TY - JOUR
AU - Tomberlin, Jeffery, K
AB - Abstract The black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), is mass produced worldwide for use in waste management and the production of an alternative protein for use as feed. However, few publications have explored its adult behavior, particularly mating, as a means to optimize egg production in colony. In addition, there is limited knowledge of appropriate marking techniques to explore the mating behavior of this insect. The goal of this study was to validate water-based acrylic paint pens as a possible marking tool for behavioral studies with adult black soldier flies. Adult black soldier flies (<24-h old) were marked with green, gold, red, or white acrylic paint. Adult longevity, as well as the number of matings (mating success) were recorded for each treatment. Regardless of marking, 1) adult females lived ~7 d, whereas males lived 9 d, 2) mating frequency peaked 2 d after initiation of the experiment, 3) approximately 30% of adults across treatments were recorded mating, and 4) egg production and hatch rate were not affected. This marking technique did not affect the parameters measured, suggesting that it is suitable for experiments requiring a reliable marking technique. Hermetia illucens, mate choice, lekking behavior, insect marking Developing an appropriate technique for marking insects in research can be a challenge as materials used can negatively impact the insect. Walker and Wineriter (1981) suggested that marking materials that are nontoxic, adhesive, quick-drying, lightweight, easy to apply, and easily visible are ideal for conducting behavioral, or mark-recapture studies with insects. Many mark-recapture studies for monitoring insect pest dispersal utilized fluorescent dusts (Hogsette 1984, Hagler and Jackson 2001, Coviella et al. 2006). Acrylic paints and genetic markers have also been widely used for monitoring dispersal of sterile insects released into the wild (Shearman et al. 2010, Gilchrist and Dominiak 2019). However, genetic markers, such as DNA microsatellites (Gilchrist and Dominiak 2019), can be cumbersome, time consuming, and expensive. Hence, cheaper and simpler method, such as acrylic paints, can be more practical. The black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), is a cosmopolitan species, well known for its potential for sustainable waste management (Banks et al. 2014, Miranda et al. 2019) and alternative feed for livestock (Newton et al. 1977, St-Hilaire et al. 2007). With the increased interest in the black soldier fly, particularly with mass production, some research has focused on the optimization of egg production (Booth and Sheppard 1984, Tomberlin and Sheppard 2002, Zhang et al. 2010, Zheng et al. 2013, Park et al. 2016, Hoc et al. 2019). Hermetia illucens displays a lekking behavior (Tingle et al. 1975, Tomberlin and Sheppard 2001), whereby males battle other males on host-plant territories with the ‘winning’ male obtaining mating privilege. Giunti et al. (2018) recently described male courtship behavior including male–male interactions and wing fanning. Mating frequency or choice, both of which can impact fertile egg production for use in industrialized settings, could be influenced by the lekking behavior. Consequently, understanding this behavior could allow for optimization of fertile egg production within a mass-rearing system. These studies could demand safe and efficient marking procedures that will not affect the fitness of the marked insects. However, a marking system has not been developed for use with the black soldier fly. This study evaluated the effect of acrylic paint as a marking technique for monitoring black soldier fly lekking behavior. Materials and Methods Acquisition of Flies Eggs collected from a black soldier fly colony maintained at the Forensic Laboratory for Investigative Entomological Sciences (F.L.I.E.S.) Facility at Texas A&M University (TAMU) in College Station, Texas, which was established in 2014, were used in these experiments. The source colony was originally established at the Coastal Plain Experiment Station, University of Georgia (Georgia), from material collected at a poultry facility in Bacon Co., Georgia, in 1998. At 0800 h, corrugated