*2.1. Mosquitoes*

Adult, female yellow fever mosquitoes, *Aedes aegypti* (Diptera: Culicidae), are a major vector of pathogens that cause animal and human diseases worldwide [17–19] and were used as a model insect for the studies that follow. *Ae. aegypti* females (Figure 1A and Figure S1) were obtained from a colony maintained in the Dearstyne Entomology Laboratory at North Carolina State University, Raleigh, NC, USA. The mosquito colony has been continuously reared for approximately 5 years and is free of pathogens. Adults were kept at 27 ◦C and 80% relative humidity with a 14:10 h light: dark photoperiod. Adults were provisioned with a 10% sucrose solution (in distilled water) *ad libitum*. To obtain eggs for colony maintenance, female mosquitoes were fed porcine blood (obtained from a local abattoir) using an *in vitro* blood-feeding device (described later). Larvae were kept under the same environmental conditions as adults and fed a porcine liver powder: brewer's yeas<sup>t</sup> mixture (2:1, wt:wt). Larval rearing water was dechlorinated using a standard aquarium dechlorinating agent.

#### *2.2. In Vitro Feeding/Bioassay System*

An *in vitro* bioassay system was developed (shown in Figure S2A) to blood feed mosquitoes for routine colony maintenance and to bioassay the barrier materials for bite resistance. The major components of the system are a blood-feeding reservoir, Plexiglas® cage, and a circulating water bath for regulating the temperature of the blood. The bloodfeeding reservoir is designed to contain the blood, fix a feeding membrane over the blood, and fix barrier materials on top of the feeding membrane for bioassays [20]. Briefly, the blood reservoir (16.5 cm length × 3.5 cm width × 0.5 cm depth) was produced with a handheld router from a rectangular block of Plexiglas® (28 cm length × 5.5 cm width × l cm thickness). A hole (4 mm diameter) was drilled at the center of the top and bottom edge through the plastic into the blood reservoir. A tap was used to cut threads into the plastic so that a valve could be screwed into the top and bottom holes. Two holes (each 4 mm diameter) were drilled from the bottom edge of the device through the plastic to the blood reservoir. A loop of stainless-steel tubing (3 mm diameter) was placed into the blood reservoir, and the tubing was inserted through the holes so that the cut ends protruded out of the plastic. Epoxy cement was used to seal the tubing in place inside the blood reservoir of the device. The ends of the tubing were connected to a circulating water bath to heat the blood.

**Figure 1.** Principle of a bite-resistant textile structure against *Aedes aegypti*. (**A**) An *Ae. aegypti* adult female feeding on the blood beneath human skin. (**B**) SEM image of a knit structure. (**C**) Example of pores formed by the filaments in the knit structure. (**D**) Heat and moisture managemen<sup>t</sup> of a fabric. (**E**) The proposed three cases for mosquito-bite resistance. (**F**) Research steps for the design of bite-resistant garments.

For blood feeding, a transparent collagen film (product code 894010.95; Devro, Inc., Columbia, SC, USA) was hydrated in distilled water and stretched over the top of the device. A gasket, cut from a sheet of cork-rubber composite (Fel-Pro, part no. 3019; AutoZone, Raleigh, NC, USA) was placed on top of the collagen film. A rectangular piece of plastic (3 mm thick) the size of the blood-feeding device was then placed on top of the gasket. The central area of both the rubber gasket and plastic frame was removed so that the collagen film is fully exposed to the mosquitoes. Metal binder clips hold the gasket and frame in place on top of the blood-feeding device, preventing leakage of blood. A 30 mL syringe filled with blood was then attached to the valve that was screwed into the top hole of the blood-feeding device. With the device tilted at a slight downward angle, the blood was slowly transferred into the reservoir. The valve attached to the bottom of the device was opened to allow air displacement as the blood is added. When the device was filled with blood, both valves were closed, and the circulating water bath was started to warm the blood to 35 ◦C.

