Six complete mitochondrial genomes of ground beetles from the Harpalinae and Carabinae (Coleoptera, Carabidae) with phylogenetic analysis based on mitogenomic data.

In this study, we employed high-throughput sequencing technology to determine the complete mitochondrial genomes of six ground beetles, encompassing five Harpalinae species and one Carabinae species. The sizes of mitochondrial genomes ranged from 15,334 to 16,972 bp, encompassing 37 genes, including 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Furthermore, each species was found to possess a putative control region. Combining with 65 published mitochondrial genome sequences of Carabidae as ingroups and four species from Trachypachidae, Gyrinidae and Dytiscidae as outgroups, we conducted phylogenetic analyses utilizing Maximum likelihood and Bayesian inference methods. Moreover, we reconstructed a species tree of Carabidae based on mitochondrial genome data using the coalescent-based species tree method (ASTRAL). The results revealed that the family Carabidae was not a monophyletic group. The subfamily Harpalinae was supported to be a monophyletic group in Maximum likelihood analysis. Although the subfamily Carabinae was found to be nonmonophyletic in the concatenation analyses under both Maximum likelihood and Bayesian inference criteria, it was identified as a monophyletic group in the species tree analysis.

Effects of dietary urea supplementation on performance of selected insect species.

Feeding costs of farmed insects may be reduced by applying alternative nitrogen sources such as urea that can partly substitute true proteins. The aim of this study was to examine the effects of different nitrogen sources on body weight (BW) and survival rate (SR) of the Jamaican field cricket (JFC, Gryllus assimilis), the house cricket (HC, Acheta domesticus), yellow mealworm larvae (YM, Tenebrio molitor) and superworm larvae (SW, Zophobas morio). Crickets were either housed individually or in groups, and larvae were group-housed. Six isonitrogenous feeds composed of 3.52% nitrogen were designed for all four insect species using four independent replicates with micellar casein: urea proportions of 100-0%, 75-25%, 50-50%, 25-75%, 0-100% and 100% extracted soybean meal. All selected insect species were able to utilise urea. However, urea as the only nitrogen source resulted in low final BW. In the HC, the JFC, and the YM on nitrogen basis urea can replace 25% of micellar casein without having any negative effects on BW and SR in comparison to the 100% micellar casein group. In the SW, a 25% urea level did not have a significant effect on final BW, but SR decreased significantly.

Clonal genomic population structure of Beauveria brongniartii and Beauveria pseudobassiana: Pathogens of the common European cockchafer (Melolontha melolontha L.).

Beauveria brongniartii is a fungal pathogen that infects the beetle Melolontha melolontha, a significant agricultural pest in Europe. While research has primarily focused on the use of B. brongniartii for controlling M. melolontha, the genomic structure of the B. brongniartii population remains unknown. This includes whether its structure is influenced by its interaction with M. melolontha, the timing of beetle-swarming flights, geographical factors, or reproductive mode. To address this, we analysed genome-wide SNPs to infer the population genomics of Beauveria spp., which were isolated from infected M. melolontha adults in an Alpine region. Surprisingly, only one-third of the isolates were identified as B. brongniartii, while two-thirds were distributed among cryptic taxa within B. pseudobassiana, a fungal species not previously recognized as a pathogen of M. melolontha. Given the prevalence of B. pseudobassiana, we conducted analyses on both species. We found no spatial or temporal genomic patterns within either species and no correlation with the population structure of M. melolontha, suggesting that the dispersal of the fungi is independent of the beetle. Both species exhibited clonal population structures, with B. brongniartii fixed for one mating type and B. pseudobassiana displaying both mating types. This implies that factors other than mating compatibility limit sexual reproduction. We conclude that the population genomic structure of Beauveria spp. is primarily influenced by predominant asexual reproduction and dispersal.

Diversity and complexity of arthropod references in haiku.

