Host Suitability for Crapemyrtle Bark Scale (Acanthococcus lagerstroemiae) Differed Significantly among Crapemyrtle Species

Simple Summary An exotic insect, crapemyrtle bark scale (CMBS, Acanthococcus lagerstroemiae), has spread across 14 states of the U.S. The infestation of CMBS has negatively impacted the growth, flowering, and even fruiting of some Lythraceae plants to various extent, including cultivars of Lagerstroemia indica, L. fauriei, and Punica granatum. This raises concerns that CMBS would threaten other crapemyrtle species and native Lythraceae plants. Understanding the host range and the host suitability for CMBS would help evaluate the potential risks to landscapes and other ecosystems. Information on the host suitability provides beneficial information for breeding resistant cultivars. In this study, we conducted a host range test on six Lagerstroemia species (L. caudata, L. fauriei ‘Kiowa’, L. indica ‘Dynamite’, L. limii, L. speciosa, and L. subcostata) and a native Lythraceae plant in California (California loosestrife, Lythrum californicum) over 25 weeks. The infestation of CMBS was observed on all the tested Lythraceae plants. The suitability for CMBS differed significantly among the Lagerstroemia species. Lagerstroemia limii was the most suitable, whereas L. speciosa was the least suitable. This study expands the current knowledge on the host range for CMBS. Our results suggest that L. speciosa could be utilized in developing new cultivars with low CMBS suitability. Abstract Crapemyrtle bark scale (CMBS, Acanthococcus lagerstroemiae), an invasive polyphagous sap-sucking hemipteran, has spread across 14 states of the United States since 2004. The infestation of CMBS has negatively impacted the flowering of ornamental plants and even the fruiting of some crops. Host identification is critical for determining potential risks in ecosystems and industries and helps develop strategic management. A host confirmation test was performed over 25 weeks using six Lagerstroemia species (L. caudata, L. fauriei ‘Kiowa’, L. indica ‘Dynamite’, L. limii, L. speciosa, and L. subcostata) and California loosestrife (Lythrum californicum). The 25-week observations confirmed all tested plants as the hosts. The repeated measures of analysis of variance (ANOVA; Tukey’s HSD, α = 0.05) indicated that the average number of CMBS females differed significantly between L. limii and L. speciosa. The highest number of the females observed on L. limii was 576 ± 25 (mean ± SE) at 17 weeks after inoculation (WAI), while the highest number was 57 ± 15 on L. speciosa at 19 WAI. In addition, L. subcostata and L. speciosa had significantly high and low numbers of males, respectively, among the Lagerstroemia species. Our results suggest that L. speciosa could be incorporated in developing new cultivars with low CMBS suitability.

. U.S. Department of Agriculture Plant Hardiness Zone Map. It is "the standard by which gardeners and growers can determine which plants are most likely to thrive at a location. This map is based on the average annual minimum winter temperature, divided into 10 °F zones" [32].

