Sizing up spotted lanternfly nymphs for instar determination and growth allometry

PLOS ONE RESEARCH ARTICLE Sizing up spotted lanternfly nymphs for instar determination and growth allometry Theodore Bien1, Benjamin H. Alexander1, Eva White1, S. Tonia Hsieh ID2, Suzanne Amador Kane ID1* 1 Physics and Astronomy Department, Haverford College, Haverford, Pennsylvania, United States of America, 2 Department of Biology, Temple University, Philadelphia, Pennsylvania, United States of America * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Bien T, Alexander BH, White E, Hsieh ST, Kane SA (2023) Sizing up spotted lanternfly nymphs for instar determination and growth allometry. PLoS ONE 18(2): e0265707. https://doi. org/10.1371/journal.pone.0265707 Editor: Frank H. Koch, USDA Forest Service Southern Research Station, UNITED STATES Received: March 3, 2022 Accepted: January 3, 2023 Published: February 2, 2023 Copyright: © 2023 Bien et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data and code required to reproduce all results and figures discussed here are accessible on Figshare (DOI: 10.6084/m9.figshare.19287389). Funding: S. Tonia Hsieh National Science Foundation CAREER award (IOS-1453106). https:// www.nsf.gov The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract A major ongoing research effort seeks to understand the behavior, ecology and control of the spotted lanternfly (SLF) (Lycorma delicatula), a highly invasive pest in the U.S. and South Korea. These insects undergo four nymphal stages (instars) before reaching adulthood, and appear to shift host plant preferences, feeding, dispersal and survival patterns, anti-predator behaviors, and response to traps and chemical controls with each stage. However, categorizing SLF life stage is challenging for the first three instars, which have the same coloration and shape. Here we present a dataset of body mass and length for SLF nymphs throughout two growing seasons and compare our results with previously-published ranges of instar body lengths. An analysis using two clustering methods revealed that 1st-3rd instar body mass and length fell into distinct clusters consistently between years, supporting using these metrics to stage nymphs during a single growing season. The length ranges for 2nd-4th instars agreed between years in our study, but differed from those reported by earlier studies for diverse locations, indicating that it is important to obtain these metrics relevant to a study’s region for most accurate staging. We also used these data to explore the scaling of SLF instar bodies during growth. SLF nymph body mass scaled with body length varied between isometry (constant shape) and growing somewhat faster than predicted by isometry in the two years studied. Using previously published data, we also found that SLF nymph adhesive footpad area varies in direct proportion to weight, suggesting that footpad adhesion is independent of nymphal stage, while their tarsal claws display positive allometry and hence disproportionately increasing grasp (mechanical adhesion). By contrast, mouthpart dimensions are weakly correlated with body length, consistent with predictions that these features should reflect preferred host plant characteristics rather than body size. We recommend future studies use the body mass vs length growth curve as a fitness benchmark to study how SLF instar development depends on factors such as hatch date, host plant, temperature, and geographic location, to further understanding of life history patterns that help prevent further spread of this invasive insect. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0265707 February 2, 2023 1 / 13 PLOS ONE Sizing up spotted lanternfly nymphs for instar determination and allometry Introduction The spotted lanternfly (SLF), Lycorma delicatula White (Hemiptera: Fulgoridae) is a planthopper native to south Asia that has become a highly invasive pest in the U.S. and South Korea. SLFs feed intensively on phloem from a wide variety of trees and other plants, stressing the hosts as well as promoting the growth of sooty mold [1]. Because SLFs threaten significant economic damage to agricultural crops, native trees, and landscape plants, a large ongoing research effort seeks to understand their development, physiology, behavior and ecology to inform methods for mitigation and control [2–4]. In this study, we discuss how clustering methods can be applied to measurements of the body mass and size of immature SLFs (nymphs) in order to improve the determination of SLF life stage and to study the scaling of previously published SLF footpart and mouthpart dimensions [5] with body size. We begin by explaining how these issues are relevant to a wide variety of topics in SLF research. After emerging, SLFs develop through five life stages separated by molting: four nymphal instars and the much larger and winged adult stage. The 4th instars are readily identified by their distinctive red, black and white spotted coloration. However, the first through third instars have similar black-and-white-spotted coloration and overall body morphology (Fig 1) [6]. Many studies of SLF behavior, ecology, and phenology have relied on determination of the nymphal stage (instar determination) in order to track how life stage influences ecology and choice of host plants [1,2], dispersal patterns [3–5,7], locomotor behaviors such as climbing and jumping [8,9], phenology and activity [10], spectral preferences [6], attraction to chemicals [11], and effectiveness of various trapping methods [12]. Thus, instar determination methods for identifying the life stage of a given specimen collected in the field are useful and important in many contexts. Several previous studies have shown how a detailed microscopic examination can reveal foot, mouth part and antenna morphological changes during development [8,13,14], providing information of great utility for how these factors influence feeding, adhesion and locomotion throughout the insect’s life cycle. In practice, the life stages of the first three SLF instars have been estimated in many studies using overall body dimensions readily measured in the field, along with previously published size ranges for each instar. In spite of this growing interest, only a few previous studies have reported measured data for the ranges of body lengths corresponding to each nymphal life stage for use in instar Fig 1. Photograph of 1st, 2nd, 3rd, and 4th instar spotted lanternfly nymphs. Double-headed arrow shows the definition of body length, L. (scale bar = 10 mm). https://doi.org/10.1371/journal.pone.0265707.g001 PLOS ONE | htt (...truncated)