3. Practical antenna design

There have been many practical antennas which are designed for the integrated antenna. Through some practical antennas, we can see that the integrated antenna may be implemented for the wireless communication. Design of the integrated antenna is dependent on the system requirement such as array structure and channel environment. In this section, we introduce various practical integrated antennas for several cases.

## 3.1 Practical integrated antenna

It is important to integrate multiple antennas with compact size, which have orthogonal radiation patterns of each other. There are practical integrated antennas without considering array structure as shown in Figure 4. It is found that the integrated antenna has two types of antenna elements which have radiation patterns of electric dipole antenna and magnetic loop antenna. Planer inverted-F antennas (PIFA) are used for the electric dipole antenna, and quarter-wavelength slot antennas are used for the magnetic loop antenna [16, 17]. It is also seen that the integrated antennas have three-dimensional structures because the array structure was not considered. Therefore, it is noted that the integrated antenna may consist of more than two antenna elements practically.

#### 3.2 Practical integrated antenna array

The integrated antenna array may be implemented as shown in Figure 5. It is found that the practical integrated antenna array is composed of Lt array elements. Each array element consists of Bt antenna elements, which are designed by utilizing a planer Yagi-Uda antenna [18].

To reduce the antenna size, we eliminated a conduct strip in front of the antenna element and designed that l3 is larger than w2 as shown in Figure 5a. The radiation


At high thermal exposure (80–141 ◦C), WCRMO-2 diet was made using a flash sterilization system (Frontier Scientific Services). Frontier's sterilizer consists of a circulating system of processing tubes that alternates between linear stretches of tubing and coiled spiraltherms that act as heat exchangers (Figure 1). To make 10 L of WCRMO-2 diet, 1.49 Kg of WCRMO-2 dry mix (WCRMO-2, Frontier Scientific Services) and 158 g of agar (7060, Frontier Scientific Services) were added to 9.26 L of cool tap water (~21 ◦C). The diet mixture was then pumped into the process lines from a tank and controlled via a metering pump that manages line pressure and flowrate of the product. The time of thermal exposure was set by manipulating the flowrate. The first heat exchanger was jacketed with hot oil set to a desired temperature. As the product flows through the coiled spiraltherm tubing, it rapidly gains thermal units from the hot oil jacketing the line. After departing the heated coil, the process line enters a series of linear switch-backs so that some excess heat is lost to atmosphere before reaching the cooling spiraltherm. At this point, the product enters a heat exchanger jacketed in cold (room temperature) water to rapidly lose thermal units and reach a desired dispense temperature. At the dispense temperature of 60 ◦C, 289.5 mL of 10% KOH (*w*/*v*) (F7633, Frontier Scientific Services) was added into the diet mixture to adjust the pH of the diet to 9, and the resulting diet solution was blended for 30 s to mix thoroughly. Subsequently, the diet solution was dispensed into a 96-well plate (3370, Corning Inc., Corning, NY, USA) using a repeater pipette (200 μL per well) in a biological cabinet (Forma 1800 Series Clean Bench, Thermo Scientific). The diet plate was then allowed to evaporate excess moisture for 10 min, stored in a refrigerator at 7 ◦C, and used for assays within 3 days.

**Figure 1.** Frontier Scientific's flash sterilization system used for the processing of insect media. From left to right: diet tank, pump, processing tubes, coiled spiraltherms, and control station.

At mild thermal exposure (50–80 ◦C), WCRMO-2 diet was made using WCRMO-2 dry mix (WCRMO-2, Frontier Scientific Services) according to the manufacturer's procedure [27]. To prepare 1 L of WCRMO-2 diet, agar (15.8 g) was added to 926 mL of purified water, and the solution was brought to a full boil using a microwave (Hamilton Beach) until agar was completely melted. The agar solution was then transferred to a blender placed in a biological safety cabinet (SG403, SterilGARD® III Advance cabinet, Sanford, ME, USA). When the agar solution cooled to designed temperatures (Table 1), 148.9 g of WCRMO-2 dry mix was added, and the mixture was blended for 10 s to mix thoroughly. Subsequently, 28.95 mL of 10% KOH (*w*/*v*) (P250, Fisher Scientific, Fair Lawn, NJ, USA) was added to increase the pH of the diet to 9, and the resulting diet solution was blended for 10 s to