CMBS and Host Range Test
The experiment was conducted in the Department of Horticultural Sciences greenhouse at Texas A&M University (30°36′31.9″ N, 96°21′1.9″ W). A set of the seven species mentioned above was enclosed in one cage and inoculated with CMBS-infested branches. The cage was replicated three times. Crapemyrtle branches infested with CMBS ( Figure  2a) were collected from the nursery at the Department of Horticultural Sciences of Texas A&M University in May 2019. Before the branches were attached to each test plant using Parafilm ® , all except five ovisacs on the branches were removed (Figure 2b). To ensure It is "the standard by which gardeners and growers can determine which plants are most likely to thrive at a location. This map is based on the average annual minimum winter temperature, divided into 10 • F zones" [32].
Because honeydew secreted from the ingested sap leads to the growth of black sooty mold covering the leaf surface and bark [31,35], CMBS threatens the growth and development of crapemyrtle and causes a reduction in aesthetic quality, resulting in concerns for landscapers and nursery growers [36]. Commercial insecticides are sometimes utilized to minimize CMBS infestations [37,38]. However, since they flower from late spring to early fall when few other resources are available [39,40], crapemyrtles are a good pollen source for pollinators, and are vital in ecosystem services benefiting humankind [41][42][43][44]. Consequently, insecticide applications on crapemyrtles to control this pest could severely affect the pollinators [45][46][47]. Non-chemical management of CMBS, such as resistance breeding and utilizing natural enemies, would be beneficial.
Natural infestations have been reported on not only crapemyrtles, but also on a wide range of plants from different families. A host plant is defined as a plant on which an insect is observed to complete its life cycle, especially with the presence of ovipositing gravid females [48,49]. Crapemyrtle bark scale exploits Punica granatum as a host, which seriously impacts the growth and fruiting of pomegranate and even leads to plant death [16,27,50,51]. It was also reported to feed on Buxus microphylla var. koreana, Celtis sinensis, Diospyros kaki, Ficus carica, Hypericum kalmianum, Ligustrum obtusifolium, Mallotus japonicus, Malus pumila, Myrtus sp., and Prunus serrulata and Rubus sp. [9,10,13,52,53]. In our previous study, infestations of CMBS were further confirmed on Malus angustifolia, Malus domestica, Chaenomeles speciosa, Diospyros rhombifolia, Heimia salicifolia, Lagerstroemia 'Spiced Plum', and twelve pomegranate cultivars [54]. Thus, CMBS is considered as a polyphagous insect with a relatively wide host range. In addition to L. indica, L. fauriei, and the interspecific hybrids, other crapemyrtle species, such as L. limii, L. subcostata, L. caudata, and L. speciosa, have been introduced into the United States as ornamental plants. To better manage CMBS in the U.S. and to help estimate its risks to ecosystems or green industries (wholesale and retail nurseries and landscape firms), further confirmation of CMBS hosts is necessary.
Currently, no CMBS-resistant crapemyrtle species or cultivars have been reported. Based on our previous observations [55], it is reasonable to predict that no L. indica, L. fauriei, or interspecific cultivars are immune to CMBS infestation. Infestation by CMBS was observed on nine crapemyrtle cultivars (Acoma, Basham's Party Pink, Catawba, Country Red, Muskogee, Natchez, Sarah's Favorite White, Sioux, and Tuscarora) in both landscapes and controlled environments. In addition, CMBS was observed on ten crapemyrtle cultivars (Biloxi, Burgundy Cotton, Chocataw, Lipan, Miami, New Orleans, Pink Ruffles, Powhatan, Royalty, and Tuskegee) in landscapes. Lythrum alatum, a plant in the same family (Lythraceae) as crapemyrtles, was reported as a CMBS host [17,30]. California loosestrife (Lythrum californicum) is native to California and is also distributed in Arizona, Kansas, New Mexico, Nevada, Oklahoma, Texas, and Utah. If Ly. californicum is indeed a host plant, its wide distribution will probably provide a continuum for spreading of CMBS. However, the suitability of Ly. californicum for CMBS is not yet known.
The aims of this study were to confirm additional plant hosts for CMBS and to test the host suitability among six Lagerstroemia species (L. caudata, L. fauriei 'Kiowa', L. indica 'Dynamite', L. limii, L. speciosa, and L. subcostata). The identification of less suitable species provides important information for breeding new CMBS-resistant cultivars.

