A Beetle In Vitro: Establishment of a Short-Term Cell Culture from the Pest Popillia japonica

Simple Summary

The Japanese beetle is an invasive insect that causes serious damage to agriculture and natural ecosystems. Until now, no means of studying its biology in the lab have been available using living cells, limiting progress in understanding this pest and controlling its invasion. In this study, we developed the first cell culture from Japanese beetle larvae using fat bodies, a tissue important for growth and energy storage. These cells were successfully grown and maintained in the laboratory over multiple cycles, showing stable appearance and healthy structure for a limited period. This new cell culture represents a short-term and accessible in vitro system for studying the beetle’s biology in controlled conditions. Although we failed to establish an immortalized cell line, they provide a useful model for investigating cytological and physiological responses to treatments, leading to understanding how the beetles can be affected by control strategies. This work lays the groundwork for future discoveries that could support better, more targeted approaches to managing this harmful species.

Abstract

The Japanese beetle (Popillia japonica Newman) is a highly invasive, polyphagous scarab causing significant agricultural and ecological damage across invaded regions. While molecular studies are gaining traction, the unavailability of P. japonica cell lines has constrained in vitro investigations. To overcome these limitations and provide a platform for controlled biological investigation, we developed the first cell culture derived from P. japonica larvae. Fat bodies from field-collected third-instar larvae were dissected and cultured. Cells initially formed floating spheroids before transitioning to adherent monolayers. Cultures remained stable over several splits, whereas a marked reduction in cell number was observed at the eighth split due to the onset of contamination. Fluorescence microscopy confirmed nuclear integrity, while transmission electron microscopy at split 5 revealed cytoplasmic features consistent with insect fat body cells, including lipid droplets. The cell culture predominantly contained trophocyte-like cells, consistent with the known cellular composition of insect fat bodies. Transcriptomic analyses comparing fresh fat bodies and cultured cells revealed moderate transcriptional divergence, with limited upregulation of genes associated with iron homeostasis and stress response, consistent with adaptive responses to in vitro conditions. While not immortalized, this cell culture offers a short-term model for studying P. japonica physiology, toxicology, host–pathogen interactions, and potential gene-targeting strategies under controlled conditions. This work represents a first step toward enabling molecular and cellular research in this economically important pest species.

1. Introduction

In the last centuries, human activities have accelerated the spread of invasive organisms, leading to a reduction in native species richness and abundance, changes in trophic networks, and alterations in ecosystem functioning, as well as significant economic losses to agriculture [1]. Among these, the Japanese beetle (Popillia japonica Newman), a polyphagous scarab native to Japan, represents a major threat to both the Old and New Worlds. Following its accidental introduction into North America in the early 20th century, the species rapidly expanded across United States and Canada, causing significant damage to agriculture [2,3]. In Europe, the first detection occurred in the Azores during the 1970s, with recent widespread outbreaks documented across the continent [4,5,6].

Due to its remarkable capacity for rapid invasion and its broad host range, feeding on over 400 plant species including turfgrass, vineyards, and fruit crops, P. japonica has caused substantial economic losses to agriculture in invaded countries [7]. As such, the beetle has been included in the A2 list quarantine pest of the European Plant Protection Organization and many control measures were taken to (a) reduce population numbers in well-established zones with phytosanitary surveillance; (b) limit the movement of the beetle’s plant hosts as much as possible; and (c) delimitate the infested areas through the establishment of risk and buffer zones (Commission Implementing Regulation (EU) 2023/1584; available at: https://eur-lex.europa.eu).

Because of the high phytosanitary risks associated with living P. japonica specimens, their use in research is restricted to specialized and regulated laboratories. This has posed a barrier to many institutions seeking to study the beetle’s biology. Nonetheless, interest in its molecular characterization is increasing. Notably, Carroll et al. [8] recently demonstrated the first gene knockout in P. japonica, while a high-quality genome assembly from an Italian population has been published [9].