cardboard (8.8 × 5 × 1.2 cm) was placed on top of a screen lid attached to a [33.0 (L) × 21.6 (W) × 30.5 (D) cm] plastic box containing 50 black soldier fly neonates feeding in 100 g of the Gainesville house fly, Musca domestica, L. (Diptera: Muscidae) diet (Hogsette 1992) with 70% water content to attract ovipositing individuals (Tomberlin et al. 2002) in the colony. At 1700 h, resulting eggs were removed from the cardboard using a 21-cm VWR disposable spatula, and placed in a 100-ml cup (Frontier Agricultural Services) with a lid and stored in a walk-in growth chamber set at 29.8 ± 0.8°C with 65.0 ± 5.3% RH and 14:10 (L:D) h photo regime. Eggs were monitored every 12 h for eclosion, and ~10,000 resulting larvae were placed in a plastic 460-ml cup (Frontier Agricultural Services) containing 40 g of the Gainesville house fly diet with 70% water content, for 4 d (Sheppard et al. 2002). Experimental Design Black soldier fly larvae (4 d) were divided into four replicates of 500 larvae per 532-ml cup (Solo Company, Lake Forest, IL), and fed 54 g of the Gainesville house fly diet (Hogsette 1992) every day until larvae reached 40% pupation (Jones and Tomberlin 2019). Cups containing larvae and diet were covered with white tulle fabric (Wal-Mart Stores Inc., Rogers, AR), secured with a rubber band, and placed individually in the center of a plastic pan [33.0 (L) × 21.6 (W) × 30.5 (D) cm]. The plastic pans were then placed in a randomized complete block design on shelves in the walk-in growth chamber previously described. On the ninth day, the 532-ml cups were then emptied into [33.0 (L) × 21.6 (W) × 30.5 (D) cm] plastic containers, which were also covered with tulle fabric secured with elastic cord, and larvae were fed as described above. Prepupae were placed in separate [33.0 (L) × 21.6 (W) × 30.5 (D) cm] plastic containers covered with tulle fabric. Two hundred emergent flies (<24-h old) were sexed and 100 were marked on their thorax with one of the randomly assigned colors: red, gold, green, or white, using 3-mm tip Garde‘n’Craft Fine Point Markers (Uchida of America Corp, Torrance, CA). Emergent black soldier flies are docile, which makes handling easy (<1 min). During the marking procedure, flies were held between the index finger and thumb with elbows resting on hard surface for balance. Unmarked adults were held in the same way, but not marked. Males and females were placed in separate 30.0 × 30.0 × 30.0-cm cages (BioQuip Products, Compton, CA) until needed. Black soldier fly adult males emerge 2 d before females (Tomberlin and Sheppard 2002). Therefore, ages of males and females were mixed in order to reach equal sex ratio. Flies were provided with water ad libitum, as adults do not need to feed. Impact of Marking Flies on Mating The study was conducted from August 2017 to February 2018. Approximately 200 black soldier fly adults of 1:1 sex ratio were released in each of four 84 (L) × 84 (W) × 132 (H)-cm cages assigned to each color treatment (Insect-A-Hide pop-up shelter, Lee Valley Tools, Ltd., New York) inside of a greenhouse. The number of mating pairs for marked and unmarked individuals was recorded for 10 min every hour from 0800 to 1700 h over a 4-d period, which is the window for such behavior to occur (Tomberlin and Sheppard 2001). Cages were misted with water (10 ml per cage) every two hours during each observation period to enhance the longevity of the adult flies (Tomberlin et al. 2002). Relative humidity, temperature, and light intensity were recorded using a HOBO data logger model U12-012 (Onset Computer, Co., Bourne, MA). Temperature inside the cages was also recorded using Acu-Rite Thermometers (Lake Geneva, WI). Two trials were conducted for the following combinations in cages, 1) marked males and unmarked females, 2) marked females and unmarked males, or 3) both marked and unmarked males and females. Color assignments to cages were randomized during each trial. A cage containing flies receiving an assigned color marking was considered a replicate (n = 4). Impact of Marking Flies on Oviposition and Hatch Rate The study was conducted from March 2018 to May 2018. Methods utilized in marking flies to monitor mating frequency were applied as described above. Two treatments