CMBS and Host Range Test
The experiment was conducted in the Department of Horticultural Sciences greenhouse at Texas A&M University (30 • 36 31.9" N, 96 • 21 1.9" W. A set of the seven species mentioned above was enclosed in one cage and inoculated with CMBS-infested branches. The cage was replicated three times. Crapemyrtle branches infested with CMBS ( Figure 2a) were collected from the nursery at the Department of Horticultural Sciences of Texas A&M University in May 2019. Before the branches were attached to each test plant using Parafilm ® , all except five ovisacs on the branches were removed (Figure 2b). To ensure successful CMBS inoculation, each test plant was tied with newly collected branches containing five fresh ovisacs again five weeks after the initial inoculation. Cages were placed on different benches, approximately 2.5 m in distance, in the greenhouse at 25 ± 5 • C and 50 ± 10% relative humidity under a 10.5 h L: 13.5 h D photoperiod. The CMBS males were recognized by snow-white tubular sacs ( Figure 2c) and females were recognized by white round spindle-shaped ovisacs [17]. The numbers of the males and females, respectively, per plant were observed weekly for the first three weeks and then counted biweekly from three weeks after the first-time inoculation (WAI) until 25 WAI. successful CMBS inoculation, each test plant was tied with newly collected branches containing five fresh ovisacs again five weeks after the initial inoculation. Cages were placed on different benches, approximately 2.5 m in distance, in the greenhouse at 25 ± 5 °C and 50 ± 10% relative humidity under a 10.5 h L: 13.5 h D photoperiod. The CMBS males were recognized by snow-white tubular sacs ( Figure 2c) and females were recognized by white round spindle-shaped ovisacs [17]. The numbers of the males and females, respectively, per plant were observed weekly for the first three weeks and then counted biweekly from three weeks after the first-time inoculation (WAI) until 25 WAI.

Statistical Analysis
The experiment was arranged in a randomized complete block design with plant species being one treatment factor. Each of the experimental units was measured biweekly for 25 weeks, so the data collection time was the second treatment factor. Each cage was a block, and there were three blocks.
Log transformation as log10((No. of CMBS) + 1) was conducted prior to data analysis. The numbers of males and females on different species over 25 weeks were analyzed as repeated measures, respectively, using analysis of variance (ANOVA) with a mixed effect in JMP Pro 15 (SAS Institute, Cary, NC, USA). Plant species and data collection time were assigned with full factorial. The blocks were included as a random effect. Then, the least squares means (LSMeans) of the number of the CMBS on species were separated using Tukey's honestly significant difference (HSD) ( = 0.05). When needed, original data prior to log transformation or reverse-transformed data were presented. Graphs were plotted

Statistical Analysis
The experiment was arranged in a randomized complete block design with plant species being one treatment factor. Each of the experimental units was measured biweekly for 25 weeks, so the data collection time was the second treatment factor. Each cage was a block, and there were three blocks.
Log transformation as log 10 ((No. of CMBS) + 1) was conducted prior to data analysis. The numbers of males and females on different species over 25 weeks were analyzed as repeated measures, respectively, using analysis of variance (ANOVA) with a mixed effect in JMP Pro 15 (SAS Institute, Cary, NC, USA). Plant species and data collection time were assigned with full factorial. The blocks were included as a random effect. Then, the least squares means (LSMeans) of the number of the CMBS on species were separated using Tukey's honestly significant difference (HSD) (α = 0.05). When needed, original data prior to log transformation or reverse-transformed data were presented. Graphs were plotted using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Host Range Confirmation
The CMBS males were first observed on L. fauriei 'Kiowa' at two WAI. Beginning at three WAI (29 May 2019), white sacs were first observed on Ly. californicum and all other Lagerstroemia species. Meanwhile, the females were first seen on Ly. californicum and all Lagerstroemia species except L. speciosa and L. subcostata at five WAI, and were observed on all species at seven WAI. Average numbers of CMBS males and females increased and peaked around 17 WAI on most species. The number of the males decreased at 19 WAI ( Figure 3) and female densities decreased at 21 WAI (Figure 4). Because the life cycle of CMBS is around six weeks [17,30], CMBS on all test plants would have completed at least one life cycle (the period roughly goes: eggs → nymphs → adult females (fertilized by males) → laying eggs), which confirmed that all test plant species were accepted by CMBS, and that they were CMBS hosts. Black sooty mold resulting from honeydew excreted by CMBS was observed on the bark or leaves of all Lagerstroemia plants at 17 WAI ( Figure 5). No black sooty mold was observed on CMBS-infected Ly. californicum.