In this context, the development of a P. japonica cell line represents a significant advancement, offering a first step towards a versatile and accessible platform for molecular and cellular studies. Indeed, insect cell lines have emerged as valuable tools for entomological research, enabling a range of experimental applications including (i) the implementation of functional genomics approaches, such as RNA interference (RNAi), to deepen the knowledge on gene functions in a controlled and replicable cellular environment [10,11]; (ii) the analysis of gene expression and signal transduction pathways, supporting advances in insect physiology and immunology [12]; (iii) the investigation of insect–virus interactions, with implications for the study of plant–pathogen transmission dynamics [12,13]; (iv) the screening of pest management strategies under controlled conditions, particularly important as insecticide resistance continues to rise [14,15]; and (v) the replication of viruses, particularly important for the production of recombinant proteins commonly used in virological applications [16].

Here, we present the first cell culture obtained from the fat bodies of P. japonica larvae. We describe the methodology used for its development and maintenance and discuss the broad potential of this tool for future research in insect molecular biology, pest control, and host–pathogen interactions.

2. Materials and Methods

2.1. Insect Sampling and Tissue Extraction

Ten third-instar larvae of P. japonica were sampled in April 2023 in the municipality of Cameri (N 45°28′46″, E 8°38′14″), in Novara province (Italy). Prior to dissection, larvae were rinsed three times in 70% ethanol. Fat bodies were dissected individually from each larva (n = 10) under sterile conditions using a Leica Wild M3C stereomicroscope (Leica; Wetzlar, Germany) and collected in ten separate tubes, each used to establish one cell culture. Adipose tissues were recovered in tubes containing an antibiotic solution of penicillin (200 U/mL) and streptomycin (0.2 mg/mL; Merck KgaA; Darmstadt, Germany), to prevent contamination from gut-associated bacteria. Cells were pelleted with a 5 min centrifuge at 500× g and rinsed with IPL-41 Insect Medium (1X) (Gibco™, Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Cytiva; Marlborough, MA, USA). To allow cell dissociation, cells were treated with trypsin 1:250 (500 mg/L) and EDTA (372 mg/L) by pipetting multiple times, as in [17].

2.2. Culture Conditions

Ten batches of cell culture were maintained up to a confluent state, in a humidified atmosphere at 27 °C, in T-75 flasks (n = 10) containing 15 mL IPL-41 Insect Medium (1X) with the addition of 10% FBS, glutamine (2 mM), penicillin (100 U/mL), streptomycin (0.1 mg/mL) and doxycycline (5 µg/mL; Merck KgaA; Darmstadt, Germany). Cells were propagated by splitting 1:4 once a week. The clones were expanded and employed up to 8 splits. Cells were periodically cryopreserved in 1 mL of FBS with 10% DMSO (Merck KgaA; Darmstadt, Germany).

2.3. Cell Counting Assessment

Cell counts were performed in duplicate at multiple time points up to split 5 (week 5). At each time point, cells from two independent T-75 flasks were detached with trypsin-EDTA and resuspended in 1 mL of IPL-41 Insect Medium, and 8 µL of the suspension were loaded into a Thoma chamber. Cell numbers were then assessed under a Nikon Eclipse E400 light microscope (Nikon Instruments Europe; Amsterdam, The Netherlands) at 10× magnification. Counting data were plotted in RStudio v. 4.4.0 using ggplot2 v. 3.5.1 [18], reporting the total number of cells ± standard deviation (SD).

2.4. Nuclei Analysis

At split 5, 5 × 10⁵ cells were seeded into a new culture flask. After a two-day incubation, the cells were detached and recovered. The culture medium was carefully removed from the flask into a 15 mL tube to minimize cell loss. Subsequently, 2 mL of trypsin 1:250 (500 mg/L) were added to the flask to fully cover the adherent cell layer, and the flask was incubated at room temperature for 2 min. The detached cells were then transferred into the tube containing the previously collected culture medium. The tube was centrifuged at 300× g for 5 min at 4 °C. After removing the supernatant, the resulting cell pellet was resuspended in 1 mL of IPL-41 Insect Medium (1X) and prepared for morphological analysis.

