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Plants and their associated insect herbivores, represent more than 50% of all known species on earth. The first step in understanding the mechanisms generating and maintaining this important component of biodiversity is to identify plant-herbivore associations. In this study we determined insect-host plant associations for an entire guild of insect herbivores using plant DNA extracted from insect gut contents. Over two years, in a tropical rain forest in Costa Rica (La Selva Biological Station), we recorded the full diet breadth of rolled-leaf beetles, a group of herbivores that feed on plants in the order Zingiberales. Field observations were used to determine the accuracy of diet identifications using a three-locus DNA barcode (rbcL, trnH-psbA and ITS2). Using extraction techniques for ancient DNA, we obtained high-quality sequences for two of these loci from gut contents (rbcL and ITS2). Sequences were then compared to a comprehensive DNA barcode library of the Zingiberales. The rbcL locus identified host plants to family (success/sequence = 58.8%) and genus (success/sequence = 47%). For all Zingiberales except Heliconiaceae, ITS2 successfully identified host plants to genus (success/sequence = 67.1%) and species (success/sequence = 61.6%). Kindt's sampling estimates suggest that by collecting ca. four individuals representing each plant-herbivore interaction, 99% of all host associations included in this study can be identified to genus. For plants that amplified ITS2, 99% of the hosts can be identified to species after collecting at least four individuals representing each interaction. Our study demonstrates that host plant identifications at the species-level using DNA barcodes are feasible, cost-effective, and reliable, and that reconstructing plant-herbivore networks with these methods will become the standard for a detailed understanding of these interactions.

Insects represent the biodiversity of the terrestrial ecosystem, and their prosperity is attributable to their association with symbiotic microorganisms. By sequestering microbial functionality into their bodies, organs, tissues, or cells, diverse insects have successfully exploited otherwise inaccessible ecological niches and resources, including herbivory enabled by utilization of indigestible plant cell wall components. In leaf beetles of the subfamily Cassininae, an ancient symbiont lineage, Stammera , whose genome is extremely reduced and specialized for encoding pectin-degrading enzymes, is hosted in gut-associated symbiotic organs and contributes to the host’s food plant digestion. Diverse insects host specific microbial symbionts that play important roles for their growth, survival, and reproduction. They often develop specialized symbiotic organs for harboring the microbial partners. While such intimate associations tend to be stably maintained over evolutionary time, the microbial symbionts may have been lost or replaced occasionally. How symbiont acquisitions, replacements, and losses are linked to the development of the host’s symbiotic organs is an important but poorly understood aspect of microbial symbioses. Cassidine leaf beetles are associated with a specific gammaproteobacterial lineage, Stammera , whose reduced genome is streamlined for producing pectin-degrading enzymes to assist the host’s digestion of food plants. We investigated the symbiotic system of 24 Japanese cassidine species and found that (i) most species harbored Stammera within paired symbiotic organs located at the foregut-midgut junction, (ii) the host phylogeny was largely congruent with the symbiont phylogeny, indicating stable host-symbiont association over evolutionary time, (iii) meanwhile, the symbiont was not detected in three distinct host lineages, uncovering recurrent losses of the ancient microbial mutualist, (iv) the symbiotic organs were vestigial but present in the symbiont-free lineages, indicating evolutionary persistence of the symbiotic organs even in the absence of the symbiont, and (v) the number of the symbiotic organs was polymorphic among the cassidine species, either two or four, unveiling a dynamic evolution of the host organs for symbiosis. These findings are discussed as to what molecular mechanisms and evolutionary trajectories underpin the recurrent symbiont losses and the morphogenesis of the symbiotic organs in the herbivorous insect group. IMPORTANCE Insects represent the biodiversity of the terrestrial ecosystem, and their prosperity is attributable to their association with symbiotic microorganisms. By sequestering microbial functionality into their bodies, organs, tissues, or cells, diverse insects have successfully exploited otherwise inaccessible ecological niches and resources, including herbivory enabled by utilization of indigestible plant cell wall components. In leaf beetles of the subfamily Cassininae, an ancient symbiont lineage, Stammera , whose genome is extremely reduced and specialized for encoding pectin-degrading enzymes, is hosted in gut-associated symbiotic organs and contributes to the host’s food plant digestion. Here, we demonstrate that multiple symbiont losses and recurrent structural switching of the symbiotic organs have occurred in the evolutionary course of cassidine leaf beetles, which sheds light on the evolutionary and developmental dynamics of the insect’s symbiotic organs and provides a model system to investigate how microbial symbionts affect the host’s development and morphogenesis and vice versa .