The barrier materials (for example, the plastic blocks shown in Figure S2C; the barrier materials tested are in toto listed in Table 1) to be evaluated for bite resistance were cut exactly to fit over the collagen film within the plastic frame. The test area for the in vitro bioassay was the same as that for the arm-in-cage studies discussed later. Masking tape, placed around the inner edges of the plastic frame, slightly overlaps the barrier. In this way, mosquitoes are prevented from gaining access to the collagen film by probing around the edges of the barrier. The blood-feeding device was inserted into a Plexiglas® bioassay cage (30 cm square on each side; Figure S2A) which contains mosquitoes for feeding (with the barrier material absent) or bioassay (when the barrier material is in place). For routine colony maintenance, the feeding membrane was not covered with barrier materials.

Prior to testing the barrier materials and inserting the blood-feeding device into the cage, 100 *Ae. aegypti* females were transferred to the bioassay cage (Plexiglas®, 30 cm on each side). Mosquitoes were starved overnight (sugar water removed from their rearing cage; females not blood fed) prior to testing. Female mosquitoes were 6–7 days of age (post emergence). Porcine blood obtained from a local abattoir was used in our bioassays. At the time of blood collection, sodium citrate was added as an anticoagulant. Just prior to initiating the bioassay, ATP (Sigma) was added to the blood (2.5 mg/mL) as a phagostimulant [20]. Each bioassay was conducted for 10 min., during which the number of times females landed and probed the barrier material was counted. A single event was recorded if a female landed and then inserted or attempted to insert her proboscis into the barrier material, regardless of whether the female probed multiple times after landing. A video recording was made of each bioassay so that the mosquitoes' responses to the surface of each barrier and probing behavior could be studied. At the end of the exposure period, mosquitoes were removed and killed in a freezer. Subsequently, each mosquito was crushed on a sheet of white paper to determine if she was able to probe through the barrier and obtain a blood meal. Blood spots on the paper were counted, and the percentage of mosquitoes that were blood fed was calculated based on the total number of mosquitoes released into the cage. The *in vitro* bioassays were repeated for each barrier material a minimum of 3 times. For routine blood feeding for colony maintenance, the number of mosquitoes in the cage was variable (50 to 200), and the feeding time extended until all of the mosquitoes that want to feed have time to feed to repletion. All bioassays and mosquito adult feeding, including the *in vitro* and *in vivo* (described later) tests, were conducted in the mosquito insectary laboratory at the Dearstyne Entomology Building of NC State University, at a temperature of 27–29 ◦C and 75–80% humidity. All tests were conducted during the photophase under florescent lighting.

#### *2.3. In Vivo Bioassay for Bite Resistance*

Measurement of the *in vitro* mosquito-bite resistance of the barrier materials was standardized in terms of the apparatus architecture (dimensions and exposed area of the feeding membrane) and blood-feeding conditions. Similarly, for the *in vivo* studies, the

dimensions of the bioassay cage and cloth area exposed for mosquito probing were the same. Our IRB for the *in vivo*, arm-in-cage studies required us to demonstrate *in vitro* bite resistance of greater than 80% for the barrier materials before conducting an *in vivo* test on the same barrier material. This restriction was to limit the potential number of mosquito bites received by the human subject. *In vivo* tests using human subjects is a more rigorous test of a fabric's bite resistance because of the volatile attractants emitted from the skin. *In vivo* testing is critical to understanding whether a textile will prevent mosquito bites. Therefore, validation of our predictive model and development of textiles for garmen<sup>t</sup> construction (discussed later) required *in vivo*, arm-in-cage studies.

**Table 1.** Barrier materials studied, their abbreviation, measured thickness and pore diameter, model prediction, and bite-resistance bioassay results.


Note: † Model prediction means the bite resistance of each fabric predicted by the bite-resistance model. "Safe" means the fabric has 100% bite protection and "unsafe" means the fabric is predicted to allow mosquito biting (based on our bite-resistance model). †† Bioassay result is an actual measurement of bite resistance. For in vitro and in vivo tests, "Pass" means the fabric demonstrated at least 95% bite protection. For the walk-in-cage test, pass means no bites.