mix thoroughly. The diet was poured into a 1-L glass beaker containing a stir-bar and placed on a stirring hot plate (CimarecTM, Thermo Scientific) set at the designed temperatures. The temperatures of diet solution were monitored using an infrared thermometer (IR002, Ryobi, Fuchu, Hiroshima, Japan) and held at the test temperatures for the designed times (5–30 min) using the hot plate. Subsequently, the diet solution was dispensed into a 96-well plate (3370, Corning Inc.) using a repeater pipette (200 μL per well), allowed to evaporate excess moisture for 10 min, stored in a refrigerator at 4 ◦C, and used for assays within 3 days.

#### *3.1. Insect Artificial Diet Bioassays*

The diet bioassays were conducted as described previously [25]. All materials used in the diet assays were surface-treated via exposure to UV light for 10 min in a biological cabinet (SterilGARD® III Advance cabinet). Each diet treatment, which is WCRMO-2 diet made at different temperatures and time of thermal exposure (Table 1), was randomly assigned to a 12-well row of the 96-well plate and replicated at least 3 times in different diet plates. Each well was infested with one WCR neonate (<24 h old) using a fine paintbrush. A sealing film (TSS-RTQ-100, Excel Scientific, Inc., Victorville, CA, USA) was used to cover the plate. For ventilation, a hole was made in the sealing film over each well using a number zero insect pin. The plates were kept in an incubator (Percival, Perry, IA, USA) at 25 ◦C in darkness for 10 days. Larval molting was recorded daily during the experiments, whereas larval weight, survival, and evidence of contamination were recorded at the end of the experiments. For larval dry weight, all live larvae in each treatment were pooled per replicate (12 possible) into 95% ethanol, dried in an oven (Binder GmbH, Tuttlingen, Germany) at 55 ◦C for 48 h, and weighed using a micro balance (MSU6.6S-000-DM, Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany).

#### *3.2. Data Processing and Statistical Analyses*

Survival and molting data were calculated by dividing the number of live larvae and successful larval molt from 1st to 2nd instar and from 1st to 3rd instar per replicate, respectively, by the initial number of larvae infested and multiplying by 100 to obtain percentages. Weight per larva (mg) was determined by dividing the dry weight by the number of larvae that survived per replicate.

For the experiment with high temperatures (80–141 ◦C) and short time of thermal exposure, the diet treatments that resulted in a "soft-set" of the media were excluded in the analyses because the soft-set form led to the death of the majority of larvae after 4 days post infestation. Because of the exclusion of these treatments, the remaining data points were not adequate to generate the response surface models of the measured responses. The remaining data were analyzed as a completely randomized experiment. The data were analyzed with analysis of variance (ANOVA) using PROC MIXED in SAS 9.4 (SAS Institute, Cary, NC, USA). Diet was the fixed effect, and replication was the random variable. Differences between the remaining treatments were determined using Fisher's least significant difference (LSD) at *p* < 0.05. The percent variables (survival and molting) were arcsine square-root transformed prior to the analysis to meet assumptions of normality and homoscedasticity, whereas untransformed data were presented as mean ± SEM.

For the experiment with mild temperatures (50–80 ◦C) and extended time of thermal exposure, polynomial equations were generated to describe the impact of two factors (temperature and time of thermal exposure) on the measured responses (larval weight, molting, and survival). The best fit model for each measured response was selected from all possible models from linear to quartic polynomials generated with Design Expert® (Stat-Ease, Inc., Minneapolis, MN, USA). Model selection was based on several criteria, including low model *p*-value, lack of fit *p*-value, low standard deviation, high R-values, and a low PRESS value [38,39]. Once more than one satisfactory model was generated, adequacy tests were performed to further evaluate the selected model, as described previously [40].

