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ABSTRACT Little is known about what factors influence the climbing ability of bed bugs, Cimex lectu- larius L. (Hemiptera: Cimicidae), in relation to the various ...
BEHAVIOR, CHEMICAL ECOLOGY

Climbing Ability of the Common Bed Bug (Hemiptera: Cimicidae) B. A. HOTTEL,1,2 R. M. PEREIRA,1 S. A. GEZAN,3 R. QING,4 W. M. SIGMUND,4 AND P. G. KOEHLER1

J. Med. Entomol. 52(3): 289–295 (2015); DOI: 10.1093/jme/tjv012

ABSTRACT Little is known about what factors influence the climbing ability of bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae), in relation to the various surfaces they encounter. We examined how sex, time since last fed, and what surfaces the bed bugs were in contact with affected their climbing performance. The effects of sex and time since fed were tested by counting the number of bed bugs able to climb a 45 slope. The pulling force was recorded using an analytical balance technique that captured the sequential vertical pulling force output of bed bugs attached to various surfaces. Recently fed female bed bugs were found to have the most difficulty in climbing smooth surfaces in comparison with males. This difference can be explained by the larger weight gained from bloodmeals by female bed bugs. A variety of vertical pulling forces were observed on surfaces ranging from sandpaper to talc powder-covered glass. For surfaces not treated with talc powder, bed bugs generated the least amount of vertical pulling force from synthetically created 0.6-mm plastron surfaces. This vast range in the ability of bed bugs to grip onto various surfaces may have implications on limiting bed bugs dispersal and hitchhiking behaviors. KEY WORDS bed bug, Cimex lectularius, locomotion, climbing ability In comparison with head lice, Pediculus humanus capitus De Geer (Phithiraptera: Pediculidae), which spend their entire lives on a host, bed bugs, Cimex lectularius L. (Hemiptera: Cimicidae), only contact the host to feed (Lehane 2005). While bed bugs are not feeding, they spend the rest of their time in harborages surrounding the location of where the host rests (Boase 2001). This temporal behavior causes bed bugs to navigate over a variety of surfaces when traveling from harborage to host and vice versa. There are a few specific morphological features in bed bug legs that aid them in navigating over various surfaces they encounter. Two simple claws are present at the end of the tarsus (Usinger 1966). In other insects, these claws are specialized in gaining traction on rough surfaces (Bullock and Federle 2011). There is also a hairy pad attachment structure called the fossula spongiosa found on the distal end of the tibia. This structure is proposed to aid locomotion in kissing bugs (Hemiptera: Reduiviidae) by enabling the adults to climb smooth surfaces (Gillett and Wigglesworth 1932, Wigglesworth 1938, Weirauch 2007); however, the fossula spongiosa was shown to not aid in the smooth

surface climbing ability of bed bugs on glass (Wigglesworth 1938). Along with glass, there are a few other surfaces that bed bugs encounter that may be difficult for them to climb. Pitfall traps of various designs have recently been found to be particularly effective at detecting and monitoring bed bugs (Wang et al. 2009a,b, 2011; Anderson et al. 2009; Singh et al. 2013). These traps rely on the inability of bed bugs to climb the smoothwalled moats to entrap wandering bed bugs. Despite the practical use of smooth surfaces for bed bug management, the importance of surface characteristics on bed bug climbing ability has not been quantified. The objectives of our study were to: 1) examine the differences in climbing ability and vertical pulling force between fed and starved male and female bed bugs, and 2) observe the differences in vertical pulling force generated by bed bugs on surfaces hypothesized to be difficult to climb. Glass, commercial bed bug pitfall trap walls, a synthetically derived surface, and two different grades of sandpaper were tested. Materials and Methods

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Department of Entomology and Nematology, University of Florida, 1881 Natural Area Dr., Gainesville, FL 32611. Corresponding author, e-mail: [email protected]. 3 School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611. 4 Department of Materials Science and Engineering, University of Florida, Gainesville, FL 32611. 2