> Arm-in-cage studies (apparatus used shown in Figure S3A) were conducted with informed consent using a protocol for use of human subjects in research approved by the NC State University Institutional Review Board (IRB #2925) [21]. The assay methodology was designed to mimic a textile worn on the forearm with the fabric in close contact with the skin. Odorants and heat from the skin can diffuse through the fabric attracting mosquitoes seeking a blood meal.

> The sleeve device (Figure S3A), constructed from bioassay textiles, exposed the cloth surface through an opening that was identical in size as was used in the *in vitro* assays. The sleeve was constructed from a polyvinyl-coated roofing membrane, Samafil® (Sika Corp., Canton, MA, USA). The sleeve was cut into a trapezoidal shape to fit a human arm and

with a 16.5 cm × 3.5 cm opening in the center that corresponds to the size and shape of the opening in the *in vitro* blood-feeding device described earlier. A plastic frame was riveted to the sleeve to keep the exposure area of the textile from deforming when the sleeve was attached to the forearm of the study participant.

In total, 100 unfed, nectar-starved *Ae. aegypti* adult females were transferred to a bioassay cage 10–30 min before being assayed, as described earlier for the *in vitro* assay. The textile to be assayed was laid over the underside of the forearm of the study participant. The sleeve was laid on top of the cloth and attached to the participant's forearm with Velcro® straps. The hand of the participant was then covered with a nitrile glove to prevent mosquito bites on the hand. The bioassay was started when the participant inserted his/her arm through a cloth sleeve into the bioassay cage. An observer counted the numbers of mosquitoes landing on the cloth and probing during a 10 min exposure period, and in some cases video recordings were made of the inserted arm only as needed for further documentation. After the bioassay was terminated, mosquitoes were examined for blood feeding by crushing them on white paper as previously described for the *in vitro* assay. Blood spots on the paper were counted, and the percentage of mosquitoes that were blood fed was calculated based on the total number of mosquitoes released into the cage. The mosquitoes used, mosquito conditioning, the number of mosquitoes, and level of replication were the same as that described for the *in vitro* assay.

#### *2.4. Walk-in-Cage Studies of Whole Garments*

A garmen<sup>t</sup> is composed of integrated fabrics and seams that have various rectilinear and curvilinear pattern pieces needed to conform to differing human body shapes. The gap distance between the garmen<sup>t</sup> and the skin varies throughout the body and can change with posture along with textile stretching, all of which can affect bite resistance. These factors affect the fabric performance regarding mechanical bite resistance and comfort, which can only be evaluated through whole-garment testing. Walk-in-cage studies provide a method for testing garments under quasi-field conditions with higher mosquito-bite pressures. We also avoided disease risks to human subjects that might occur using wild mosquito populations in a field test.

Garments (Figure S6A,B, described later in detail, and all the garments tested are listed in Table 1) were tested in a walk-in enclosure (2 m height × 4 m length × 4 m width) constructed from polypropylene screens (mesh size 1.8 mm; Lumite Company, Alto, GA, USA) that were sewn together to form a cage. The test cage had a zippered opening and was supported with a 2 inch × 4 inch wooden frame. The bottom edges of the panels were taped to the cement floor to prevent mosquitoes from escaping. The cage was covered with white bed sheets and then an outer layer of black plastic to block external light. Light inside the cage was provided by a single 35 W fluorescent tube placed at each corner suspended from the ceiling. Prototype garments were worn by a human subject with informed consent with an approved research protocol (IRB# 9075) from the NC State University Institutional Review Board. For the prototype base layer garment, the subject's head and neck were protected by a bee veil, the hands were covered by nitrile gloves and the feet covered with shoes. Each pant's leg was taped to the shoe to prevent biting at the margin between the pants and shoe. For the prototype NCSU shirt, the subject wore three pairs of pants that combined were 100% bite proof; otherwise everything was the same as for the base layer.