were examined in this study. To account for limited number of cages, adult males and females within a cage were marked with either green and white or red and gold on their thorax or remained unmarked in control cages. For each color combination, two replicates of each treatment and the control were used in each of two trials. Methods for harvesting eggs were based on those previously described by Tomberlin and Sheppard (2002). Following the initiation of the experiment, eggs were collected daily on days three through five which coincided with peak oviposition behavior by the black soldier fly (Tomberlin and Sheppard 2002). A corrugated cardboard block (5 (L) × 2.54 (W) × 1.27 (H) cm) was placed on top of a 400-ml plastic box containing 300 g Gainesville diet with 70% water content placed in the center of each cage. The corrugated cardboard block was replaced daily at 0700 h. Egg clutches present in the cardboard were removed using a 21-cm VWR disposable spatula and individually weighed on an Ohaus Scout Pro Balance Scale (Ohaus Corporation, Parsippany, NJ). In order to estimate the number of eggs gravimetrically (Booth and Sheppard 1984), 200 eggs were counted to determine a standard weight and calculated the total number of eggs laid during the experiment. Resulting eggs were kept in a 59-ml cup and placed in the walk-in growth chamber. Egg production by day was calculated to give total number of eggs produced. Hatch rate for each treatment was calculated by dividing the number of viable larvae hatched after 4 d by the number of eggs produced. Longevity Study The impact of this marking system on adult longevity was determined. Adults were obtained using the method described above. Methods for producing adults previously described were used. Fifteen of each initial emergent male and female flies (<24-h old) were assigned to each color treatment. The same number of adults remained unmarked and served as controls. Adults were placed individually in 59-ml cups, capped with a breathable lid, labeled, and placed in the walk-in growth chamber previously described. They were provided water ad libitum via a cotton ball placed through the lid (Tomberlin et al. 2002), and mortality was recorded daily. Statistics All statistical analyses were performed using R version 3.5.1. A Shapiro–Wilk test for normal distribution, and Levene Test for homogeneity of variance were implemented to test for normality assumptions, and the standard deviation was used to quantify variance. A paired t-test was used to compare number of mating pairs by marked and unmarked males or females. A repeated measures one-way analysis of variance (ANOVA) was used to compare number of mating pairs by marked and unmarked males and females. A two-way ANOVA was implemented to determine whether color and day influenced mate choice of marked and unmarked individuals. Oviposition data, including oviposition over time, did not meet normality assumptions; therefore a Kruskal–Wallis test was used to compare the number of eggs produced by marked females with unmarked ones. Pearson’s product–moment correlation analysis was used to determine level of correlation between mating frequency and egg production with temperature, RH, and light intensity. For the longevity study, parametric assumptions were met; therefore, an ANOVA was used to test for differences between colors and adult longevity. Alpha for all analyses was set at 0.05. Results Impact on Mating: Marked Males Only The temperature range inside cages was 20.9–32.2°C (mean 26.6°C ± 3.7). Greenhouse temperatures ranged from 19.8 to 35.0°C (mean 25.1°C ± 4.8); and RH from 47.7 to 60.6% (mean 52.5% ± 3.2). Mating frequency was not correlated to temperature (r = 0.1012, df = 58, P = 0.4416) or RH (r = 0.0291, df = 58, P = 0.8251), but it was positively correlated with light intensity (r = 0.3200, df = 58, P = 0.0126). Overall, there was no significant difference in mate choice by marked and unmarked males (t = 0.84615, df = 1, P = 0.5529; Table 1). Number of mating pairs did not differ per day between marked and unmarked males (F = 3.614, df = 1, P = 0.1300; Table 1). Additionally, color did not affect mating frequency between marked and unmarked males (F = 0.0380, df = 3, P = 0.9890; Table 