Test Insects. A lab strain of C. lectularius collected in 1973 from Ft. Dix, N.J. was used for all experiments. Bed bug colonies were kept in screw-top lidded 300-ml plastic jars (Mold-Rite Plastics, Plattsburgh, NY). Folded filter paper (Fisher Scientific, Pittsburg, PA) was added as harborage. Jar lids were modified to allow colonies to feed while being contained by cutting a 7.5-cm-diameter hole in the center of the lids and

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Fig. 1. General procedure of creating super hydrophobic plastron hairy surfaces. Reprinted (adapted) with permission from (Hsu and Sigmund 2010). Copyright (2014) American Chemical Society.

placing a 90-mm nylon mesh (Amazon Supply, Seattle, WA) over the opening. Bed bugs were fed on live chickens by inverting the jar and placing it under the wing (University of Florida Institutional Animal Care and Use Committee #201303836_01). Jared colonies were fed once a week. Colonies were kept at a temperature of around 24 C and exposed to natural light cycles. Surfaces Tested. Surfaces tested included glass microscope slides (Premium Microsope Slides Plain; Fisher Scientific, Pittsburgh, PA), glass microscope slides with a talc powder application (Johnson’s baby powder; Johnson & Johnson Consumer Companies Inc., Skillman, NJ), P600 grit sand paper (Imperial Wetordry Sandpaper; 3 M, St. Paul, MN), and P60 grit sand paper (Aluminum Oxide Sandpaper; 3 M, St. Paul, MN), and commercial bed bug trap surfaces including ClimbUp insect interceptor smooth surface inner walls (Susan McKnight Inc, Memphis, TN), ClimbUp insect interceptor inner walls with talc powder, Verifi bed bug detector smooth surface inner pitfall walls (FMC Professional Solutions, Philidelphia, PA), and BlackOut bed bug detector smooth surface inner walls (Protect-A-Bed, Wheeling, IL). Microscope slides were reused for each experiment and were cleaned using a 70% aqueous solution of ethanol. Other surfaces were disposed of and replaced after all available area was used once. Super-hydrophobic plastron hairy surfaces were also included in the experiment (Hsu and Sigmund 2010). These were prepared using a template molding technique. A track-etched produced polycarbonate (PC) membrane (ISOPORE, Millipore Inc, Billerica, MA) with two different pore sizes (diameter ¼ 0.6 mm and 3.0 mm) was chosen for our experiment. Polypropylene (PP) plastic sheets (File jacket No. 85781, SMEAD Co.) were used as the substrate. The PP plastic sheets were cut into 1-cm2 squares and cleaned with a high-pressure blower to remove any surface dusts. Together with the PC membrane, the two plastic pieces were placed between two glass slides and fixed using four binder clips to apply pressure. A vacuum oven (VO914A, Lindberg/Blue M; Thermo Electron Corporation, Asheville, NC) was heated to 190 C beforehand for the casting process. Vacuum pressure 1 kPa was applied on samples during the heat treatment to remove air in pores. The whole casting process took 10 min. After the heat treatment, surfaces were removed and cooled down to room temperature and the PC membrane was directly peeled off from the substrate (Fig. 1). Pulling Force on Test Surfaces. The amount of force generated from the parallel contact of adult male

Fig. 2. Setup of pulling force experiment. A tethered bed bug, C. lectularius, mounted to a piece of clay was placed onto an analytical balance. The balance was zeroed and a test surface was brought into contact with the bed bug’s legs. The sequential change in mass was recorded onto a computer for 5 min.

and female bed bugs on different surfaces were quantified using a procedure described previously (Betz 2002; Fig. 2). All surfaces were mounted vertically to a wooden platform. Sandpaper, commercial trap walls, and plastron surfaces were mounted onto a microscope slide before being attached to the wooden platform. Individual C. lectularius specimens were tethered to 4-cm-long polyester paintbrush bristles (Great American Marketing, Valencia, CA). Tethering was accomplished by gluing (Loctite Super Glue; Henkel Corporation, Rocky Hill, CT) one tip of a bristle onto the first and second abdominal segments of a specimen. The other end of the bristle extended beyond the posterior end of the insect and the loose end of the bristle was inserted 1 cm deep into a 25.3 g ball of modeling clay. The tethered bed bug was then placed onto an analytical balance (Model GA 200-D; OHAUSE, Parsippany, NJ) that was tarred to zero. One of the test surfaces was slowly moved into contact with all six tarsi of the tethered bed bug (Fig. 2). Once the tarsi contacted the surface, changes in mass on the balance were recorded on a computer for 5 min using data acquisition software (Advance Serial Port Monitor; AGG Software, Seatle, WA). A mass reading was taken an average of 2.5 times per second. The data were then converted from mass to force measured in millinewtons (mN) using the formula: F ¼ ma (where F ¼ force (mN), m ¼ mass (g), and a ¼ acceleration (m2/s)). The acceleration due to gravity (  9.81 m2/s) was used. Response variables taken from each bed bug include the maximum force and the mean force. In addition, the sum of the amount of force over the change in time for each reading, also known as sum of the impulses, was recorded. The mean force of the top three peaks generated and the total number of peaks observed by