At the beginning of the trial in the bioassay cage, 200, 6–7-day-old, unfed adult female *Ae. aegypti* were released by the test subject. The condition of the mosquitoes was described earlier. In the bioassay cage, the subject stood motionless with arms at her/his sides for 10 min and then sat with arms crossed for an additional 10 min on a waist-high stool (no back support). In a sitting position, the fabric was stretched at the knees, elbows, and shoulders. These two postures mimicked how a garmen<sup>t</sup> would be worn for mosquito protection. The postures caused the garmen<sup>t</sup> to deform, changing the gap distance between the fabric and skin on different parts of the body, thus potentially affecting bite-resistance performance. Assays were conducted during the photophase at 25–28 ◦C and a relative humidity of approximately 30–40%. At the end of each trial, the subject exited the bioassay cage, and all mosquitoes were collected with a mechanical aspirator and killed in a freezer. After removing the garment, the test subject's skin was examined for mosquito bites with the assistance of another researcher. Areas of the body where bites occurred were recorded so that the corresponding areas of the garmen<sup>t</sup> could be reinforced to prevent bites in subsequent prototypes. Mosquitoes were collected, frozen, and examined for blood feeding by crushing them on white paper, as described earlier. Each garmen<sup>t</sup> was evaluated in a minimum of three separate trials conducted on different days.

#### *2.5. Model Rationale and Mosquito Morphometrics*

Blood feeding of mosquitoes on humans involves physical interactions between the mosquito's external morphology associated with the head and exposed skin, requiring a combination of insect behaviors allowing the mouthparts to penetrate the cornified, squamous epithelium and insert into the host blood vessels near the skin surface. When a textile is placed over the skin, the fabric restricts access to the skin and affects mosquito landing and probing behaviors. This creates another compliment of physical interactions between the textile and the mosquito that affects differently how the mosquito also interacts with the skin below. These physical parameters of the mosquito's head and mouth parts impose three-dimensional limits, defined by their shape and size, on a mosquito's ability to penetrate the textile and the skin. Understanding these limits and the mechanics of biting affected by the physical structure of cloth and the morphometrics of the mosquito's feeding structures can be used to develop textiles to optimally resist blood feeding, as well as providing optimal comfort without the need for insecticides or repellents.

The mosquito proboscis (Figure S1A,B) is a collection of interlocking needle-like mouthparts (stylet in shape) covered by a sheath, the labium. The stylets consist of the labrum (Figure S1C,D), a pair of mandibles, a pair of maxillae, and a hypopharynx extending from the floor of the mouth between the mandibles and maxillae. The rigid, pointed labrum tip is shown in Figure S1D and is the first part of the proboscis that makes contact with skin to initiate biting. The other mouth parts are used to advance the insertion into the skin and for channeling blood to the mouth. Preventing labrum penetration and/or contact with the skin prevents blood feeding.

Our model to describe the physical interactions between a mosquito and a barrier material is divided into three Cases that represent the process of fabric penetration to obtain a blood meal and how the mosquito interacts with different textile surfaces. For our Case 1 model (Figure 1E), the dimension of the labrum (the largest mouthpart needed for penetration of the skin and blood feeding) is a critical attribute of the mosquito's mouthparts. To measure its dimensions, the labrum from 20 adult female mosquitoes (described before) was dissected using needle-point forceps, then gold coated using a SC7620 Mini Sputter Coater (Quantum Design GmbH, Darmstadt, Germany), visualized using a Phenom G1 desktop scanning electron microscope (SEM; Thermo Fisher Scientific Inc., Waltham, MA, USA) in the Phenom SEM and Forensic Textile Microscopy Laboratory at NC State University, and the measurements of maximum labrum diameter (D), labrum tip angle (*α*), and tip length (*L*tip) taken from these images. To avoid body shrinkage from dehydration, the mosquitoes were killed by freezing, and the mouth parts were quickly dissected and gold coated.

For the model for Case 2 and Case 3 (Figure 1E), 20 adult females were used for measurements of the head diameter ( *D*head) and antenna length (*L*antenna), not including the flagella branches and proboscis length (*L*proboscis), using a Nikon SMZ-1000 Zoom Stereo Microscope fitted with an ocular micrometer (Nikon Metrology, Inc., Brighton, MI, USA) in the Phenom SEM and Forensic Textile Microscopy Laboratory at NC State University. To avoid body shrinkage from dehydration, the mosquitoes were killed by freezing and then morphometric measurements were immediately taken. The mosquito anatomy that was measured is shown in (Figure S1B,C).