#### **4. Results**

#### *4.1. Diet Quality with High Thermal Exposure and Short Time of Thermal Exposure*

Exposure to high temperatures (110–141 ◦C), even if constrained to a small window of time (0.9–2.3 s), had significant deleterious effects on the quality of WCRMO-2 diet for feeding WCR larvae. The WCRMO-2 diet exposed to the high temperatures for the brief time exposure resulted in larval weight significantly smaller compared to the control diet, WCRMO-2 diet made at a temperature of 65 ◦C for 10 min (*p* < 0.0001, *F6,13* = 15.88. Figure 2a). Average larval dry weight on the diet treatments ranged from 0.30 mg to 0.39 mg, while average dry weight on the control diet was 0.71 mg, an approximately 2-fold difference. There was no significant difference in dry weight on all diet treatments. Significant delays in larval molt to the 2nd instar were observed when they were reared on all diet treatments compared with the control diet (Figure 2b). There were significantly fewer 2nd instar larvae on the diet treatments than on the control diet after 5 days post infestation when larvae began to molt to 2nd instar. At day 5 post infestation, 20.6% of larvae had molted to 2nd instar on the control diet, whereas nearly 0% of 2nd instar larvae on all diet treatments (*p* < 0.0001, *F6,32* = 37.60). At day 7 post infestation, nearly 100% of the larvae had molted to 2nd instar on the control diet, significantly higher than that of all diet treatments (*p* < 0.0001, *F6,32* = 19.82). There was no significant difference in percent larvae molted to 2nd instar in the diet treatments (range from 39.3%–48.4%) at day 7 post infestation. No larvae that had molted to the 3rd instar were observed on the diet treatments by 10 days. Larval survivorship on all diet treatments ranged from 95.7% to 100%, which was not significantly different from survivorship on the control diet (*p* = 0.7764, *F6,13* = 0.53, Figure 2c).

**Figure 2.** Effects of high thermal exposure (110–141 ◦C) and short time intervals of thermal exposure (0.8–2.3 s): dry weight (**a**), percent successful completion of molt to 2nd instar (**b**), and percent survival (**c**). Western corn rootworm larvae were reared on WCRMO-2 diet for 10 days. Means with bars followed by different letters are significantly different (*p* < 0.05). Means ± SEM.

#### *4.2. Diet Quality with Mild Thermal Exposure and Extended Time of Thermal Exposure*

The two-level factorial experiment yielded significant response surface models for larval weight (*p* < 0.0015, *F3,7* = 16.27), molt to 2nd instar (*p* = 0.0410, *F3,7* = 4.76), molt to 3rd instar (*p* = 0.0100, *F3,7* = 8.46) and a marginally significant model for survival (*p* = 0.0531, *F3,7* = 4.23) (Table 2). Models for weight, molt to 3rd instar, and survival had insignificant lack of fit, whereas there was a significant lack of fit for molt to 2nd instar due to a very small value of pure error. The relationships between the two factors tested (mild thermal exposure and extended time of exposure) and the quality of WCRMO-2 diet were revealed in contour plots (Figure 3). The contour plots generated from the models of responses measured (weight, survival, and molting) displayed the performance of larvae when reared on the WCRMO-2 diet prepared at the mild temperatures for the extended duration. The magnitude of the response variables is coded in color and can be envisioned as perpendicular to the page, as indicated by labelled isobars.

**Table 2.** *p*-values, regression coefficients, and response surface-model fitting diagnostic statistics for western corn rootworm responses to a 2-factorial experiment at mild thermal exposure (50–80 ◦C) and extended time of thermal exposure (5–30 min). A: time of thermal exposure, B: temperature.

**Figure 3.** *Cont*.

**Figure 3.** Effects of mild thermal exposure (50–80 ◦C) for extended times of thermal exposure (5–30 min). Contour plots of dry weight (**a**), percent successful completion of molt to 2nd instar (**b**) and percent successful completion of molt to 3rd instar (**c**), and percent survival (**d**). Western corn rootworm larvae were reared on WCRMO-2 diet for 10 days. Color bars display the magnitude of the measured responses.