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each bed bug was also recorded. The mass of each bed bug was also recorded. The pulling force of 10 different male and female bed bugs, which were fed three days before the experiment, were tested on microscope glass slides, 60 grit sandpaper, and 600 grit sandpaper. Only males were tested on glass microscope slides covered in talc powder, ClimbUp insect interceptor trap walls, ClimbUp insect interceptor trap walls covered in talc powder, BlackOut trap walls, Verifi trap walls, 0.6-mm plastron surface, and 3.0-mm plastron surface. Individuals were not reused in any of these experiments. Readings for the top three pulling force peaks per bed bug were not included due to the lack of peaks on surfaces with very low force readings (ClimbUp þ Talc, Glass þ Talc, and 0.6-mm plastron surface). Ability to Climb Glass. The ability of bed bugs to climb smooth surfaces was tested on glass microscope slides attached to wood blocks cut at a 45 angle (Fig. 3). In order to keep specimens on a linear path while climbing the glass, each specimen was tethered with a 4-cm-polyester paintbrush bristle that was inserted into a plastic straw glued to the center a microscope slide. Bed bugs were tethered by gluing one bristle end to the center of the abdomen with the other end of the bristle extending over the anterior end of the insect. The tethered bed bug was then placed 3 cm away from the stationary straw. The bristle was inserted into the straw (Fig. 3). Bed bugs were allowed 5 min to reach the straw. A success was recorded if the bed bug could reach the straw. Bed bugs that fell while climbing were placed back to the original starting position and those that were immobile were stimulated by a frayed paintbrush bristle stroke on the abdomen. Bed bugs used in this experiments were fed either 4, 72, and 336 h before being tested. In order to evaluate the effects of sex and postfeeding time on climbing ability 15 C. lectularius males and 15 females were tested at each postfeeding time. Statistical Analyses. Principal component analysis (PCA) was used to combine correlated response variables for male and female experiments on 60 grit sandpaper, 600 grit sandpaper, and glass surfaces. The response variables used in the PCA included the maximum pulling force, mean pulling force, impulse, and

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the total number of peaks generated by each bed bug. A scree plot was used to determine the appropriate number of principal components to retain for further analysis. Principal component scores created from these response variables were used to judge if there was a difference between male and female bed bug pulling force output. Principal component scores for male and female bed bugs were analyzed by Hotelling T-squared test at a ¼ 0.05. Response variables were log-transformed and then Z-score standardized to meet normality and equal variance assumptions before analysis. PCA was also performed on the maximum pulling force, mean pulling force, impulse, and the total number of peak readings for surfaces tested on male bed bugs alone. Surfaces tested on male bed bugs included microscope glass slides, microscope glass slides covered in talc powder, ClimbUp walls, ClimbUp walls with talc powder, 0.6-mm plastron surfaces, 3.0-mm plastron surfaces, BlackOut walls, and Verifi walls. Pulling force data on surface treatment tests were log-transformed to meet normality assumptions in later analysis. Z-score standardization was applied to the log-transformed data before the PCA. A scree plot was used to determine the number of principal components adequate to explain the variation in the data. A biplot of the PCA was also produced to aid in the interpretation of the PCA results as described in Kohler and Luniak (2005). Principal component factor scores grouped by test surfaces were analyzed by a multivariate analysis of variance (MANOVA) using Wilk’s lambda (k) at a ¼ 0.05. Hotelling’s T-squared statistic was used for nine planned contrast between test surface groups at a ¼ 0.05. A Dunn–Sidak correction of a at 0.006 was used on all significant comparisons to control for Type I errors. Glass angle climbing results were analyzed using Fisher’s exact test with calculated mid P-value (Agresti 2007). The climbing success at different feeding times within each sex was compared. The climbing success for each sex at each postfeeding time was also compared. We hypothesized that there was a positive relationship between the time since fed and climbing success (H0 : h ¼ 1; Ha : h > 1Þ and between climbing success and sex ðH0 : h ¼ 1; H a : h > 1Þ. Here, h