Host Range Confirmation
The CMBS males were first observed on L. fauriei 'Kiowa' at two WAI. Beginning at three WAI (29 May 2019), white sacs were first observed on Ly. californicum and all other Lagerstroemia species. Meanwhile, the females were first seen on Ly. californicum and all Lagerstroemia species except L. speciosa and L. subcostata at five WAI, and were observed on all species at seven WAI. Average numbers of CMBS males and females increased and peaked around 17 WAI on most species. The number of the males decreased at 19 WAI ( Figure 3) and female densities decreased at 21 WAI (Figure 4). Because the life cycle of CMBS is around six weeks [17,30], CMBS on all test plants would have completed at least one life cycle (the period roughly goes: eggs → nymphs → adult females (fertilized by males) → laying eggs), which confirmed that all test plant species were accepted by CMBS, and that they were CMBS hosts. Black sooty mold resulting from honeydew excreted by CMBS was observed on the bark or leaves of all Lagerstroemia plants at 17 WAI ( Figure 5). No black sooty mold was observed on CMBS-infected Ly. californicum.

The Suitability for CMBS Differed Significantly among the Lagerstroemia Species
The number of CMBS reflects the host suitability for CMBS among Lagerstroemia species. There was no interaction between species and time affecting the number of CMBS males (F = 1.42; df = 55,132; p = 0.0558). The fixed-effect test showed that the main factors, plant species (F = 3.96; df = 5,12; p = 0.0236) and time (F = 50.1; df = 11,132; p < 0.0001), had significant effects on the number of CMBS males. Based on the 25-week comparison results using Tukey's HSD (Table 2), the LSMeans of the average number of the males on L.

The Suitability for CMBS Differed Significantly among the Lagerstroemia Species
The number of CMBS reflects the host suitability for CMBS among Lagerstroemia species. There was no interaction between species and time affecting the number of CMBS males (F = 1.42; df = 55,132; p = 0.0558). The fixed-effect test showed that the main factors, plant species (F = 3.96; df = 5,12; p = 0.0236) and time (F = 50.1; df = 11,132; p < 0.0001), had significant effects on the number of CMBS males. Based on the 25-week comparison results using Tukey's HSD (Table 2), the LSMeans of the average number of the males on L.

The Suitability for CMBS Differed Significantly among the Lagerstroemia Species
The number of CMBS reflects the host suitability for CMBS among Lagerstroemia species. There was no interaction between species and time affecting the number of CMBS males (F = 1.42; df = 55,132; p = 0.0558). The fixed-effect test showed that the main factors, plant species (F = 3.96; df = 5,12; p = 0.0236) and time (F = 50.1; df = 11,132; p < 0.0001), had significant effects on the number of CMBS males. Based on the 25-week comparison results using Tukey's HSD (Table 2), the LSMeans of the average number of the males on L. speciosa was significantly lower than on the other six species (L. limii and L. subcostata, L. fauriei 'Kiowa' and L. indica 'Dynamite', and L. caudata). However, the LSMeans among these other species had no significant difference over the 25 weeks. According to the average number of CMBS (Figure 3), the highest number of the males on L. subcostata was 1057 ± 107 (mean ± SE) at 17 WAI, whereas the highest number on L. speciosa was 45 ± 29 (mean ± SE) at 19 WAI. Table 2. The least squares means of the male and female Acanthococcus lagerstroemiae and sex ratio (male-female) on different Lagerstroemia species within 25 weeks after inoculation.

Plant Species
No. Males (95% CI z ) No. Females (95% CI) Sex Ratio z CI = confidence intervals. y Log transformation as log 10 ((No. of CMBS) +1) was conducted prior to data analysis. The original number of CMBS and the reverse-transformed 95% CI values are presented. The numbers within a single column indicated by the same letter are not significantly different within 25 weeks, as compared by Tukey's honestly significant difference (HSD) (α = 0.05).