Models for weight, molt to 2nd instar, and molt to 3rd instar revealed that temperature had significant effects on these measured responses, while no significant effect on weight and molt was observed due to time of thermal exposure. *p* values of temperature were 0.0012, 0.0224, and 0.0051 in the models for weight, molt to 2nd instar, and molt to 3rd instar, respectively (Table 2). Non-linear effects of temperature on weight, molt to 2nd instar, and molt to 3rd instar were found (Figure 4). WCRMO-2 diet prepared a temperature of approximately 60 ◦C yielded the maximum larval weight and percent molt, whereas there were significantly negative effects on weight and molting when WCRMO-2 diet was produced at temperatures above 75 ◦C (Figures 3a–c and 4).

**Figure 4.** *Cont*.

**Figure 4.** Nonlinear effects of mild thermal exposure (50–80 ◦C) for 15 min on dry weight (**a**), percent successful completion of molt to 2nd instar (**b**), and percent successful completion of molt to 3rd instar (**c**). Western corn rootworm larvae reared on WCRMO-2 diet for 10 days.

A model for survival indicated marginally significant effects of time exposure (*p* = 0.0582) and the interaction between thermal exposure and time exposure (*p* = 0.0596). However, larval survivorship on all diet treatments was >95% (Figure 3d). A similar pattern was found in the experiment with high thermal exposure and short time of thermal exposure (Figure 2c). Consequently, survival was not considered as an important criterion for evaluation of the effects of the treatments.

#### *4.3. Contamination*

All experiments had minor contamination (<3%) except for diet treatments that experienced a soft-set of the media that were excluded from the analyses. No evidence for a relationship between contamination and two experimental factors (thermal exposure and time of thermal exposure) was determined. Similarly low contamination rates were observed previously [27].

#### **5. Discussion**

Published WCR diets require antibiotics as diet preservatives to alleviate bacterial contamination [24,25,27]. However, ingestion of antibiotics results in changes in WCR gut microbiota [31], thereby possibly interfering with the determination of the susceptibility of WCR to insecticide toxins using diet bioassays. The availability of a WCR diet free of antibiotics would facilitate research programs of this important pest. In diet processing, heating is required as one of the most important parts for activating gelling agents (e.g., agar) to stabilize diets and promote the form of suitable textures for insect feeding. This can be also used as an extremely effective means for destroying microbial contaminants derived from diet ingredients and preparation [34]. To further the goal of developing a diet free of antibiotics for WCR, we explored the effects of the thermal exposure from 50–141 ◦C for the short time (<3 s) and extended time (10–30 min) on the quality of WCRMO-2 diet based on life history parameters of WCR larvae fed on the treated diets. By using geometric and mathematical approaches, we further characterized the influence of both thermal exposure and time of thermal exposure, allowing determination of the optimum conditions (temperature and time) for making WCRMO-2 diet.

Our results indicated that the exposure to the high thermal exposure (110–141 ◦C) for brief intervals (0.9–2.3 s) caused detrimental effects on the performance of larvae on

WCRMO-2 diet, indicating a possible reduction in nutrients required for WCR growth and development due to the high thermal exposure even if constrained for the short duration. WCR larvae fed on the treated diets exhibited significant reductions in weight and molting compared to those fed on the control diet made at the mild temperature of 65 ◦C for 10 min, whereas no difference in larval survivorship was observed between the diet treatments and the control diet. Heating is known to provide many benefits, including destruction of microbial contaminants, activation of gelling agents, increasing protein digestibility, denaturation of digestive inhibitors and harmful enzymes (e.g., phenol oxidases, lipo-oxygenase), increasing flavor, and acceleration of desirable chemical reactions [34]. However, severe overheating can cause reduction of protein digestibility, nutrient destruction (e.g., ascorbic acid, unsaturated lipid), destruction of vitamins, and formation of complexes (sugar amino acid products) [41]. A similar pattern of negative effects of heating on the quality of insect diets was previously reported for a diet of *Chrysoperla carnea* Stephens (Neuroptera: Chrysopidae) [42]. Initially, Vanderzant [43] developed a liquid diet for *C. canea* larvae that contains soy and casein hydrolysates as the main protein sources along with fructose, vitamins (ascorbic acid, B-vitamins), and other diet ingredients. This formulation was successfully used for the production of 18 generations of *C. canea*, but it did not promote the growth of *C. canea* after autoclaving at 121 ◦C [42]. The author successfully developed an improved diet that can be autoclaved by removing ascorbic acid (a heat-sensitive ingredient), replacing fructose with sucrose, and adding yeast hydrolysate and casein. These substitutions effectively compensated the loss of heat-sensitive nutrients. Only minor difference in percent adult recovery from larvae between the improved diet with and without autoclaving, which was 39% and 34%, respectively, was observed [42]. However, there was approximately 2-fold reduction in the percentage of adult recovery from larvae when *C. canea* larvae were reared on the standard diet [43] compared with the heat sterilized diet [42]. Griffin et al. [44] compared percent adult recovery from larvae of the boll weevils, *Anthonomus grandis* Boheman (Coleoptera: Curculionidae), reared on artificial diets that were made high temperatures of 130 ◦C, 138 ◦C, 144 ◦C, and 151 ◦C for 30 s using a flash-type sterilizer. They found that no significant effects of these temperatures on the percentage of adult recovery from larvae, though the highest temperature of 151 ◦C yielded fewer and smaller weevils than other temperatures. Our ongoing efforts to develop a heat-sterilized diet for WCR facilitating fewer or no antibiotics focus on the identification of alternative ingredients (e.g., yeast hydrolysate, casein) that can be compensated for the loss of nutrition in WCRMO-2 diet due to the overheating.