p1=ð1p1Þ represents the odds ratio p2=ð1p2Þ , where p1 and p2 equal the probabilities of success of row 1 and row 2 in a 2  2 contingency table respectively (Agresti 2007). A two-factor ANOVA model was used to analyze the effects of sex and the time after feeding on bed bug weight. Weight was log-transformed to meet normality assumptions. R statistical software was used to analyze data (R Development Core Team 2012) together with the following R packages: ggplot2, vegan, ca, MASS, ade4, and candisc.

Results Fig. 3. Setup of glass climbing experiment. A tethered bed bug, C. lectularius, was placed onto a glass microscope slide at a 45 angle. The plastic bristle, used to tether the bed bug, was inserted into a plastic straw to guide the bed bug.

Pulling Force of Test Surfaces. A sample output of one of the vertical pulling force readings can be viewed in Fig. 4. The PCA yielded two components

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Fig. 4. Sample force reading of a male bed bug, C. lectularius, clinging vertically to glass. The maximum peak in the center, the area under the graph (impulse), the top three peaks (T1, T2, and T3), and the average of the total positive force were used in the PCA.

(PC1 and PC2) that collectively explained 90.9% of the total variance for the results of the male and female readings on 60 grit sand paper, 600 grit sandpaper, and glass. We did not observe a statistically significant difference between male and female bed bugs concerning their pulling force output represented by PC1 and PC2 (Hotelling’s T-squared ¼ 0.034, approximate F2,57 ¼ 0.971, P ¼ 0.385). Only males were tested on additional surfaces because of the lack of statistical significance found between the pulling force output of male and female bed bugs. PCA on surfaces tested with male bed bugs also generated two components (PC1 and PC2) consisting of the mean force, max force, impulse, and the total number of peak readings per bed bug. PC1 and PC2 collectively explained 92.4% of the total variance in the data. In a biplot, the correlation of the response variables is approximated by the cosine of the angle made between each arrow. Individual force output observations in the opposite direction of a response variable arrow represent lower values and vice versa. A strong positive correlation of the max force, mean force, and impulse response was found in the biplot of the PCA (Fig. 5). The peak number of counts was also positively correlated with the other response variables but the relationship was not as strong. The mean force, max force, and impulse vectors oriented farther from PC1 origin of the biplot than the PC2 origin. This indicated that these response variables carried more weight on PC1 than PC2. Because force vectors are heavily weighted in

PC1, PC1 could be considered a summation of the pulling force readings. The count peak vector, however, oriented farther from the PC1 origin than the PC2 origin and therefore carried more weight on PC2. PC2 could be termed as the climbing performance explained by the peak counts. The MANOVA yielded a statistically significant difference among the surfaces tested in relation to the principal component factor scores of PC1 and PC2 (Wilk’s k ¼ 0.248, approximate F18,176 ¼ 9.842, P < 0.001; Fig. 6). On examination of the roughest surfaces, 60 and 600 grit sandpaper, PC1 and PC2 factor scores found that there was not a statistically significant difference between the two surfaces (Hotelling’s T-squared ¼ 0.44316, approximate F2,17 ¼ 3.767, P ¼ 0.044). Differences were also not found between comparisons of glass and any of the smooth plastic commercial trap surfaces (Verifi P ¼ 0.583, Blackout P ¼ 0.927, and ClimbUp P ¼ 0.784). The addition of talc to glass elicited a significant difference when compared with plain glass (Hotelling’s T-squared ¼ 3.590, approximate F2,17 ¼ 30.52, P < 0.001). Similarly, adding talc to a ClimbUp trap surface also resulted in a statistically significant difference when compared with a plain ClimbUp trap surface (Hotelling’s T-squared ¼ 6.505, approximate F2,17 ¼ 55.30, P < 0.001). There was a surprisingly large difference between the two plastron surfaces (Hotelling’s T-squared ¼ 4.889, approximate F2,17 ¼ 37.509, P < 0.001). Plastron 0.6-mm surface was not found to be statistically different from the glass

