Topical Application of Synthetic Hormones Terminated Reproductive Diapause of a Univoltine Weed Biological Control Agent

Simple Summary Yellow starthistle (Centaurea solstitialis) is an invasive annual plant that has infested multi-million ha in the western United States, causing multi-billion dollars of losses and management costs. The rosette weevil (Ceratapion basicorne) has recently been approved for biological control of this weed. However, this weevil reproduces only once a year, which hinders mass-rearing for release. Here, we tested whether insect hormones can break the reproductive diapause of female weevils. We found that applying two insect hormones, 20E and methoprene, can consistently terminate diapause of female weevils to enable them to lay eggs. Thus, topically applying insect hormones could be used to induce females to lay eggs at any time of year, which would permit rearing more than one generation per year. This could greatly increase the number of weevils produced per year in a mass-rearing program to accelerate the release of rosette weevils to help suppress yellow starthistle in the United States. These insects would revert to having one generation per year after release in synchrony with their host plant. Abstract Classical biological control is an important method for controlling invasive alien weeds. Univoltine insects can be highly effective biological control agents of annual weeds because they are well synchronized with their host plant. However, having only one generation per year makes it difficult and slow to multiply them in the laboratory for initial field releases. If it were possible to terminate reproductive diapause early, then we could rear multiple generations per year, which would greatly increase annual production. We used a recently approved biocontrol agent, Ceratapion basicorne (a univoltine weevil), for yellow starthistle (Centaurea solstitialis) as a model system to study the use of two insect hormones, 20-hydroxyecdysone (20E) and methoprene, to terminate reproductive diapause. Methoprene (1 μg applied topically) terminated reproductive diapause of female weevils, whereas doses of 0.0, 0.01 and 0.1 μg did not. The combination of methoprene and 20E had a stronger effect and induced an increase in eggs (1.51 ± 0.16 eggs/day, mean ± SE) compared with a methoprene only group (1.00 ± 0.13 eggs/day), and a control group (0.21 ± 0.04 eggs/day). Thus, topical application of these hormones should enable us to rear the weevil out of its normal season and produce more than one generation per year, which will increase productivity of mass-rearing it for field release. Once released in the field, the insect would continue as a univoltine agent that is well-synchronized with its host plant.


Introduction
Classical biological control, the use of host-specific natural enemies, is an important strategy for controlling invasive alien plants [1]. Once new classical biocontrol agents are terminated the reproductive diapause of Colorado potato beetle in the laboratory [31]. Similarly, the combined application of 20E and methoprene had a greater effect on termination of reproductive diapause of female face flies (Musca autumnalis De Geer, Diptera: Muscidae) than treatment of either hormone alone [26]. Termination of reproductive diapause by regulating the abovementioned hormones has been demonstrated in the following insect orders: Coleoptera [31][32][33][34], Diptera [35], Hemiptera [36], Hymenoptera [37] and Lepidoptera [38][39][40]. However, to the best of our knowledge, the idea of using synthetic hormones to terminate reproductive diapause has not been examined regarding helping to rear biocontrol agents for weeds.
The purpose of this study is to measure the effect of two synthetic hormone analogs to terminate the reproductive diapause of female C. basicorne, which would enable them to start laying fertile eggs and facilitate rearing weevils in the laboratory for the field release. We measured the effect of (1) different concentrations of methoprene and (2) preceding the methoprene treatment with 20E to stimulate an increase in adult feeding and oviposition of C. basicorne.

Insect Rearing and Plant Material
Adult C. basicorne emerged from potted yellow starthistle plants in the greenhouse at ambient photoperiod and temperature from 02/03/2020 to 04/03/2020. They were placed in transparent plastic cups with organdy screen tops, and provided cut yellow starthistle leaves inserted in sealed water vials for food.
Adults were observed mating when held at room temperature. Weevils were held in a refrigerator (Whirlpool, Benton Harbor, MI, USA) at 5 • C under 24 h darkness, and were removed for a few hours one day a week to allow them to feed and to minimize cold stress [41], until further use. During these warming periods, males copulated with females even though the latter may have still been in reproductive diapause (not able to oviposit). Tests were conducted at the following conditions (LD 12:12, 21 • C: 17 • C) and 60-80% (RH) in an incubator (Percival, Perry, IA, USA), which simulates spring conditions at which reproduction has previously been observed [20].