A catalog is presented of species of the subfamily Cassidinae (Coleoptera, Chrysomelidae) deposited in the Insect Collection of the College of Agriculture, Yangtze University in Jingzhou, Hubei, China. A complete list of all specimens for each species is provided, together with the label data. A total of 1,207 adult specimens of Cassidinae were examined, representing 53 species, 13 genera, and six tribes. Fourteen species are recorded in Hubei Province for the first time: Aspidomorpha (s. str.) transparipennis (Motschulsky, 1860), Basiprionota gressitti Medvedev, 1957, Callispa dimidiatipennis Baly, 1858, Cassida australica (Boheman, 1855), C. expansa Gressitt, 1952, C. juglans Gressitt, 1942, C. pallidicollis Boheman, 1856, C. postarcuata (Chen & Zia, 1964), C. quinaria (Chen & Zia, 1964), C. rati Maulik, 1923, C. sigillata (Gorham, 1885), Glyphocassis (Hebdomecosta) spilota (Gorham, 1885), Hispellinus chinensis Gressitt, 1950, and H. moerens (Baly, 1874). Five species are newly recorded from Chongqing: Aspidomorpha (s. str.) difformis (Motschulsky, 1860), Cassida sauteri (Spaeth, 1913), C. vespertina Boheman, 1862, Gly. (Hebdomecosta) spilota (Gorham, 1885), and Dactylispa chinensis Weise, 1905. Furthermore, Callispa almora Maulik, 1923 is new to Zhejiang Province, Platypria aliena Chen & Sun, 1962 is new to Guangxi Zhuang Autonomous Region, and Cassida rubiginosa Müller, 1776 is new to Sichuan Province.

Abstract Zatrephina lineata (Coleoptera: Chrysomelidae) is a phytophagous insect, mainly of plants of the genera Ipomoea and Mikania. The objective was to study the development, survival and to describe the life stages of Z. lineata fed on leaves of Ipomoea pes-caprae. Biological observations were made daily with the aid of a stereoscopic microscope and the instars of this insect identified by the exuvia left between one moulting and the next. The duration of development and survival of the egg, larva and pupa stages and the first, second, third, fourth and fifth instars and of the nymph stage of Z. lineata differed, but not between sexes of this insect. The duration of development of Z. lineata was longer in the larval stage and in the fifth instar, and its survival greater in the egg and pupa stages and in the first and fifth instars. Zatrephina lineata eggs, cream-colored, are ellipsoid and deposited in groups on the adaxial surface of older I. pes-caprae leaves. The larvae of this insect go through five instars, with the first three being gregarious with chemo-behavioral defenses. The exarated pupae of Z. lineata, light yellow in color and with an oval shape flattened dorsoventrally, attach to the abaxial surface of the I. pes-caprae leaves. The shape of adults of this insect is oval, straw yellow in color with lighter longitudinal stripes and females are slightly larger than males. Resumo Zatrephina lineata (Fabricius, 1787) (Coleoptera: Chrysomelidae) é um inseto fitófago, principalmente de plantas dos gêneros Ipomoea e Mikania. O objetivo foi estudar o desenvolvimento, a sobrevivência e descrever as fases de vida de Z. lineata alimentada com folhas de Ipomoea pes-caprae. Observações biológicas foram feitas diariamente com auxílio de microscópio estereoscópico e os ínstares desse inseto identificados pela exúvia deixada entre uma muda e outra. A duração do desenvolvimento e sobrevivência dos estágios de ovo, larva e pupa e dos primeiro, segundo, terceiro, quarto e quinto ínstares e do periodo ninfal de Z. lineata diferiu, mas não entre os sexos deste inseto. A duração do desenvolvimento de Z. lineata foi maior na fase larval e no quinto ínstar, e sua sobrevivência maior nas fases de ovo e pupa e no primeiro e quinto ínstares. Os ovos de Z. lineata, de cor creme, são elipsoides e depositados em grupos na superfície adaxial das folhas mais velhas de I. pes-caprae. As larvas desse inseto passam por cinco ínstares, sendo os três primeiros gregários com defesas quimio-comportamentais. As pupas exaradas de Z. lineata, de cor amarelo claro e formato oval achatado dorsoventralmente, fixam-se na superfície abaxial das folhas de I. pes-caprae. O formato dos adultos deste inseto é oval, de cor amarelo palha com listras longitudinais mais claras e as fêmeas são ligeiramente maiores que os machos.