The Effect of the Species-Time Interaction on the Weeks after Inoculation (WAI) when the Number of CMBS Increased Significantly Compared to the Previous Week on Different Species
The number of males on L. limii, L. subcostata, or L. indica 'Dynamite' did not increase significantly compared to the previous week until 11 WAI (L. fauriei 'Kiowa' at 13 WAI) ( Table 3 and Table S1). There was no significant increase in the number of the males on L. caudata or L. speciosa between consecutive weeks during the 25-week experiment. The number of CMBS females on L. limii, L. subcostata, L. fauriei 'Kiowa', or L. indica 'Dynamite' did not become significantly higher compared to the previous week until 11 WAI (Table 3  and Table S1), and then, there was no significant change in insect densities among these crapemyrtle species. The number on L. caudata or L. speciosa did not increase significantly compared to the previous week until 17 WAI, representing six weeks later than the more suitable crapemyrtle species.

Discussion
The hosts confirmed in this study validated Lagerstroemia indica, which agrees with the CMBS hosts listed in Kozar's findings [53], and L. fauriei (mentioned as L. japonica in the host list [15,53]). Moreover, this study added four additional Lagerstroemia species (L. limii, L. caudata, L. speciosa, and L. subcostata) and Ly. californicum as CMBS hosts.
One important finding is that L. speciosa is not suitable for the growth and development of CMBS. Among all tested crapemyrtle species, L. speciosa supported CMBS's growth and development the least, as indicated by the lowest numbers of male and female CMBS ( Table 2). The highest number of males on L. speciosa was nearly 23-fold less than that on L. subcostata. The largest peak of the females on L. speciosa was 10-fold less than that on L. limii. A previous feeding preference study found that L. speciosa was the least preferred host for crapemyrtle aphids [5]. Thus, it is reasonable to predict that L. speciosa is not suitable for the growth and development of phloem-sap hemipterans.
An interesting observation from this study was the different sex ratio of CMBS on different crapemyrtle species. Even though the number of male CMBS on L. speciosa did not differ significantly over the 25 weeks (Table 3), the number of females (fertilized by male CMBS) on L. speciosa increased significantly from 15 to 17 WAI, which was six weeks later than on the more suitable species (L. subcostata, L. fauriei 'Kiowa', and L. indica 'Dynamite'). The sex ratio (male-female) of CMBS on L. speciosa (1.6:1) was much lower than on other tested species, such as L. subcostata (2.8:1), which may restrict the occurrence of severe CMBS infestation on L. speciosa for a period. Herbivore sex ratios can be affected by environmental factors, host plant defensive chemistry, and nutrient availability [66][67][68][69][70]. Our results showed different sex ratios of CMBS on different crapemyrtle species, which hinted at the importance of the male insect's contribution to individual reproduction or the population dynamics via the quality of nuptial gifts [71,72].
Different levels of host suitability could be attributed to, but not limited to, physical properties [6], a balance between stimulation and deterrence [73,74], and some secondary metabolites of the plant [75,76]. For example, many alkaloids, terpenoids, flavonoids, sterols, and polyphenols in different structural types have been isolated from various parts of different Lagerstroemia species, such as L. indica, L. subcostata, L. fauriei, and L. speciosa [77][78][79]. Currently, there is no report on the association between CMBS suitability and plant compounds. To further understand the CMBS-host interaction, one important future direction would be to investigate the role of plant compounds on host suitability and biological parameters of A. lagerstroemiae, which would help improve the integrated pest management for CMBS.

Conclusions
This study confirmed L. limii, L. caudata, L. speciosa, L. subcostata, and Ly. californicum as CMBS hosts in addition to the previously reported L. indica 'Dynamite' and L. fauriei. Importantly, these Lagerstroemia species showed significantly different suitability to CMBS. Lagerstroemia speciosa was the least suitable for CMBS, as indicated by the lowest numbers of CMBS males and females, and can be utilized as a parental plant for breeding new CMBS-resistant cultivars.
Supplementary Materials: The following are available online at https://www.mdpi.com/2075-4 450/12/1/6/s1, Table S1: The effect of the species-time interaction on the weeks after inoculation (WAI) when the number of CMBS males increased significantly compared to the previous week on different species, Table S2: The effect of the species-time interaction on the weeks after inoculation (WAI) when the number of CMBS females increased significantly compared to the previous week on different species.