Haiku are short poems, each composed of about 10 words, that typically describe moments in nature. People have written haiku since at least the 17th century, and the medium continues to be popular with poets, amateurs, educators, and students. Collectively, these poems represent an opportunity to understand which aspects of nature-e.g., which taxa and biological traits-resonate with humans and whether there are temporal trends in their representation or the emotions associated with these moments. We tested this potential using a mix of linguistic and biological methods, in analyses of nearly 4,000 haiku that reference arthropods. We documented the taxa and the life history traits represented in these poems and how they changed over time. We also analyzed the poems for emotion and tone. Our results reveal a mix of predictable trends and compelling surprises, each of which stand to potentially inform engagement strategies. At least 99 families of arthropods, in 28 orders, are represented in these haiku. The eight most commonly referenced taxa, from highest to lowest number of references, include: Lepidoptera, Hymenoptera, Diptera, Coleoptera, Araneae, Orthoptera, Hemiptera, and Odonata. Several common, conspicuous orders were never referenced, including Trichoptera, Plecoptera, and Megaloptera. The most commonly referenced traits relate to ecology (especially habitat, phenology, time of day), behavior (especially sound production), phenotype (especially color), and locomotion (especially flight). The least common traits in haiku relate to arthropod reproduction and physiology. Our analyses revealed few obvious temporal trends in the representations of taxa, biological traits, or emotion and tone. The broader implications of these results and possible future directions are discussed.

Seeds of nonhost species as sources of toxic compounds for the cowpea weevil Callosobruchus maculatus (F.).

Cowpea weevil, Callosobruchus maculatus, is the primary pest of stored cowpea seeds. The management of this infestation currently relies on insecticides, resulting in environmental pollution and selection of insecticide-resistant pests. Consequently, research efforts are being devoted to identify natural insecticides as sustainable and environment friendly alternatives for the control of C. maculatus. In this study, we explore the toxic effects of the nonhost seeds Parkia multijuga, Copaifera langsdorffii, Ormosia arborea, Amburana cearensis, Lonchocarpus guilleminianus, Sapindus saponaria, and Myroxylon peruiferum, on the cowpea weevil C. maculatus. Notably, all nonhost seeds led to reductions between 60 and 100% in oviposition by C. maculatus females. Additionally, the larvae were unable to penetrate the nonhost seeds. Artificial seeds containing 0.05% to 10% of cotyledon flour were toxic to C. maculatus larvae. Approximately 40% of larvae that consumed seeds containing 0.05% of O. arborea failed to develop, in contrast to control larvae. Proteomic analysis of A. cearensis and O. arborea seeds identify revealed a total of 371 proteins. From those, 237 are present in both seeds, 91 were exclusive to O. arborea seeds, and 43 were specific to A. cearensis seeds. Some of these proteins are related to defense, such as proteins containing the cupin domain and 11S seed storage protein. The in silico docking of cupin domain-containing proteins and 11S storage protein with N-acetylglucosamine (NAG)4 showed negative values of affinity energy, indicating spontaneous binding. These results showed that nonhost seeds have natural insecticide compounds with potential to control C. maculatus infestation.

Potential of Thuja occidentalis L. Essential Oil and Water Extracts against Field Crop Pests.

Thuja occidentalis L. essential oil (EOTO) and its compounds, such as terpinyl acetate, bornyl acetate, and ß-thujone, are claimed to be highly effective against some storage pests, sanitary insects, or pests of fruit trees, while data about its use in protecting field crops are very scarce. There is also a lack of information in the literature about the insecticidal value of water extracts from T. occidentalis (WETOs). Both essential oils (EOs) and water extracts (WEs) from various plants have advantages and disadvantages in terms of their use as insecticides. EOs are generally more effective, but their preparation is more complicated and quite expensive. In turn, WEs are simple to prepare and cheap, but they often have limited effectiveness. Moreover, significant differences in responses exist depending on the species of the donor plant, the method of preparing the extract, its concentration, the species of the pest being controlled, the developmental stage, and even the gender of the pest. The goals of the research were to assess the effect of EOTO and WETOs prepared from dry and fresh matter on the mortality, feeding, and body mass changes of important crop pests, i.e., the black bean aphid, pea leaf weevil, and Colorado potato beetle (CPB), respectively, as well as on the mortality and voracity of non-target organism Asian lady beetle young larvae. EOTO showed significant aphicidal activity with LC50 = 0.8267% and 0.2453% after 42 h of the experiment for nymphs and wingless females of black bean aphid, respectively. Adults of CPB were more resistant to EOTO than aphids, with LC50 values for females equal to 1.5327% and 1.3113% after 48 h and after 72 h of the experiment. There was no significant effect of EOTO on CPB foraging. Calculated LC50 values for pea leaf weevil adults were lower than those for CPB (0.9638% and 0.8573% for males after 12 h and 24 h, respectively). In the case of this pest, a clear reduction in foraging was obtained, with higher concentrations of EOTO resulting in more pronounced reductions in foraging behavior. Concentrations of EOTO above 0.5%, which showed efficacy against the aphid, were lethal to 3-day-old larvae of the Asian lady beetle. WETOs, in turn, showed significant potential in inhibiting adult pea leaf weevil feeding, with very low or no effectiveness in reducing A. fabae and CPB, respectively.