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Fig. 5. A PCA biplot displaying the relationships of the response variables (black arrows) and individual observations of male bed bug, C. lectularius, vertical pulling force output on various surfaces tested. Individual surface observations are distinguished by grey letters: A ¼ P60 grit sandpaper, B ¼ P600 grit sandpaper, C ¼ Blackout trap inner wall, D ¼ ClimbUp trap inner wall, E ¼ Glass, F ¼ 0.6-diameter plastron surface, G ¼ 3.0-diameter plastron surface, H ¼ Verifi trap inner wall, I ¼ ClimbUp trap inner wall þ talc powder, and J ¼ Glass þ Talc powder. The X and Y axes represent the principal component (PC) factor loadings and the secondary X and Y axes represent the PC factor scores. Principal component 1 (PC1, 78%) and principal component 2 (PC2, 14%) collectively explain 92% of the variation in the data set.

surface treated with talc when using a Dunn–Sidak correction of 0.006 (Hotelling’s T-squared ¼ 0.7675, approximate F2,17 ¼ 6.524, P ¼ 0.008) but a statistically significant difference was found when compared with plain glass (Hotelling’s T-squared ¼ 2.133, approximate F2,17 ¼ 18.13, P < 0.001). Ability of Bed Bugs to Climb Glass. Both male and female bed bugs climbed the 45 glass slope (Table 1). An association between the time after feeding and climbing success on glass could not be found in male bed bugs (P > 0.05). Significantly fewer female bed bugs were able to climb 45 glass four hours after feeding than 336 hours after feeding (P ¼ 0.042). All other comparisons of climbing success were not statistically significant (P > 0.05). When examining the associations of sex and climbing success for each time after feeding, a significant difference was only found between male and female bed bugs that had fed within 4 h (P ¼ 0.009). At this time after feeding, male bed bugs climbed better than female bed bugs. Sex, time after

feeding, and the interaction between sex and time after feeding were all significant in the ANOVA analysis on P < 0.001; bed bug weight (F1,66 ¼ 63.57, F2,66 ¼ 133.80, P < 0.001; F2,66 ¼ 13.49, P < 0.001). Mean female bed bug weight was higher than mean male weight at 4 and 72 h after feeding but not at 336 h after feeding. Female to male weight ratios for 4, 72, and 336 h after feed were 1.580, 1.594, and 1.042, respectively. Discussion Similarly designed experiments measuring the pulling force of arthropods only analyzed the maximum pulling force readings the arthropods produced on a given surface (Betz 2002, Heethoff and Koehrner 2007). The maximum pulling force readings are extreme values and could be viewed as outliers in the data. We hypothesized that these values represented an overestimate of the actual climbing ability of a bed bug.

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Fig. 6. Male bed bug, C. lectularis, median principal component 1 factor scores (bold black lines) for each surface tested. The upper and lower portions of each box represent the 1st (Q1) and 3rd (Q3) quartile of the data. The notches are calculated by the formula: þ/1.58 x (Q3-Q1) / Hn. Empty circles represent data outliers. Higher scores represent a lesser ability of the bed bugs to grip a given surface. Table 1. Climbing success of bed bugs, C. lectularius, on glass at a 45 angle Gender Male Female

Time after fed (h)

Weight (g)

Success (%)

4 72 336 4 72 336

0.008b 0.006c 0.005c 0.020a 0.009b 0.005c

100a 100a 100a 58b 75ab 92a

Data were analyzed using a Fisher’s exact test. Factors with the same letters are not significant at P < 0.05. n ¼ 12.