Methoprene and 20-Hydroxyecdysone
Methoprene was purchased from Sigma Aldrich (33375-100MG, Sulpelco, St. Louis, MO, USA). Methoprene was dissolved in acetone (≥99.9%, 650601-1L, Sigma Aldrich, St. Louis, MO, USA) to make a stock solution under a fume hood. The stock solution was made by adding 0.892 mL of acetone to a vial containing 100 mg of methoprene, which is equivalent to 100 mg/mL. The stock solution was serially diluted to produce concentrations of 1.0, 0.1 and 0.01 µg/µL. Topical application of 1 µL of each solution was applied on the abdomen of female C. basicorne by two squirts from a 0.5 µL Hamilton microsyringe (22184-U, 86250, Hamilton, Reno, NV, USA) under a stereo-microscope. An equal volume of acetone (1 µL) was topically applied to the control group.
In addition, 20-Hydroxyecdysone was purchased (H5142-5MG, Sigma Aldrich, St. Louis, MO, USA). Instead of dissolving in methanol as used in other studies [42][43][44], we dissolved 20E in the acetone to be consistent with the methoprene experiment. Another study also showed that the topical application of 20E dissolved in acetone worked on the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) [39]. Instead of direct injection or oral administration, we chose to use topical application for two reasons. Injection involves piercing the insect, which we thought would be very harmful, especially for a small hard-bodied weevil (abdomen < 2 mm long). Second, oral administration was not an appropriate method for this species, because adults feed inside the leaves, so it would not be possible to apply a known dose. Topical application of 20E using alcohols or acetone have previously been used successfully on other types of insects [45][46][47]. We used 1 µg of 20E because 20E (1 µg) and methoprene (1 µg) caused 92% of diapausedestined female face flies, Musca autumnalis, to become reproductively active [26]. We conducted four different experiments to test the relationship between methoprene and 20E to terminate the reproductive diapause of female C. basicorne in the laboratory.

Experiment 1: Methoprene Dose-Response Experiment
Female weevils went through the following environmental conditions before conducting Experiment 1: (1) an overwinter condition at 5 • C:5 • C under a 0:24 h light:dark photoperiod for four weeks, (2) a late spring condition at 22 • C:17 • C and 12:12 h LD for a week and adjusted to 22 • C:17 • C and 15:09 h LD for three weeks, and (3) an aestivation condition at 30 • C:22 • C and 15:09 h LD for eight weeks. We conducted oviposition experiments during the late spring condition to the aestivation condition, but none of the females oviposited in yellow starthistle leaves. To identify the concentration of methoprene that terminates diapause of females, we used the following concentrations: 1.0, 0.1, 0.01 µg of methoprene dissolved in 1 µL of acetone, and a control (=1 µL of acetone only) on Day 0 (4 female weevils for control; 5 females each for 1.0, 0.1 and 0.01 µg of methoprene). Each female was placed in a container with a cut yellow starthistle leaf for food and oviposition site and held in an incubator (I-30BLL, Percival Scientific Inc., Perry, IA, USA) with four Philips 17-watt Alto II linear fluorescent tube light bulbs in the following environmental conditions: LD 12:12, 21 • C:17 • C and 60-80% RH. The leaf was replaced every day. We recorded the number of feeding holes and eggs for each female per day. Experiment 1 was conducted from 06/17/2020 to 07/01/2020.