With diet ingredients in WCRMO-2 formulation, we demonstrated that a temperature of 60 ◦C yielded the highest larval performance on WCRMO-2 diet, while a time exposure of less than 30 min did not have significant impacts on the quality of WCRMO-2 diet. The exposure of WCRMO-2 diet to temperatures below 55 ◦C or above 65 ◦C resulted in a reduction in larval weight and molting compared to WCRMO-2 diet made at the optimum temperature. The significant losses in nutrients needed for WCR larval growth and development were observed when the WCRMO-2 diet was heated over 75 ◦C. Although most insect diets are often made at temperatures of 65–70 ◦C [24] that are likely used to avoid the loss of nutrition of heat-sensitive ingredients (e.g., ascorbic acid, vitamins, lipids), no information on the effects of relative low temperatures (50–80 ◦C) on insect diets is available. This study adds to the limited number of studies characterizing the effects of thermal exposure and time of thermal exposure, especially at mild temperatures, on insect diets. Some insect diets that have been reported to be heat-tolerant are usually produced at temperatures of 121 ◦C for 15–20 min by autoclaving or 141 ◦C for a few seconds by flash sterilizing [37,44]. It is noteworthy that WCR is nearly monophagous on maize roots and can survive on a few grass species [45]. This pest may require specific nutrients, and their bioavailability in WCRMO-2 diet is significantly reduced when the diet is exposed to temperatures over 75 ◦C.

Diet assays determining susceptibility of corn rootworm larvae typically utilizes a 96-well microtiter plate format [46–48]. Typically, each well is filled with 200 μL of diet and overlaid with toxins and followed by an infestation of a single neonate larva and sealing the well. This assay involves a labor-intensive filling process. A high-throughput system designed to run and analyze assays on a large scale, factorially increasing the number of compounds screened in the case of discovery, or the number of assays completed in the case of insect resistance management (IRM) studies would greatly facilitate research programs of this important pest. Research with lepidopteran insects has shown the great value of high-throughput systems for mass production and diet bioassays. In fact, utilizing a flash sterilizer coupled to a form-fill seal machine allowed the mass production of 3 million corn earworm pupae, *Helicoverpa zea* (Boddie) (Lepidoptera: Noctuidae), annually [49]. More recently, the high-throughput system can produce 25,000 rearing units per hour containing artificial diet and eggs of *Trichoplusia ni* (Hubner) (Lepidoptera: Noctuidae) through the use of automation [37]. For these systems, in addition to a flash sterilizer and a form-fill seal machine, a heat-sterilized diet is one of the key components. WCR has proven to be one of the most challenging pests in North America [50]. Many management strategies have been developed for controlling WCR (crop rotation, soil insecticides, Bt maize), but this pest has evaded nearly all management tactics in recent years [10–17]. Recently, WCR has been reported to evolve resistance to the newest management technology, RNA interference (RNAi) [51]. Future research could aim to leverage automation and robotics technology to establish the high-throughput system for WCR that would accelerate discovery efforts related to novel insecticide compounds and their related products.