1), and color*day was not significant (F = 0.289, df = 3, P = 0.8320; Table 1). Table 1. Total number of black soldier fly mating pairs ± SD (n = 4; n = 200; 100 ♂ 100 ♀) observed in all experiments where males were marked with a single color on their thorax Day . MM + UF ± SD . . . . UM + UF ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.50 3.00 ± 0.50 5.00 ± 0.50 3.00 ± 0.50 3.00 ± 0.06 2.00 ± 0.77 6.00 ± 0.50 4.00 ± 0.50 2 5.00 ± 0.50 7.00 ± 0.50 3.00 ± 2.00 6.00 ± 0.50 4.00 ± 0.77 6.00 ± 0.67 3.00 ± 0.50 5.00 ± 1.00 3 1.00 ± 0.50 1.00 ± 0.67 0.00 ± 0.50 1.00 ± 0.67 1.00 ± 1.27 1.00 ± 0.50 1.00 ± 0.50 1.00 ± 0.50 4b – – – – – – – – Total 8.00 ± 1.50 11.00 ± 1.67 8.00 ± 2.50 10.00 ± 1.67 8.00 ± 2.10 9.00 ± 1.94 10.00 ± 1.50 10.00 ± 2.00 Day . MM + UF ± SD . . . . UM + UF ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.50 3.00 ± 0.50 5.00 ± 0.50 3.00 ± 0.50 3.00 ± 0.06 2.00 ± 0.77 6.00 ± 0.50 4.00 ± 0.50 2 5.00 ± 0.50 7.00 ± 0.50 3.00 ± 2.00 6.00 ± 0.50 4.00 ± 0.77 6.00 ± 0.67 3.00 ± 0.50 5.00 ± 1.00 3 1.00 ± 0.50 1.00 ± 0.67 0.00 ± 0.50 1.00 ± 0.67 1.00 ± 1.27 1.00 ± 0.50 1.00 ± 0.50 1.00 ± 0.50 4b – – – – – – – – Total 8.00 ± 1.50 11.00 ± 1.67 8.00 ± 2.50 10.00 ± 1.67 8.00 ± 2.10 9.00 ± 1.94 10.00 ± 1.50 10.00 ± 2.00 Adults were observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 25.1°C ± 4.8 and 52.5% RH ± 3.2. MM, marked males; UF, unmarked females; UM, unmarked males; UF, unmarked females. aColor treatments. bDay 4 showed no mating observations. Open in new tab Table 1. Total number of black soldier fly mating pairs ± SD (n = 4; n = 200; 100 ♂ 100 ♀) observed in all experiments where males were marked with a single color on their thorax Day . MM + UF ± SD . . . . UM + UF ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.50 3.00 ± 0.50 5.00 ± 0.50 3.00 ± 0.50 3.00 ± 0.06 2.00 ± 0.77 6.00 ± 0.50 4.00 ± 0.50 2 5.00 ± 0.50 7.00 ± 0.50 3.00 ± 2.00 6.00 ± 0.50 4.00 ± 0.77 6.00 ± 0.67 3.00 ± 0.50 5.00 ± 1.00 3 1.00 ± 0.50 1.00 ± 0.67 0.00 ± 0.50 1.00 ± 0.67 1.00 ± 1.27 1.00 ± 0.50 1.00 ± 0.50 1.00 ± 0.50 4b – – – – – – – – Total 8.00 ± 1.50 11.00 ± 1.67 8.00 ± 2.50 10.00 ± 1.67 8.00 ± 2.10 9.00 ± 1.94 10.00 ± 1.50 10.00 ± 2.00 Day . MM + UF ± SD . . . . UM + UF ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.50 3.00 ± 0.50 5.00 ± 0.50 3.00 ± 0.50 3.00 ± 0.06 2.00 ± 0.77 6.00 ± 0.50 4.00 ± 0.50 2 5.00 ± 0.50 7.00 ± 0.50 3.00 ± 2.00 6.00 ± 0.50 4.00 ± 0.77 6.00 ± 0.67 3.00 ± 0.50 5.00 ± 1.00 3 1.00 ± 0.50 1.00 ± 0.67 0.00 ± 0.50 1.00 ± 0.67 1.00 ± 1.27 1.00 ± 0.50 1.00 ± 0.50 1.00 ± 0.50 4b – – – – – – – – Total 8.00 ± 1.50 11.00 ± 1.67 8.00 ± 2.50 10.00 ± 1.67 8.00 ± 2.10 9.00 ± 1.94 10.00 ± 1.50 10.00 ± 2.00 Adults were observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 25.1°C ± 4.8 and 52.5% RH ± 3.2. MM, marked males; UF, unmarked females; UM, unmarked males; UF, unmarked females. aColor treatments. bDay 4 showed no mating observations. Open in new tab Impact on Mating: Marked Females Only The temperature range inside cages was 22.2–36.1°C (mean 29.2°C ± 4.1). Greenhouse temperatures ranged from 19.2 to 34.8°C (mean 25.8°C ± 4.5), while RH ranged from 42.6 to 61.8 % (mean 50.7% ± 5.4). As with marked males, analyses indicated no correlation between temperature (r = −0.0380, df = 58, P = 0.7731) or RH (r = −0.0552, df = 58, P = 0.6751) and mating frequency, whereas it was positively correlated with light intensity (r = 0.2591, df = 58, P = 0.0458). No difference in mate choice of marked and unmarked females (t = 0.53612, df = 1, P = 0.6867; Table 2) was observed. Number of mating pairs of marked or unmarked females did not differ per day of the experiment either (F = 1.8820, df = 1, P = 0.242; Table 2). Additionally, color did not affect mating frequency (F = 0.0780, df = 3, P = 0.9680; Table 2), and color*day was not significant (F = 0.0780, df = 3, P = 0.9680; Table 2). Table 2. Total number of black soldier fly mating pairs ± SD (n = 4; n = 200; 100 ♂ 100 ♀) observed in all experiments where females were marked with a single color on their thorax Day . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 3.00 ± 0.92 2.00 ± 1.17 2.00 ± 1.59 3.00 ± 0.64 3.00 ± 1.66 3.00 ± 0.50 6.00 ± 0.50 4.00 ± 0.80 2 5.00 ± 1.67 4.00 ± 1.29 4.00 ± 1.47 5.00 ± 1.96 4.00 ± 0.50 5.00 ± 0.67 3.00 ± 0.50 6.00 ± 1.67 3 2.00 ± 1.50 1.00 ± 1.40 3.00 ± 1.27 1.00 ± 1.82 2.00 ± 0.77 0.00 ± 0.50 1.00 ± 0.50 0.00 ± 0.50 4b – – – – – – – – Total 10.00 ± 4.09 7.00 ± 3.86 9.00 ± 4.33 9.00 ± 4.42 9.00 ± 2.93 8.00 ± 1.67 10.00 ± 1.50 10.00 ± 2.97 Day . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 3.00 ± 0.92 2.00 ± 1.17 2.00 ± 1.59 3.00 ± 0.64 3.00 ± 1.66 3.00 ± 0.50 6.00 ± 0.50 4.00 ± 0.80 2 5.00 ± 1.67 4.00 ± 1.29 4.00 ± 1.47 5.00 ± 1.96 4.00 ± 0.50 5.00 ± 0.67 3.00 ± 0.50 6.00 ± 1.67 3 2.00 ± 1.50 1.00 ± 1.40 3.00 ± 1.27 1.00 ± 1.82 2.00 ± 0.77 0.00 ± 0.50 1.00 ± 0.50 0.00 ± 0.50 4b – – – – – – – – Total 10.00 ± 4.09 7.00 ± 3.86 9.00 ± 4.33 9.00 ± 4.42 9.00 ± 2.93 8.00 ± 1.67 10.00 ± 1.50 10.00 ± 2.97 Adults were observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 25.8°C ± 4.5 and 50.7% RH ± 5.4. MF, marked females; UM, unmarked males; UF, unmarked females; UM, unmarked males. aColor treatments bDay 4 showed no mating observations. Open in new tab Table 2. Total number of black soldier fly mating pairs ± SD (n = 4; n = 200; 100 ♂ 100 ♀) observed in all experiments where females were marked with a single color on their thorax Day . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 3.00 ± 0.92 2.00 ± 1.17 2.00 ± 1.59 3.00 ± 0.64 3.00 ± 1.66 3.00 ± 0.50 6.00 ± 0.50 4.00 ± 0.80 2 5.00 ± 1.67 4.00 ± 1.29 4.00 ± 1.47 5.00 ± 1.96 4.00 ± 0.50 5.00 ± 0.67 3.00 ± 0.50 6.00 ± 1.67 3 2.00 ± 1.50 1.00 ± 1.40 3.00 ± 1.27 1.00 ± 1.82 2.00 ± 0.77 0.00 ± 0.50 1.00 ± 0.50 0.00 ± 0.50 4b – – – – – – – – Total 10.00 ± 4.09 7.00 ± 3.86 9.00 ± 4.33 9.00 ± 4.42 9.00 ± 2.93 8.00 ± 1.67 10.00 ± 1.50 10.00 ± 2.97 Day . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . 1 3.00 ± 0.92 2.00 ± 1.17 2.00 ± 1.59 3.00 ± 0.64 3.00 ± 1.66 3.00 ± 0.50 6.00 ± 0.50 4.00 ± 0.80 2 5.00 ± 1.67 4.00 ± 1.29 4.00 ± 1.47 5.00 ± 1.96 4.00 ± 0.50 5.00 ± 0.67 3.00 ± 0.50 6.00 ± 1.67 3 2.00 ± 1.50 1.00 ± 1.40 3.00 ± 1.27 1.00 ± 1.82 2.00 ± 0.77 0.00 ± 0.50 1.00 ± 0.50 0.00 ± 0.50 4b – – – – – – – – Total 10.00 ± 4.09 7.00 ± 3.86 9.00 ± 4.33 9.00 ± 4.42 9.00 ± 2.93 8.00 ± 1.67 10.00 ± 1.50 10.00 ± 2.97 Adults were observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 25.8°C ± 4.5 and 50.7% RH ± 5.4. MF, marked females; UM, unmarked males; UF, unmarked females; UM, unmarked males. aColor treatments bDay 4 showed no mating observations. Open in new tab Impact on Mating: Marked Males and Females Combined The temperature range inside cages was 20.0–31.6°C (mean 24.3°C ± 2.9). Greenhouse temperatures ranged from 17.7 to 29.4°C (mean 23.4°C ± 3.1); and RH from 40.7 to 64.0% (average 52.4% ± 8.3). As with the previous tests, there was no significant correlation between mating frequency and temperature (r = 0.0683, df = 58, P = 0.6041) or RH (r = 0.1325, df = 58, P = 0.3125), but it was positively correlated with light intensity (r = 0.3040, df = 58, P = 0.0182). No significant difference (F = 0.8663, df = 3, P = 0.4682) of mate choice was found for marked or unmarked males and females (Table 3). Number of marked and unmarked mating pairs of males and females did not differ for experiment day (F = 1.2790, df = 1, P = 0.3210; Table 3). Marker colors had no significant impact on mate choice by either sex (F = 0.0250, df = 3, P = 0.9940; Table 3), and color*day was not significant (F = 0.0430, df = 3, P = 0.9870; Table 3). Table 3. Total number of black soldier fly mating pairs ± SD (n = 4; n = 200; 100 ♂ 100 ♀) observed in all experiments where males and females were marked with a single color on their thorax Day . MM + MF ± SD . . . . MM + UF ± SD . . . . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.67 2.00 ± 0.95 2.00 ± 0.67 4.00 ± 0.81 2.00 ± 0.67 3.00 ± 0.97 3.00 ± 1.28 4.00 ± 0.93 3.00 ± 1.01 2.00 ± 0.50 4.00 ± 1.21 3.00 ± 0.93 5.00 ± 0.81 4.00 ± 1.17 3.00 ± 0.77 3.00 ± 1.13 2 5.00 ± 0.95 5.00 ± 0.57 6.00 ± 0.67 5.00 ± 1.07 3.00 ± 1.13 4.00 ± 0.77 4.00 ± 0.67 5.00 ± 0.50 5.00 ± 1.03 5.00 ± 1.03 3.00 ± 0.93 6.00 ± 0.81 4.00 ± 1.13 4.00 ± 0.93 5.00 ± 0.95 6.00 ± 1.07 3 1.00 ± 0.95 2.00 ± 0.57 1.00 ± 0.81 0.00 ± 0.50 3.00 ± 0.82 2.00 ± 0.50 1.00 ± 0.67 1.00 ± 0.77 1.00 ± 0.50 1.00 ± 0.23 2.00 ± 0.67 1.00 ± 0.50 2.00 ± 0.50 1.00 ± 0.50 2.00 ± 0.23 2.00 ± 0.67 4b – – – – – – – – – – – – – – – – Total 8.00 ± 2.57 9.00 ± 2.09 9.00 ± 2.15 9.00 ± 2.38 8.00 ± 2.62 9.00 ± 2.24 8.00 ± 2.62 10.00 ± 2.20 9.00 ± 2.54 8.00 ± 1.76 9.00 ± 2.81 10.00 ± 2.24 11.00 ± 2.44 9.00 ± 2.60 10.00 ± 1.95 11.00 ± 2.87 Day . MM + MF ± SD . . . . MM + UF ± SD . . . . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.67 2.00 ± 0.95 2.00 ± 0.67 4.00 ± 0.81 2.00 ± 0.67 3.00 ± 0.97 3.00 ± 1.28 4.00 ± 0.93 3.00 ± 1.01 2.00 ± 0.50 4.00 ± 1.21 3.00 ± 0.93 5.00 ± 0.81 4.00 ± 1.17 3.00 ± 0.77 3.00 ± 1.13 2 5.00 ± 0.95 5.00 ± 0.57 6.00 ± 0.67 5.00 ± 1.07 3.00 ± 1.13 4.00 ± 0.77 4.00 ± 0.67 5.00 ± 0.50 5.00 ± 1.03 5.00 ± 1.03 3.00 ± 0.93 6.00 ± 0.81 4.00 ± 1.13 4.00 ± 0.93 5.00 ± 0.95 6.00 ± 1.07 3 1.00 ± 0.95 2.00 ± 0.57 1.00 ± 0.81 0.00 ± 0.50 3.00 ± 0.82 2.00 ± 0.50 1.00 ± 0.67 1.00 ± 0.77 1.00 ± 0.50 1.00 ± 0.23 2.00 ± 0.67 1.00 ± 0.50 2.00 ± 0.50 1.00 ± 0.50 2.00 ± 0.23 2.00 ± 0.67 4b – – – – – – – – – – – – – – – – Total 8.00 ± 2.57 9.00 ± 2.09 9.00 ± 2.15 9.00 ± 2.38 8.00 ± 2.62 9.00 ± 2.24 8.00 ± 2.62 10.00 ± 2.20 9.00 ± 2.54 8.00 ± 1.76 9.00 ± 2.81 10.00 ± 2.24 11.00 ± 2.44 9.00 ± 2.60 10.00 ± 1.95 11.00 ± 2.87 Adults were observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 28.4°C ± 5.3 and 60.1% RH ± 5.5. MM, marked males; MF, marked females; UF, unmarked females; and UM, unmarked males. aColor treatments. bDay 4 showed no mating observations. Open in new tab Table 3. Total number of black soldier fly mating pairs ± SD (n = 4; n = 200; 100 ♂ 100 ♀) observed in all experiments where males and females were marked with a single color on their thorax Day . MM + MF ± SD . . . . MM + UF ± SD . . . . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.67 2.00 ± 0.95 2.00 ± 0.67 4.00 ± 0.81 2.00 ± 0.67 3.00 ± 0.97 3.00 ± 1.28 4.00 ± 0.93 3.00 ± 1.01 2.00 ± 0.50 4.00 ± 1.21 3.00 ± 0.93 5.00 ± 0.81 4.00 ± 1.17 3.00 ± 0.77 3.00 ± 1.13 2 5.00 ± 0.95 5.00 ± 0.57 6.00 ± 0.67 5.00 ± 1.07 3.00 ± 1.13 4.00 ± 0.77 4.00 ± 0.67 5.00 ± 0.50 5.00 ± 1.03 5.00 ± 1.03 3.00 ± 0.93 6.00 ± 0.81 4.00 ± 1.13 4.00 ± 0.93 5.00 ± 0.95 6.00 ± 1.07 3 1.00 ± 0.95 2.00 ± 0.57 1.00 ± 0.81 0.00 ± 0.50 3.00 ± 0.82 2.00 ± 0.50 1.00 ± 0.67 1.00 ± 0.77 1.00 ± 0.50 1.00 ± 0.23 2.00 ± 0.67 1.00 ± 0.50 2.00 ± 0.50 1.00 ± 0.50 2.00 ± 0.23 2.00 ± 0.67 4b – – – – – – – – – – – – – – – – Total 8.00 ± 2.57 9.00 ± 2.09 9.00 ± 2.15 9.00 ± 2.38 8.00 ± 2.62 9.00 ± 2.24 8.00 ± 2.62 10.00 ± 2.20 9.00 ± 2.54 8.00 ± 1.76 9.00 ± 2.81 10.00 ± 2.24 11.00 ± 2.44 9.00 ± 2.60 10.00 ± 1.95 11.00 ± 2.87 Day . MM + MF ± SD . . . . MM + UF ± SD . . . . MF + UM ± SD . . . . UF + UM ± SD . . . . . Greena . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . Green . Gold . Red . White . 1 2.00 ± 0.67 2.00 ± 0.95 2.00 ± 0.67 4.00 ± 0.81 2.00 ± 0.67 3.00 ± 0.97 3.00 ± 1.28 4.00 ± 0.93 3.00 ± 1.01 2.00 ± 0.50 4.00 ± 1.21 3.00 ± 0.93 5.00 ± 0.81 4.00 ± 1.17 3.00 ± 0.77 3.00 ± 1.13 2 5.00 ± 0.95 5.00 ± 0.57 6.00 ± 0.67 5.00 ± 1.07 3.00 ± 1.13 4.00 ± 0.77 4.00 ± 0.67 5.00 ± 0.50 5.00 ± 1.03 5.00 ± 1.03 3.00 ± 0.93 6.00 ± 0.81 4.00 ± 1.13 4.00 ± 0.93 5.00 ± 0.95 6.00 ± 1.07 3 1.00 ± 0.95 2.00 ± 0.57 1.00 ± 0.81 0.00 ± 0.50 3.00 ± 0.82 2.00 ± 0.50 1.00 ± 0.67 1.00 ± 0.77 1.00 ± 0.50 1.00 ± 0.23 2.00 ± 0.67 1.00 ± 0.50 2.00 ± 0.50 1.00 ± 0.50 2.00 ± 0.23 2.00 ± 0.67 4b – – – – – – – – – – – – – – – – Total 8.00 ± 2.57 9.00 ± 2.09 9.00 ± 2.15 9.00 ± 2.38 8.00 ± 2.62 9.00 ± 2.24 8.00 ± 2.62 10.00 ± 2.20 9.00 ± 2.54 8.00 ± 1.76 9.00 ± 2.81 10.00 ± 2.24 11.00 ± 2.44 9.00 ± 2.60 10.00 ± 1.95 11.00 ± 2.87 Adults were observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 28.4°C ± 5.3 and 60.1% RH ± 5.5. MM, marked males; MF, marked females; UF, unmarked females; and UM, unmarked males. aColor treatments. bDay 4 showed no mating observations. Open in new tab Impact of Marking on Egg Production The temperature inside cages was 28.6–31.8°C (mean 29.7°C ± 3.3). Greenhouse temperatures ranged from 26.9 to 30.4°C (mean 28.4°C ± 5.3); and RH from 56.7 to 69.6% (mean 60.1% ± 5.5). Correlation analyses indicated positive correlation with temperature (r = 0.3698, df = 29, P = 0.0463) and RH (r = 0.3758, df = 29, P = 0.0434) on egg production, but not with light intensity (r = −0.1289, df = 29, P = 0.5031). No significant trial effect (H = 0.2273, df = 2, P = 0.8925), or