Phytochemical screening and insecticidal activities of some medicinal plants against the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae).

This study emphasizes the phytochemical study of some locally available botanicals against maize weevils. Nine plant parts were collected from six plant species. The test plant powder (200 g) was suspended sequentially in 600 ml of petroleum ether, chloroform, acetone, methanol, and distilled water for 72 h with frequent agitation. Different concentrations of the crude extracts were applied to maize seeds at rates of 10 ml, 15 ml and 20 ml per 100 g. All treatments with different extracts at different rates of application showed significant differences (p < 0.05) in the cumulative mean percentage mortality of the maize weevil. The seed extract of Maesa lanceolata and Croton macrostachyus and the leaf extract of Clausena anisata showed cumulative percent mortality ranged 95.32-98.02% in 28 days after treatment application. There was no significant difference (p > 0.05) among all treatments for the prevention of F1 progeny emergence. In all extracts, Clausena anisata showed 100% inhibition of F1 progeny emergence. All treatments significantly reduced seed weight loss and damage. The treated maize seeds were germinated with an acceptable germination quality. In conclusion, an increased dosage of the extract resulted in significant mortality in maize weevils. The seed extracts of Maesa lanceolata and Croton macrostachyus and Clausena anisata leaf extract were observed to be the most promising botanical in protecting stored maize against maize weevil.

Morphological diversity in true and false crabs reveals the plesiomorphy of the megalopa phase.

Brachyura and Anomala (or Anomura), also referred to as true and false crabs, form the species-rich and globally abundant group of Meiura, an ingroup of Decapoda. The evolutionary success of both groups is sometimes attributed to the process of carcinization (evolving a crab-like body), but might also be connected to the megalopa, a specific transitional larval phase. We investigate these questions, using outline analysis of the shields (carapaces) of more than 1500 meiuran crabs. We compare the morphological diversity of different developmental phases of major ingroups of true and false crabs. We find that morphological diversity of adults is larger in false crabs than in true crabs, indicating that taxonomic diversity and morphological diversity are not necessarily linked. The increasing morphological disparity of adults of true and false crabs with increasing phylogenetic distance furthermore indicates diverging evolution of the shield morphology of adult representatives of Meiura. Larvae of true crabs also show larger diversity than their adult counterparts, highlighting the importance of larvae for biodiversity studies. The megalopa phase of Meiura appears to be plesiomorphic, as it overlaps between true and false crabs and shows little diversity. Causes may be common evolutionary constraints on a developmental phase specialized for transitioning.

Vertically inherited microbiota and environment modifying behaviours conceal genetic variation in dung beetle life history.