**Author Contributions:** M.P.H. contributed to writing the first draft of the manuscript. M.P.H., M.G.V., T.A.C. and B.E.H. conceptualized the study. M.P.H., M.G.V. and B.E.H. developed the methodology. M.P.H., A.E.P. and R.W.G. performed the experiments. M.P.H. collected data, performed the analyses, and provided the visualization. M.G.V., K.S.S. and B.E.H. provided materials required for the experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** Research for this study, in part, was funded by the Small Business Innovation Research (SBIR) Program at the National Institute for Food and Agriculture (NIFA) (grant number 2020-33610-31703).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank Julie Barry, Amanda Ernwall, Michelle Gregory at USDA-ARS (Columbia, Missouri), and David Tague at University of Missouri for technical assistance. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA or the University of Missouri.

**Conflicts of Interest:** Michael Vella is an employee of Frontier Scientific Services, Newark, Delaware. All other authors declare no competing interests.

#### **References**

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ISBN 978-3-0365-2493-1


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The authors would like to thank the Distributed European Infrastructure for Supercomputing Applications (DEISA) for allowing of computational resources under the Extreme Computing Initiative (BIOGLASS Project). CINECA computing centre is also kindly acknowledged.

Colleagues V. Bolis (Dip. DiSCAFF, University of Eastern Piedmont) and G. Martra (Dip. Chimica IFM, University of Torino) are acknowledged for fruitful discussion and for providing the HA samples, synthesized and kindly supplied by ISTEC-CNR (Faenza, Italy).

R. Dovesi, B. Civalleri and the CRYSTAL team (Dip. Chimica IFM, University of Torino) are thanked for discussion and continuous support with the use of the code.

Part of the results on carbonated HA has been obtained by G. Ulian during his Master Degree Thesis, entitled "DFT study of carbonated defect in hydroxyapatite", 2010, University of Torino.

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**15**

*1,2USA 3Turkey*

**The Use of Vibration Principles to Characterize**

**the Mechanical Properties of Biomaterials**

*Whistler Carbohydrate Research Center, Purdue University, West Lafayette, IN*

*3Department of Food Engineering, Middle East Technical University, Ankara*

Mechanical properties are a primary quality factor in many materials ranging from liquids to solids including foods, cosmetics, certain pharmaceuticals, paints, inks, polymer solutions, to name a few. The mechanical properties of these products are important because they could be related to either a quality attribute or a functional requirement. Thus, there is always a need for the development of testing methods capable to meet various material

There is a wide range of mechanical tests in the market with a wide price range. However, there is an increasing interest in finding new methods for mechanical characterization of materials specifically capable to be adapted to in-line instruments. Acoustic/vibration methods have gained considerable attention and several instruments designed and built in government labs (e.g. Pacific Northwest National Laboratory and Argon National

To measure mechanical properties of material a number of conventional techniques are available, which in some cases may alter or change the sample during testing (destructive testing). In other tests the strains/deformations applied are so small that the test can be considered non-destructive. Both types of test are based on the application of a controlled strain and the measurement of the resulting stress, or viceversa. Different types of

Depending on the type of material, different conventional techniques utilized to measure its mechanical properties can be grouped as viscosity measurement tests (liquid properties), viscoelasticity measurement tests (semiliquid/semisolid properties), and elastic

Acoustics based techniques can be used for all types of material and the following sections discuss in detail how these techniques have been adapted and used to measure materials whose properties range from liquids to solids. Some of the applications discussed in this chapter are based on the basic impedance tube technique. Applications of this technique for

characterization requirements from both the industry and basic research.

deformations, e.g. compression, shear, torsion are used to test these materials.

Laboratory) have been made commercially available.

measurement tests (solid properties).

**1. Introduction**

Osvaldo H. Campanella1, Hartono Sumali2,

*2Sandia National Laboratories, Albuquerque, NM*

*1Agricultural and Biological Engineering Department and*

Behic Mert3 and Bhavesh Patel1