Fluorescent marking
Dispersal of SLF adults was tracked using a fluorescent marking system (FMS), which has been demonstrated to be applicable for multiple insect species including SLF nymphs21,22,24. To mark the SLF, either red, yellow, or blue fluorescent paint (#1166R, #1166Y, #1166B, BioQuip Products, USA) was diluted with distilled water (1:4). The mixture was then gently sprayed three times (ca. 20 mg each time) on each SLF individual using a mist sprayer from a distance of 30–50 cm (SI 2). Throughout the field survey, a handheld ultraviolet (UV) laser (PX 600 mW, class IIIB purple laser, 405 nm, Big Lasers, USA) was used to detect fluorescent-marked SLF individuals25.
Effect of fluorescent marking on SLF
Prior to field survey, the potential effects of fluorescent marking on the survivorship and flight behavior of SLF adults (sex ratio 1:1) were evaluated. SLF adults were collected using sweeping nets (BioQuip Products, USA) from Gyeonggi-do, South Korea (37°47′85.95″ N, 127°11′64.58″E) in September 2020. Two hours after fluorescent marking of SLF, both fluorescent-marked and unmarked SLF were subjected to survivorship and flight behavior assessment.
Survivorship of insects was measured on two A. altissima trees (ca. 2 m in height) located in Gachon University, South Korea (37°45′38.50″N, 127°13′37.75″E). Two fluorescent-marked and two unmarked insects were placed in a cylindrical mesh cage [25 × 30 cm (radius × height)] enclosing a tree branch; a total of 20 groups were tested (n = 40). Then, survivorship of SLF was determined once every two days until no individuals were alive. Survivorship was compared between fluorescent-marked and unmarked SLF using Kaplan-Meir survivorship analysis (JMP 12, SAS Institute Inc., USA).
The effects of fluorescent marking on flight behavior were evaluated in an open space (986 m2) in Gachon University, South Korea (37°45′08.37″N, 127°12′79.69″E) at 26 ± 1 °C and a relative humidity of 30 ± 5%. To induce flight of SLF adults, a wooden square rod [3 × 3 × 100 cm (width × length × height)] was established upright at the center of the arena. The SLF adult was placed individually 10 cm away from the top on the wooden square rod. To minimize any unnecessary stimuli from experimenter, SLF flight was induced by following the same sequence: once the insect climbed up the rod and oriented itself staying still to a random direction, then an experimenter carefully positioned at the back of the insect and gently pecked the forewings using tweezers to initiate its flight33,34. Pecking was intended to mimic predatory behavior of birds. Once the insect jumped away, an operator followed the individual until it landed on the ground (n = 30). The experiment was conducted for 2 h between 13:00–15:00 and marked and unmarked SLF were randomly tested during the evaluation. The number of pecks to initiate the flight, flight duration, and flight distance of SLF were compared using t-test (JMP 12, SAS Institute Inc., USA).
Field study sites
Dispersal patterns of SLF adults in A. altissima patches and their oviposition patterns were investigated in multiple A. altissima patches located along two streams in Gyeonggi-do, South Korea: Tan stream in Seongnam-si (37°48′01.80″N, 127°11′56.03″E) and Gyeongan stream in Gwangju-si (37°41′54.21″N, 127°27′12.37″E). Both Tan and Gyeongan streams run along suburban residential areas in their respective cities, with pedestrian lanes built along the streams. We selected seven A. altissima patches as study patches when more than 10 SLF adults were found per patch (Fig. 3). In the study patch, all SLF individuals or ca. up to 30 adults were florescent-marked. In addition, when the number of SLF adults was less than 10 from an A. altissima patch, those patches were designated as neighboring patches (Fig. 3). Dispersal and oviposition of SLF adults were monitored from both study and neighboring patches during the study.
In Tan stream, four study patches (patches A–D) and one neighboring patch, which were distributed over ca. 1760 m, were selected (Fig. 3a). Areas around the patches were generally covered with grass and shrubs, and the areas were occasionally managed by local administration. Deciduous trees were regularly planted along the pedestrian lanes. There were a total of four, four, 61, and 47 A. altissima trees in patches A to D, respectively (Table 2). Compared with Tan stream, A. altissima patches were located closely to each other in Gyeongan stream: three study patches (patches E–G) and three neighboring patches were spread over only ca. 90 m (Fig. 3b). Vegetation surrounding A. altissima patches consisted of grasses and small shrubs as well as deciduous trees planted along the border of residential area nearby. There were a total of 69, nine, and 53 A. altissima trees in patches E to G, respectively (Table 2). Unlike Tan stream, 45% of A. altissima trees had trunks having cut off by local administration in Gyeongan stream (Table 2; Fig. 5).
Dispersal pattern of SLF on A. altissima
Three fluorescent paint colors were used to mark SLF individuals in the study patches (Fig. 3; SI 2). Insects that took off during marking were captured and excluded from the experiment. Among the selected study patches, SLF adults were generally distributed throughout each patch, while SLF adults were observed only from one out of 61 A. altissima trees in patch C. As a result, in Tan stream, 15 (color of paint used to fluorescent-marking; red), 31 (yellow), 11 (blue), and 32 (red) adults were marked from patches A to D, respectively, whereas in Gyeongan stream, 30 (red), 30 (blue), and 33 (yellow) adults were marked from patches E to G, respectively. Starting from September 14th, 2020 in Tan stream and September 18th in Gyeongan stream, fluorescent-marked SLF adults on A. altissima trees in both study and neighboring patches were counted with a UV laser twice a week (Fig. 3). Survey continued until no individuals were observed from the study patches.
Oviposition pattern of SLF on A. altissima
Oviposition pattern of SLF was surveyed on all A. altissima trees in the study patches in December in both streams (Table 2). For the survey, SLF egg masses were categorized into three types as follows: egg mass with waxy layer, egg mass without waxy layer, and scattered eggs (SI 3). Eggs that were not covered with waxy layer and did not form aggregates were categorized as scattered (SI 3). In the field, A. altissima trees were visually inspected to identify SLF egg mass, and the number of egg masses and their distances from the ground were recorded. In addition, the number of eggs per egg mass was recorded for egg masses located < 2.5 m above the ground. When egg masses were covered with waxy layer, brushes were used to gently remove the powder-like waxy layer.
To characterize the A. altissima trees surveyed for SLF egg mass, we recorded the following morphological characteristics: tree height, DRC, and cutting status. Heights of A. altissima trees < 2 m were measured using an iron steel measuring tape, and those > 2 m were estimated with reference to the investigator’s height. DRC of A. altissima was determined by measuring the circumference of the stem at the root collar using a tape measure. In case of multi-stemmed A. altissima trees, the diameter of each stem was first determined individually, and the DRC of the tree was calculated using the following equation:
$$ DRC = sqrt {mathop sum limits_{i = 1}^{n} d_{i}^{2} } $$
where n is the number of stems and di denotes the stem diameter of the A. altissima tree.
The effects of A. altissima height, DRC, their interactions, and cutting status of the trees on the number of egg masses were analyzed using a generalized linear mixed model (GLMM) assuming a Poisson distribution with a log link function (JMP 12, SAS Institute Inc., USA). Patch was included as random effect in the model. Due to concerns over collinearity between tree height and DRC, variance inflation factor (VIF) was calculated: VIF > 5 generally indicates collinearity35,36. VIF between height and DRC was 1.56, and therefore the two variables were included together in the GLMM model.
Policy statement
Experiments involving Ailanthus altissima were conducted in compliance with relevant institutional, national, and international guidelines and legislation.
Source: Ecology - nature.com