trial by treatment effect (H = 6.561, df = 3, P = 0.0872) was found on number of eggs. Neither color (H = 5.303, df = 2, P = 0.0706) nor marking affected (H = 0.1448, df = 1, P = 0.7036) number of eggs or hatch rate (H = 0.0123, df = 1, P = 0.9115) (Table 4). In addition, egg production by day for marked and unmarked cages showed no significant differences (H = 0.9631, df = 4, P = 0.9153; Table 4). Table 4. Total number of eggs ± SD, and hatch ± SD (n = 4; n = 200; 100 ♂ 100 ♀) by marked and unmarked adults observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 28.4°C ± 5.3 and 60.1% RH ± 5.5 Day . Experiment 1a . . Experiment 2b . . . Marked . Unmarked . Marked . Unmarked . 1 4457.00 ± 589.14 5691.00 ± 667.75 3211.00 ± 791.14 3515.00 ± 712.44 2 9984.00 ± 1669.56 11,934.00 ± 1354.79 5784.00 ± 2416.21 4748.00 ± 1810.54 3 2516.00 ± 658.94 1532.00 ± 323.11 3401.00 ± 716.56 1708.00 ± 946.71 4 – – – – Total Number of Eggs 16,957.00 ± 1597.14 19,157.00 ± 2845.45 12,395.00 ± 1885.18 9971.00 ± 1298.52 Total Hatch Rate (%) 62.00 ± 5.97 69.00 ± 8.67 71.00 ± 6.27 64.00 ± 4.25 Day . Experiment 1a . . Experiment 2b . . . Marked . Unmarked . Marked . Unmarked . 1 4457.00 ± 589.14 5691.00 ± 667.75 3211.00 ± 791.14 3515.00 ± 712.44 2 9984.00 ± 1669.56 11,934.00 ± 1354.79 5784.00 ± 2416.21 4748.00 ± 1810.54 3 2516.00 ± 658.94 1532.00 ± 323.11 3401.00 ± 716.56 1708.00 ± 946.71 4 – – – – Total Number of Eggs 16,957.00 ± 1597.14 19,157.00 ± 2845.45 12,395.00 ± 1885.18 9971.00 ± 1298.52 Total Hatch Rate (%) 62.00 ± 5.97 69.00 ± 8.67 71.00 ± 6.27 64.00 ± 4.25 aColor combinations of green and white for marked adults. bColor combinations of red and gold for marked adults. Open in new tab Table 4. Total number of eggs ± SD, and hatch ± SD (n = 4; n = 200; 100 ♂ 100 ♀) by marked and unmarked adults observed in 84 (L) × 84 (W) × 132 (H) cm cages in a greenhouse maintained at 28.4°C ± 5.3 and 60.1% RH ± 5.5 Day . Experiment 1a . . Experiment 2b . . . Marked . Unmarked . Marked . Unmarked . 1 4457.00 ± 589.14 5691.00 ± 667.75 3211.00 ± 791.14 3515.00 ± 712.44 2 9984.00 ± 1669.56 11,934.00 ± 1354.79 5784.00 ± 2416.21 4748.00 ± 1810.54 3 2516.00 ± 658.94 1532.00 ± 323.11 3401.00 ± 716.56 1708.00 ± 946.71 4 – – – – Total Number of Eggs 16,957.00 ± 1597.14 19,157.00 ± 2845.45 12,395.00 ± 1885.18 9971.00 ± 1298.52 Total Hatch Rate (%) 62.00 ± 5.97 69.00 ± 8.67 71.00 ± 6.27 64.00 ± 4.25 Day . Experiment 1a . . Experiment 2b . . . Marked . Unmarked . Marked . Unmarked . 1 4457.00 ± 589.14 5691.00 ± 667.75 3211.00 ± 791.14 3515.00 ± 712.44 2 9984.00 ± 1669.56 11,934.00 ± 1354.79 5784.00 ± 2416.21 4748.00 ± 1810.54 3 2516.00 ± 658.94 1532.00 ± 323.11 3401.00 ± 716.56 1708.00 ± 946.71 4 – – – – Total Number of Eggs 16,957.00 ± 1597.14 19,157.00 ± 2845.45 12,395.00 ± 1885.18 9971.00 ± 1298.52 Total Hatch Rate (%) 62.00 ± 5.97 69.00 ± 8.67 71.00 ± 6.27 64.00 ± 4.25 aColor combinations of green and white for marked adults. bColor combinations of red and gold for marked adults. Open in new tab Impact of Marking on Adult Longevity No significant trial effect (F1,1 = 0.48, P = 0.4891), or trial by treatment effect (F3,3 = 0.96, P = 0.4105), was found on adult longevity. Neither color (F3,3 = 0.91, P = 0.4345) nor marking affected (F1,1 = 0.17, P = 0.6787) adult longevity (Table 5). Sex did not significantly (F3,3 = 1.41, P = 0.2407) impact longevity either. Table 5. Mean adult longevity of marked and unmarked black soldier fly adults ± SE (n = 4) . Sex (mean ± SE) . . . . Color . MF . UF . MM . UM . Green 7.23 ± 0.34Aa 7.26 ± 0.34Aa 8.66 ± 0.34Aa 8.96 ± 0.34Aa Gold 7.13 ± 0.34Aa 7.30 ± 0.34Aa 8.86 ± 0.34Aa 8.50 ± 0.34Aa Red 7.10 ± 0.31Aa 7.20 ± 0.31Aa 9.03 ± 0.31Aa 9.00 ± 0.31Aa White 7.53 ± 0.30Aa 7.23 ± 0.30Aa 9.56 ± 0.30Aa 9.06 ± 0.30Aa . Sex (mean ± SE) . . . . Color . MF . UF . MM . UM . Green 7.23 ± 0.34Aa 7.26 ± 0.34Aa 8.66 ± 0.34Aa 8.96 ± 0.34Aa Gold 7.13 ± 0.34Aa 7.30 ± 0.34Aa 8.86 ± 0.34Aa 8.50 ± 0.34Aa Red 7.10 ± 0.31Aa 7.20 ± 0.31Aa 9.03 ± 0.31Aa 9.00 ± 0.31Aa White 7.53 ± 0.30Aa 7.23 ± 0.30Aa 9.56 ± 0.30Aa 9.06 ± 0.30Aa Larvae were reared in a walk-in growth chamber set at 29.8°C ± 0.8 with 65.0% RH ± 5.3 and 14:10 (L:D) conditions. Capital letters within a column indicate no significant (P > 0.05) differences