Experiment 2: Application of 1 µg Methoprene to Unresponsive Females
To evaluate whether 1 µg methoprene induces previously unresponsive females in Experiment 1 to lay eggs, we divided unresponsive females in Experiment 1 into two groups. The first group was topically treated with 1 µg methoprene dissolved in 1 µL acetone and the control group received only 1 µL acetone (n = 6 female weevils). "Unresponsive females" are defined as females that did not lay any eggs after one of the treatments used in Experiment 1. Because 1 µg methoprene made some females lay eggs in Experiment 1, females that previously received the 1 µg dose but did not lay eggs were excluded. Females in both groups were held with an excised leaf of the rosette stage of yellow starthistle that was replaced daily for two weeks. We recorded the number of feeding holes and eggs for each female per day. Because additional light sources increase the number of eggs in the mass-rearing process [48], we added an additional incandescent light bulb (75 watts, BR30, Philips, Somerset, NJ) with a 24-h mechanical timer inside the same incubator used in Experiment 1 (LD 12:12, 21 • C:17 • C). Experiment 2 was conducted from 07/16/2020 to 07/30/2020.

Experiment 3: The effect of 20E on the Methoprene Treatment
To investigate the role of 20E applied two days before application of methoprene, we conducted three treatments: (1) acetone + acetone [AA: control], (2) acetone + methoprene [AM], and 20E + methoprene [2M]. A 20E alone application was not tested because of the small number of females available and because 20E alone failed to terminate reproductive diapause in Colorado potato beetles [31]. Females had been held in the cold conditions (LD 0:24, 5 • C) for 12 weeks, pulled out and oviposition was checked in the laboratory at ambient temperature and photoperiod from 1 April 2020 to 23 April 2020, but none of them laid eggs on rosette leaves of yellow starthistle. They were then placed back to the refrigerator (LD 0:24, 5 • C) for six months. Other newly emerged female weevils in April 2020 were placed in the refrigerator for approximately six months. Both groups of females were used in Experiment 3, and the environmental conditions were the same as Experiment 2. Eight weevils were tested for each treatment. The first application (1 µg of 20E/µL acetone or acetone only) was conducted on Day 0, followed by the second application (1 µg of methoprene/µL acetone or acetone only) on Day 2. A yellow starthistle leaf was replaced every two days for 24 days. We recorded the number of feeding holes and eggs for each female every two days. The hatching rate of eggs among three Insects 2021, 12, 834 5 of 14 treatments was measured by dissecting cut leaves under the microscope 6-8 days after the day when the weevils were changed. We also compared the fecundity with that of non-diapausing females [21] to examine the effects of hormonal treatment on female C. basicorne. Experiment 3 was conducted from 24 October 2020 to 17 November 2020. After Experiment 3, each reproductively active female was placed on potted yellow starthistle plants every two days for them to oviposit to rear adult weevils as described in [21].

Experiment 4: Application of 2M on Underperforming and Unresponsive Females
To test whether the 2M treatment can induce egg laying on underperforming and unresponsive females used in Experiment 3, we applied 20E (1 µg) followed in 2 days by methoprene (1 µg) to underperforming and unresponsive females using the same methods as in Experiment 3. We define "underperforming females" as female weevils that oviposited several days and stopped oviposition (=a short oviposition period) or that oviposited less than 1 egg per day more than two consecutive days. "Unresponsive females" are defined as females that did not lay any eggs after one of the treatments used in Experiment 3. A total of 18 weevils were used with the same environmental conditions as Experiment 3. We recorded the number of feeding holes and eggs in each female every two days. Experiment 4 was conducted from 19 November to 21 December 2020.

Statistical Analysis
Because the normality of residuals was significantly different on the Shapiro-Wilk test, we used the Kruskal-Wallis test as a non-parametric test to examine whether different methoprene concentrations affected the number of feeding holes and eggs in Experiment 1 and whether the absence/presence of 20E and/or methoprene altered the number of feeding holes and eggs in Experiment 3. The two-parameter Hill equation (where a is the inflection point and n is the Hill coefficient, which controls the curvature) was fit to the data using nonlinear regression.
feeding holes/maximum feeding holes = 1/(1 + (a/dose) n ) In addition, the paired Wilcoxon signed-rank test was used to test whether the number of both feeding holes and eggs was different after the preoviposition period among individual females used in experiments 1 vs. 2 (e.g., methoprene retreatment from Day 8 to 14) and experiments 3 vs. 4 (e.g., 20E plus methoprene retreatment from Day 8 to 24). The unpaired T-test was conducted to test whether feeding holes and eggs were different among treatments in Experiment 2. The Chi-square test was used to test whether proportions of ovipositing females, number of eggs, hatching rates, and adult mortality were different among three treatments.