Five subfamilies within Chrysomelidae (leaf beetles) have larvae that retain their feces as a coat or armor which serves for thermoregulation, camouflage, or barrier to enemies. The construction, retention and repair of these fecal structures are associated with specialized larval morphologies in the tortoise beetles (subfamily Cassidinae) and in the Cryptocephalinae + Lamprosomatinae (Camptosomata), but morphology associated with fecal encrustations on larvae in the Blepharida -group flea beetles (Galerucinae: Alticini) and in Criocerinae have not been examined. Experiments with live larvae of Podontia quatuordecimpunctata (L., 1767) (or sineguelas leaf beetle, SLB; Blepharida group) reveal the anus opens dorsally and deposits feces directly to the larva’s dorsum; the armor is maintained and is reconstructed. Scanning electron microscopy reveals integumental microtrichia that presumably hold on the feces. This invasive beetle has become an introduced tree-crop pest in the Philippines, so ongoing research seeks to mitigate its population. Insecticidal chemical assays show that fecal armor does not fully protect SLB larvae but delays potency slightly. The study recommends rotating the insecticides (Imidacloprid, Cypermethrin, and Buprofezin) to prevent the development of resistance. Specialized morphology for fecal retention is known in Cassidinae, Camptosomata and is now documented in the Blepharida group. Such morphology and the fecal-building behavior can offer additional phylogenetic information for these beetles.

Body size is a key trait influencing life history and ecological adaptation, and sexual size dimorphism (SSD) reflects divergent selective pressures acting on males and females. In morphologically conserved insect groups such as Cassidinae leaf beetles, the external similarity between sexes often impedes accurate dimorphism assessment. To address this, we conducted a systematic morphometric study of ten Cassidinae species from the Nanling Mountains—the largest east–west mountain system in southern China—where we definitively assigned sex via genital dissection. We measured body weight, body length, body width, length–width ratio, and corresponding wing traits. Across all species, SSD was consistently female biased, with statistically significant but subtle differences in most traits; body weight exhibited the greatest relative disparity. While this pattern aligns with the fecundity advantage hypothesis, direct fecundity data were not collected. Crucially, interspecific allometric analyses revealed that the scaling of male and female body sizes was statistically indistinguishable from that of isometry, providing no significant support for Rensch’s rule in this female-biased system. Our findings offer foundational insights into SSD evolution in cryptically dimorphic, herbivorous beetles and highlight the need for phylogenetically informed studies across broader geographic and taxonomic scales.