across color treatments within a sex; lowercase letters indicate no significant (P > 0.05) differences between sexes within the same color treatment. MF, marked females; UF, unmarked females; MM, marked males; UM, unmarked males. Open in new tab Table 5. Mean adult longevity of marked and unmarked black soldier fly adults ± SE (n = 4) . Sex (mean ± SE) . . . . Color . MF . UF . MM . UM . Green 7.23 ± 0.34Aa 7.26 ± 0.34Aa 8.66 ± 0.34Aa 8.96 ± 0.34Aa Gold 7.13 ± 0.34Aa 7.30 ± 0.34Aa 8.86 ± 0.34Aa 8.50 ± 0.34Aa Red 7.10 ± 0.31Aa 7.20 ± 0.31Aa 9.03 ± 0.31Aa 9.00 ± 0.31Aa White 7.53 ± 0.30Aa 7.23 ± 0.30Aa 9.56 ± 0.30Aa 9.06 ± 0.30Aa . Sex (mean ± SE) . . . . Color . MF . UF . MM . UM . Green 7.23 ± 0.34Aa 7.26 ± 0.34Aa 8.66 ± 0.34Aa 8.96 ± 0.34Aa Gold 7.13 ± 0.34Aa 7.30 ± 0.34Aa 8.86 ± 0.34Aa 8.50 ± 0.34Aa Red 7.10 ± 0.31Aa 7.20 ± 0.31Aa 9.03 ± 0.31Aa 9.00 ± 0.31Aa White 7.53 ± 0.30Aa 7.23 ± 0.30Aa 9.56 ± 0.30Aa 9.06 ± 0.30Aa Larvae were reared in a walk-in growth chamber set at 29.8°C ± 0.8 with 65.0% RH ± 5.3 and 14:10 (L:D) conditions. Capital letters within a column indicate no significant (P > 0.05) differences across color treatments within a sex; lowercase letters indicate no significant (P > 0.05) differences between sexes within the same color treatment. MF, marked females; UF, unmarked females; MM, marked males; UM, unmarked males. Open in new tab Discussion Using the acrylic paints as a marking system had no impact on the mating success, egg production, hatch rate, or adult longevity of the black soldier fly (Table 1). Color used to mark adults did not affect mate choice either. Number of successful mating pairs by marked and unmarked adults did not differ over time. In spite of the frequent misting process in the black soldier fly cages (Tomberlin et al. 2002), colors remained unaffected throughout the study. The acrylic paints have not been useful for other insects due to negatively impacted marked individuals. In some instances, the meticulous application of paints to each individual can be time consuming (Walker and Wineriter 1981). In other cases, these paints impacted insect mobility, and only lasted for 48 h (Mattioli and Walsh 2008). None of these issues were observed in the current study. In addition, and while not a specific aim of this project, the impact of temperature and RH were investigated a posteriori given the data were already recorded. We determined a high degree of variation in light intensity (200–42,000 lux) and RH (40.7–64.0%) was experienced in the greenhouse over the course of the trials. Fortunately, the acrylic paints were able to withstand such variation and were detectable on the flies throughout the experiments. As far as mating frequency, it was only significantly correlated to light intensity. This does not come as a surprise as such factors are known to play a role in such behavior (Tomberlin and Sheppard 2002). In addition, temperature and RH were significantly correlated with egg production, as found in similar work (Holmes et al. 2012). Such information should be considered when establishing a mass-production site in order to stabilize production. Furthermore, future research is sorely needed to determine appropriate ranges for each of these factors as a means to optimize production in industrialized facilities. This study determined acrylic paint marking of adult black soldier flies did not affect their mating success, egg production, hatch rate, or adult longevity. Therefore, we recommend more exploration into the use of acrylic paint markers for black soldier fly mating studies in controlled environments. The use of acrylic paint markers can also be an inexpensive and effective method in mark-recapture studies. Acknowledgments We would like to thank Dr. Hojun Song at Texas A&M University for providing suggestions on experiment design. 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TI - Validation of Acrylic Paint as a Marking Technique for Examining Mating Success of the Black Soldier Fly (Diptera: Stratiomyidae)
JF - Journal of Economic Entomology
DO - 10.1093/jee/toaa129
DA - 2019-01-31
UR - https://www.deepdyve.com/lp/oxford-university-press/validation-of-acrylic-paint-as-a-marking-technique-for-examining-nlpIvr7Qi2
DP - DeepDyve
ER -