Experiment 2: Application of 1 µg Methoprene to Unresponsive Females
Application of 1 µg of methoprene to females previously used in the control, 0.1 and 0.01 µg of methoprene treatments in Experiment 1 stimulated a gradual increase in the number of feeding holes from Day 1 to Day 9, which remained higher (21.64 ± 1.94; mean ± SE) than the feeding holes made by females in the control group from Day 8 to 14 (3.87 ± 0.46; unpaired T-test: T = 8.9, p < 0.0001, Figure 2A). Linear regression for the preoviposition period (Days 1-7) for the control was Y  Figure 2B) from Day 8 to 14, whereas the number of feeding holes was not different in the control group (W = −17.00, p = 0.0938, Figure 2C).

Experiment 4: Application of 2M on Underperforming and Unresponsive Females
The feeding holes were similar among the three retreated groups from Day 8 to 24 (Z = 0.3751, p > 0.05, Figure 4A): the acetone + acetone group (Pre AA) (8.19 ± 1.30 feeding holes/day, mean ± SE), the acetone + methoprene group (Pre AM) (8.92 ± 0.96 feeding holes/day), and the 20E + methoprene group (Pre 2M) (10.27 ± 1.92 feeding holes/day). There was no difference in the number of feeding holes between underperforming and unresponsive females in Experiment 3 and their performance in Experiment 4 (paired Wilcoxon signed-rank test, W = 19, p = 0.3750; Figure 4B). Oviposition by the Pre AA group started on Day 8, versus Day 10 for the Pre AM group, and Day 12 for the Pre 2M group ( Figure 4C). The egg production during the oviposition period from Day 8 to 24 was not different among three groups (Z = 0.28, p > 0.05; Figure 4C): the Pre AA group (0.43 ± 0.07 eggs/day), the Pre AM group (0.52 ± 0.14 eggs/day), and the Pre 2M group (0.81 ± 0.31 eggs/day, mean ± SE). All females treated with 1 μg of 20E and 1 μg of methoprene initiated oviposition regardless of their previous treatments (W = 46, p < 0.05; Figure 4D). The mortality was 16.7% for the Pre AA group, 40% for the Pre AM group After Experiment 3, six female weevils treated with hormones (two females from the AM group and four females from the 2M group) oviposited viable eggs on potted plants for additional two months. The egg hatching rate was not explicitly examined on potted plants because it was difficult to check the 1st instar larvae inside the plant without damaging them. However, we reared out newly emerged weevils (n = 170) from the potted yellow starthistle plants. These adults were held at summer photoperiod for three months (at 19-24 • C) on cut yellow starthistle leaves, but did not oviposit, indicating that they were in reproductive diapause.

Discussion
Our results demonstrated that doses of 0.01, 0.1 and 1.0 μg of methoprene progressively increased feeding by adult females. However, only the dose of 1 μg of methoprene stimulated oviposition. The fact that the egg production only occurred in 60% of females treated with 1 μg of methoprene in Experiment 1 suggests that 1 μg of methoprene is