Diverse organisms actively manipulate their (sym)biotic and physical environment in ways that feed back on their own development. However, the degree to which these processes affect microevolution remains poorly understood. The gazelle dung beetle both physically modifies its ontogenetic environment and structures its biotic interactions through vertical symbiont transmission. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess how environment modifying behaviour and microbiome transmission shape heritable variation and evolutionary potential. We found that depriving larvae of symbionts and environment modifying behaviours increased additive genetic variance and heritability for development time but not body size. This suggests that larvae's ability to manipulate their environment has the potential to modify heritable variation and to facilitate the accumulation of cryptic genetic variation. This cryptic variation may become released and selectable when organisms encounter environments that are less amenable to organismal manipulation or restructuring. Our findings also suggest that intact microbiomes, which are commonly thought to increase genetic variation of their hosts, may instead reduce and conceal heritable variation. More broadly, our findings highlight that the ability of organisms to actively manipulate their environment may affect the potential of populations to evolve when encountering novel, stressful conditions.

The first chromosome-level genome of the stag beetle Dorcus hopei Saunders, 1854 (Coleoptera: Lucanidae).

Stag beetles (Coleoptera: Lucanidae) represent a significant saproxylic assemblage in forest ecosystems and are noted for their enlarged mandibles and male polymorphism. Despite their relevance as ideal models for the study of exaggerated mandibles that aid in attracting mates, the regulatory mechanisms associated with these traits remain understudied, and restricted by the lack of high-quality reference genomes for stag beetles. To address this limitation, we successfully assembled the first chromosome-level genome of a representative species Dorcus hopei. The genome was 496.58 Mb in length, with a scaffold N50 size of 54.61 Mb, BUSCO values of 99.8%, and 96.8% of scaffolds anchored to nine pairs of chromosomes. We identified 285.27 Mb (57.45%) of repeat sequences and annotated 11,231 protein-coding genes. This genome will be a valuable resource for further understanding the evolution and ecology of stag beetles, and provides a basis for studying the mechanisms of exaggerated mandibles through comparative analysis.

The paralobe: A new diagnostic and synapomorphic character for the genera Paulipalpina Gnaspini & Peck, 1996 and Parapaulipalpina Gnaspini, 1996 (Leiodidae: Cholevinae: Ptomaphagini).

In insect taxa with homogeneous external morphology, genital structures often emerge as essential traits for interspecific differentiation. In the tribe Ptomaphagini (Coleoptera, Leiodidae, Cholevinae), precise identification often depends on analyzing the male genital morphology, even at the genus level. Here, we present a new character for diagnosing the genera Paulipalpina Gnaspini & Peck, 1996 and Parapaulipalpina Gnaspini, 1996. This feature, which we dub 'paralobe', is a projection arising from the internal surface of the right lobe of the aedeagal apex. Based on its absence in other beetles, including other Ptomaphagini, we recognize it as a putative synapomorphy for those genera. The recognition of this previously overlooked structure adds important information for understanding the sequence of changes that occurred in the male genitalia among the genera of Ptomaphagini.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation.

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

The efficacy of green silica nanoparticles synthesized from rice straw in the management of Callosobruchus maculatus (Col., Bruchidae).

Rice straw, a byproduct of harvesting rice, must be disposed of by farmers in a variety of ways, including burning, which is hazardous for the environment. To address this issue, the straw needs to be utilized and turned into valuable products. One such product is nano-silica (SNPs), which will be synthesized and investigated in our study as a safe alternative to chemical insecticides. Rice straw-derived SNPs were synthesized using the Sol-Gel method. The contact toxicity of SNPs on Callosobruchus maculatus, a major pest of cowpea seeds, has been assessed. The size of synthesized SNPs was determined by transmission electron microscopy to be ~ 4 nm. The SNPs estimated LC50 on C. maculatus adults was 88.170 ppm after 48h exposure. By raising the tested concentration, SNPs treatment increased the mortality%, which reached 100% at 200 ppm exposures. Additionally, SNPs at LC50 treatment decreased adult longevity and the average number of emerged adults. The findings also verified that SNPs had no phytotoxic effects on the cowpea seeds germination. Rather, their application improved seed germination efficacy. This study proposed that rice straw can be utilized to manufacture highly efficient SNPs which can be efficiently employed to preserve stored grains from C. maculatus infestation.

Girdling behavior of the longhorn beetle modulates the host plant to enhance larval performance.