Therefore, a macro examination of the pulling force readings (maximum force, average force, impulse, average top three peak forces, and the number of peak counts) was used to more accurately represent the true pulling force performance of a specimen on a test surface. The maximum force, impulse, and average force were highly correlated with each other as seen in the biplot. The number of peak counts, however, was not found to be as highly correlated with the other response variables and can be seen as a dominant factor in PC2. Given these findings, measurements of maximum force alone are not adequate enough to portray the true performance of a bed bug pulling on a vertical surface. Although female bed bugs have been observed to be only 17% larger than males, females will ingest over twice the amount of blood as males do (Araujo et al. 2009). In our experiments females and males performed equally well on glass vertical pulling force trials, but recently fed males performed better than females at climbing a glass slope. Because females took a disproportionately larger bloodmeal than males, the added weight from a larger bloodmeal was likely the cause of the reduced female climbing ability. All of the recent commercially available bed bug pitfall traps and traps used in research experiments have been made of plastic (Wang et al. 2009b, Anderson

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et al. 2009, Singh et al. 2013) whereas historically glass was a preferred surface for traps. Glass was observed to be superior to plastic in preventing the escape of epigaeic beetles from pitfall traps (Luff 1975) and catching more stored product beetles (Obeng-Ofori 1993). Surprisingly there was not a statistically significant difference in the pulling force readings (PC1) and the number of peak counts (PC2) among glass and the commercial plastic trap surfaces tested (P ¼ 0.784). Given these results, it maybe equally difficult for bed bug to escape from a plastic and glass trap. The addition of a powder or dust to reduce the ability of an insect to grip a surface has been demonstrated in aphids (Lees and Hardie 1988). Talc powder is sometimes applied on bed bug pitfall traps as an additional measure to prevent bed bugs from escaping (Wang et al. 2009a, 2013); however, regular talc powder reapplication is necessary to ensure continued bed bug entrapment (Wang and Cooper 2011). Talc powder greatly reduced the pulling force readings and the number of peak counts on glass surfaces and ClimbUp trap inner wall. Applications of talc powder to other smooth plastic surfaces would also likely reduce the ability of bed bugs to grip these surfaces. There has been some research on the use of synthetic plant-derived hooked trichome stalks to trap bed bugs. Although bean leaf trichomes have been shown to entrap bed bugs, synthetic trichomes only hindered bed bug movement temporarily (Szyndler et al. 2013). We examined the use of hair-like fibers much smaller than synthetic hooked trichomes to prevent bed bug movement. Instead of entangling bed bugs’ legs in these fibers, the 0.6-mm plastron surface reduced the ability of bed bug tarsi to grip the contacted surface. The reason for the superior performance of 0.6-mm over 3.0-mm plastron surfaces is likely because the 0.6mm fibers are smaller in diameter and less dense than the 3.0-mm fibers (Hsu and Sigmund 2010). Super hydrophobic plastron surfaces with 0.6-mm diameter fibers were not found to be equal in performance to the talc powder-treated smooth surfaces, but were found to greatly reduce bed bug vertical pulling force when compared with glass. Because of their enhanced ability to reduce bed bugs’ grip while clinging onto a surface without the added maintenance of applying talc powder, 0.6-mm plastron surfaces may be of particular interest for bed bug pitfall trap design. Bed bugs encounter a variety different objects, surfaces, and materials while on their quests for food or harborages. Different factors can affect the ability of bed bugs to navigate over objects. We found that feeding status, sex, and climbing surface are important factors affecting bed bugs’ movement. The ability or inability of bed bugs to climb these surfaces could have influence on the distribution and dispersal of bed bugs in the environment, and therefore, on their monitoring and control through the use of traps. Bed bugs are unlikely to be found in areas where they are unable to climb. Based on this research, plastic materials that resist bed bug movement could be manufactured to make items, such as mattress covers or suitcases, more resistant to harboring bed bugs.

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HOTTEL ET AL.: Cimex lectularius CLIMBING ABILITY Acknowledgments

We would like to thank Seth McNeill for his assistance in setting up the analytical balance technique and Josh Westin for his help in editing figures. We would also like to thank Benjamin Baiser for looking over an earlier version of the manuscript. We would finally like to thank Susan McKnight Inc. for their financial contributions to the project.

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