Animal constructions are the outcomes of complex evolutionary, behavioural, and ecological forces. A brief review of diverse animal builders, the materials used, and the functions they provide their builders is provided to develop approaches to studying faecal-based constructions and faecal-carrying in leaf beetles (Coleoptera: Chrysomelidae). Field studies, rearing, dissections, photography, and films document shields constructed by larvae in two species in two tribes of the subfamily Cassidinae, Calyptocephala attenuata (Spaeth, 1919) (Spilophorini), and Cassida sphaerula Boheman, 1853 (Cassidini). Natural history notes on an undetermined Cassidini species and Stolas cucullata (Boheman, 1862) (Tribe Mesomphaliini) outline the life cycle of tortoise beetles and explain terms. Commonly, the cassidine shield comprises exuviae onto which faeces are daubed, producing a pyramidal-shaped shield that can cover most of the body (up to the pronotum). In Cal. attenuata the larval shield comprises only exuviae, while in Cass. sphaerula , instar 1 initiates the shield by extending its telescopic anus to apply its own faeces onto its paired caudal processes; at each moult the exuvia is pushed to the caudal process base but remains attached, then more faeces are applied over it. The larva’s telescopic anus is the only tool used to build and repair the shield, not mouthparts or legs, and it also applies chemicals to the shield. Pupae in Cal. attenuata retain part of the exuviae-only shield of instar VI, while pupae in Cass. sphaerula retain either the entire 5 th instar larval shield (faeces + all exuviae) or only the 5 th larval exuvia. The caudal processes are crucial to shield construction, shield retention on the body, and as materials of the central scaffold of the structure. They also move the shield, though the muscular mechanism is not known. Altogether the faecal + exuviae shields may represent a unique morpho-behavioural synapomorphy for the crown-clade Cassidinae (10 tribes, ~ 2669 species) and may have been a key innovation in subsequent radiation. Defensive shields and domiciles may help explain the uneven radiation of chrysomelid subfamilial and tribal clades.

Predator–prey interactions may be responsible for enormous morphological diversity in prey species. We performed predation experiments with morphological manipulations (ablation) to investigate the defensive function of dorsal spines and explanate margins in Cassidinae leaf beetles against three types of predators: assassin bugs (stinger), crab spiders (biter), and tree frogs (swallower). There was mixed support for the importance of primary defense mechanisms (i.e., preventing detection or identification). Intact spined prey possessing dorsal spines were more likely to be attacked by assassin bugs and tree frogs, while intact armored prey possessing explanate margins were likely to avoid attack by assassin bugs. In support of the secondary defense mechanisms (i.e., preventing subjugation), dorsal spines had a significant physical defensive function against tree frogs, and explanate margins protected against assassin bugs and crab spiders. Our results suggest a trade‐off between primary and secondary defenses. Dorsal spines improved the secondary defense but weakened the primary defense against tree frogs. We also detected a trade‐off in which dorsal spines and explanate margins improved secondary defenses against mutually exclusive predator types. Adaptation to different predatory regimes and functional trade‐offs may mediate the diversification of external morphological defenses in Cassidinae leaf beetles. We revealed defensive function of dorsal spines and explanate margins in Cassidinae leaf beetles and detected functional trade‐offs. These results suggest that adaptation to different predatory regimes and functional trade‐offs contribute to the diversification of external morphologies in herbivorous insects.

The complete mitochondrial genome of Basiprionota bisignata (Boheman, 1862) (a species of leaf beetles) was successfully sequenced, annotated, and analyzed in this study. This mitochondrial genome is a circular DNA molecule of 16,069 bp in size with 78.5% AT content, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an AT-rich region (control region). The gene order is consistent with the putative ancestral arrangement of insects. All PCGs are initiated by ATN (A/T/C/G) condons and terminated with TAA/G or their incomplete form single T-. All tRNAs can be folded into common clover leaf secondary structures, except for trnS1. The phylogenetic tree was reconstructed using maximum likelihood analysis, and the topology recovered the monophyly of Cassidinae and the sister relationship between Basiprionota and the clade (Thlaspida + Aspidomorph).