements, but movement still present, even if just twitching of extremities), or dead (completely immobile; full definitions in Morrison et al. 2018). This was performed under a stereomicroscope (SMZ18, Nikon Inc., Tokyo, Japan). A total of n = 5 replicate cohorts were tested per combination of treatments (dust regime, netting type, exposure time, post-exposure holding duration, and species).
Spillage assay
To evaluate whether netting could be used to protect sites of spillage, we performed a spillage assay in the laboratory. For this assay, only netting without insecticide but identical to LLIN and 0.34% alpha-cypermethrin LLIN (BASF) was used. Netting was placed covering a single layer of 35 g of whole organic hard winter wheat (Heartland Mills, Marienthal, KS, USA) in a 9 × 9 cm square Petri dish. A control treatment included a single layer of positive control or alpha-cypermethrin LLIN placed in a Petri dish without food. Cohorts of 20 mixed-sex R. dominica or T. castaneum adults were exposed continuously to the netting for 48 h. After that period, the conditions of the adults were recorded as alive (moving normally), affected (sluggish movements, unable to right themselves when fallen, or twitching body parts), or dead (completely immobile) according to established definitions in (Morrison et al., 2018). After sieving adults, we placed the grain from the Petri dish in a separate vial (11 × 4.9 cm H:D) for six weeks to check for progeny production, including the number of larvae, pupae, and adults. A total of n = 7 replicate cohorts were tested per combination of treatments.
Interception assay
To determine whether LLIN can prevent horizontal dispersal of stored product insects to sites of spillage, we performed an interception assay. A single layer of organic, whole wheat (Heartland Mills, Marienthal, KS, USA) was placed in a 245 × 245 mm large square Petri dish (Item# 431111, Corning Inc., Corning, NY, USA). In the center of the dish, a 2.5 x 24.5 cm (W × L) strip of netting was added on top of the wheat. A total of 50 mixed-sex R. dominica or T. castaneum adults were added to the middle of zone 1 (e.g., release zone; Figure 1). The remainder of the dish was equally divided into four more zones, including: release side before netting (zone 2), net zone (zone 3), non-release side after netting (zone 4), distal zone after netting (zone 5). Each zone was 2.5 cm in width. After release, adults were given 24 h to disperse. At the end of the period, the condition of adults was recorded as alive, affected, or dead, and the number of insects dispersing to each zone was recorded. There were a total of n = 5 replicate cohorts per treatment combination.
References
Morrison III, W.R., Wilkins, R.V., Gerken, A.R., Scheff, D.S., Zhu, K.Y., Arthur, F.H. and Campbell, J.F., 2018. Mobility of adult Tribolium castaneum (Coleoptera: Tenebrionidae) and Rhyzopertha dominica (Coleoptera: Bostrichidae) after exposure to long-lasting insecticide-incorporated netting. Journal of Economic Entomology, 111(5), pp.2443-2453.