Discussion
Our results demonstrated that doses of 0.01, 0.1 and 1.0 µg of methoprene progressively increased feeding by adult females. However, only the dose of 1 µg of methoprene stimulated oviposition. The fact that the egg production only occurred in 60% of females treated with 1 µg of methoprene in Experiment 1 suggests that 1 µg of methoprene is near the threshold for terminating diapause of female C. basicorne that had experienced only four weeks of cold. However, the dose-response curve suggests that increases in feeding rate occur slowly above about 0.4 µg, so it is possible that a dose lower than 1.0 may induce oviposition. Previous studies indicated that at least eight weeks of cold was required to terminate diapause of C. basicorne [49]. The dose-response of oviposition after the methoprene treatment was also reported in other insects. For example, topically applied 1 µg of methoprene dissolved in acetone induced 67% of female face flies, Musca autumalis, to become reproductively active vs. 38% for 0.5 µg [26]. One hundred % of female moths of Caloptilia fraxinella Ely (Lepidoptera:) developed vitellogenic oocytes after topical doses of 0.1 µg vs. 19% for 0.01 µg [38]. Experiment 2 demonstrated that 1 µg of methoprene could induce oviposition in 100% of females that previously failed to respond to 0.0, 0.01 or 0.1 µg of methoprene in Experiment 1. Females started ovipositing sooner, and produced more eggs per day in Experiment 2 (0.81 ± 0.28) than Experiment 1 (0.50 ± 0.17) (T = 3.38, df = 57, p < 0.01). A possible explanation for these differences could be the increased light intensity used in Experiment 2. Alternatively, these females were two weeks older and had been at warm temperature two weeks longer than in Experiment 1. In Experiment 2, feeding rate increased linearly during the preoviposition period (Days 1-7) and then gradually decreased during the first seven days of the oviposition period. A similar decrease in feeding rate was observed in naturally ovipositing females [21]. The artificially applied methoprene presumably acted like JH to stimulate egg maturation of diapausing females of C. basicorne, which enabled them to lay eggs [27,31,36,50].
We did not test 20E alone because it was previously demonstrated that 20E alone failed to terminate reproductive diapause of Colorado potato beetles [31], and we did not have enough insects available to test this treatment simultaneously with the other treatments. Treating 20E 2 days before methoprene increased both feeding holes and eggs compared to the control and the methoprene only treatments in Experiment 3, which shows that 20E further helped terminate reproductive diapause of female C. basicorne. Oviposition started on the same day for the AM and 2M treatments in Experiment 3, which suggests that the effect of 20E was to produce more eggs rather than reduce the time to initiate oviposition of C. basicorne. The higher egg production by the AM group compared with the AA group is consistent with the theory that exogenous JH treatment stimulates egg maturation; however, exogenous JH also inhibits CA activity and increases JH esterase activity, which degrades the exogenous JH [28,31]. Thus, application of methoprene alone is expected to produce only a temporary effect. However, 20E is thought to block activation of JH esterase, and it may also stimulate production of JH [23,31]. This is consistent with the result that the number of feeding holes and oviposition were higher for the 2M (20E + methoprene) treatment than the AM (methoprene) treatment in Experiment 3. Although 37.5% of females in the AA group became reproductively active without any hormone treatment, none of the eggs from the AA group was viable, indicating that the level of this activity was far below normal. Nevertheless, oviposition by some control females indicates that this cohort was approaching the end of diapause, which may have been a consequence of the long period of time held at cold dark conditions before testing.
We conclude that 1 µg of methoprene alone was able to induce females to oviposit viable eggs, but that the combination of 20E (1 µg) followed by methoprene (1 µg) was the best treatment for artificially terminating reproductive diapause of female C. basicorne, because it increased oviposition rate by 51% above that of methoprene alone. Our findings can be used in two ways in mass-rearing biocontrol agents. First, to increase the number of biocontrol agents to release by artificially terminating reproductive diapause when necessary, which would permit producing more than one generation per year in the laboratory. Adults produced out of season could be stored at cold temperature (5 • C) until needed for release in the spring [53]. Second, by treating reproductively inactive females a week before field release at the time of year when they should be ovipositing, they will become reproductively active and attack the host plant. Therefore, this tool could both increase annual production of a univoltine agent in the laboratory and ensure that individuals being released are reproductively active, which should enhance establishment rate of univoltine biocontrol agents for invasive plants.