BACKGROUND: Preingestive behavioral modulations of herbivorous insects on the host plant are abundant over insect taxa. Those behaviors are suspected to have functions such as deactivation of host plant defenses, nutrient accumulation, or modulating plant-mediated herbivore interactions. To understand the functional consequence of behavioral modulation of insect herbivore, we studied the girdling behavior of Phytoecia rufiventris Gautier (Lamiinae; Cerambycidae) on its host plant Erigeron annuus L. (Asteraceae) that is performed before endophytic oviposition in the stem. RESULTS: The girdling behavior significantly increased the larval performance in both field monitoring and lab experiment. The upper part of the girdled stem exhibited lack of jasmonic acid induction upon larval attack, lowered protease inhibitor activity, and accumulated sugars and amino acids in compared to non-girdled stem. The girdling behavior had no effect on the larval performance of a non-girdling longhorn beetle Agapanthia amurensis, which also feeds on the stem of E. annuus during larval phase. However, the girdling behavior decreased the preference of A. amurensis females for oviposition, which enabled P. rufiventris larvae to avoid competition with A. amurensis larvae. CONCLUSIONS: In conclusion, the girdling behavior modulates plant physiology and morphology to provide a modulated food source for larva and hide it from the competitor. Our study implies that the insect behavior modulations can have multiple functions, providing insights into adaptation of insect behavior in context of plant-herbivore interaction.

Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

Visualized in-sensor computing.

In artificial nervous systems, conductivity changes indicate synaptic weight updates, but they provide limited information compared to living organisms. We present the pioneering design and production of an electrochromic neuromorphic transistor employing color updates to represent synaptic weight for in-sensor computing. Here, we engineer a specialized mechanism for adaptively regulating ion doping through an ion-exchange membrane, enabling precise control over color-coded synaptic weight, an unprecedented achievement. The electrochromic neuromorphic transistor not only enhances electrochromatic capabilities for hardware coding but also establishes a visualized pattern-recognition network. Integrating the electrochromic neuromorphic transistor with an artificial whisker, we simulate a bionic reflex system inspired by the longicorn beetle, achieving real-time visualization of signal flow within the reflex arc in response to environmental stimuli. This research holds promise in extending the biomimetic coding paradigm and advancing the development of bio-hybrid interfaces, particularly in incorporating color-based expressions.

Symbiosis: Did bacteria bias the beetle big bang?

The massive species richness of certain taxonomic groups has long enchanted evolutionary biologists, but even within such groups there are biases in cladogenesis. A study of Metazoa's greatest radiation - the beetles - points to metabolic symbioses with bacteria as a possible driver of enhanced diversification in herbivorous clades.

Defenses of whirligig beetles against native and invasive frogs.

Many native insects have evolved defenses against native predators. However, their defenses may not protect them from non-native predators due to a limited shared history. The American bullfrog, Aquarana catesbeiana (Anura: Ranidae), which has been intentionally introduced to many countries, is believed to impact native aquatic animals through direct predation. Adults of whirligig beetles (Coleoptera: Gyrinidae), known for swimming and foraging on the water surface of ponds and streams, reportedly possess chemical defenses against aquatic predators, such as fish. Although whirligig beetles potentially encounter both bullfrogs and other frogs in ponds and lakes, the effectiveness of their defenses against frogs has been rarely studied. To assess whether whirligig beetles can defend against native and non-native frogs, we observed the behavioral responses of the native pond frog, Pelophylax nigromaculatus (Anura: Ranidae), and the invasive non-native bullfrog, A. catesbeiana, to native whirligig beetles, Gyrinus japonicus and Dineutus orientalis, in Japan. Adults of whirligig beetles were provided to frogs under laboratory conditions. Forty percent of G. japonicus and D.orientalis were rejected by P. nigromaculatus, while all whirligig beetles were easily consumed by A. catesbeiana. Chemical and other secondary defenses of G. japonicus and D. orientalis were effective for some individuals of P. nigromaculatus but not for any individuals of A. catesbeiana. These results suggest that native whirligig beetles suffer predation by invasive non-native bullfrogs in local ponds and lakes in Japan.