Cells were stained following a modified protocol based on [19]. Specifically, DAPI (Merck KgaA; Darmstadt, Germany) was diluted in PBS (1:1000) and used as a nuclear counterstain. It was added to the cell suspension, vortexed briefly, and incubated for 10 min. A drop of Fluoromount™ Aqueous Mounting Medium (Merck KgaA; Darmstadt, Germany) was placed on a microscope slide, and several drops of the stained cell suspension were added. Cell images were acquired using a Leica Leitz DMRB (Leica; Wetzlar, Germany) light microscope equipped with an AxioCam MRc5 digital camera (Zeiss; Göttingen, Germany).

2.5. Molecular Validation

To confirm the species-specific origin of the cultured cells, DNA was extracted at the third split using the Wizard^® Genomic DNA Purification Kit (Promega; Madison, WI, USA) with the following modifications. A total of ~1 × 10⁶ cells were treated with 1 mL of trypsin 1:250 (500 mg/L) and centrifuged at 13,000× g. After removing the supernatant, cells were resuspended and washed by adding 200 μL of PBS. After a second centrifugation, cells were processed following the manufacturer’s instructions. The mitochondrial cox1 barcode gene was amplified using specific primers COX1_PJ_67F (forward; 5′-GGAAGTTGAGCAGGAATAG) and COX1_PJ_679R (reverse; 5′-AAATGTAGAGAATGGGGTC) designed based on the publicly available mitogenome reference [4]. Amplifications were obtained in a BioRad T100 thermal cycler (Bio-Rad Laboratories, Inc.; Hercules, CA, USA) under the following conditions: 95 °C 30 s, 50 °C 30 s, 72 °C 60 s × 40 cycles with an initial denaturation step of 95 °C 3 min and a final elongation of 72 °C 7 min. PCR products were visualized in an agarose gel, purified, and Sanger-sequenced (BMR Genomics; Padua, Italy) as described in [20]. Electropherograms were manually curated with Sequencher 4.4.2 software (Gene Codes; Ann Arbor, MI, USA) and their identity was confirmed through BLASTn v. 2.15.0 [21].

2.6. Transmission Electron Microscopy

Following the protocol described in Section 2.4, cells were recovered, detached, and resuspended in IPL-41 Insect Medium (1X) during split 5. A total of 600 µL of phosphate buffer (PB) with 2.5% glutaraldehyde (0.1 M) was added to the pelleted cells after careful removal of the culture medium, and the suspension was incubated for 2 h at 4 °C. After two washes in 600 µL of PB, the cells were post-fixed in 600 µL 1% osmium tetroxide for 1 h at 4 °C. After thorough rinsing, the material was dehydrated in a graded ethanol series (50–100%), then transferred into a 1:1 mixture of propylene oxide and Epon-Araldite resin (SERVA Electrophoresis; Heidelberg, Germany) and cured overnight at 4 °C. All centrifugation steps were carried out at 500× g for 5 min. The next day, the material was transferred into a more concentrated mixture of propylene oxide and Epon-Araldite resin (1:2). After a two-hour incubation at room temperature, the selected material was transferred into pure Epon-Araldite resin. Finally, the material was embedded in small silicone molds to form resin blocks, which were polymerized for 48 h in an oven at 60 °C.

Semithin sections (70 nm) of the cells, obtained using a Reichert Ultracut ultramicrotome (Leica; Wetzlar, Germany), were stained with 1% uranyl acetate followed by lead citrate and observed using a FEI Tecnai G2 SPIRIT (Thermo Fisher Scientific; Hillsboro, OR, USA) transmission electron microscope operating at an accelerating voltage of 120 kV, equipment with CMOS camera TVIPS F216 (TVIPS; Gilching, Germany).

2.7. RNA Extraction and Transcriptome Analysis

To assess potential biases in transcript expression between cultured cells and fresh fat body tissues, three frozen larvae were dissected to isolate fat bodies, and total RNA was extracted using the QIAGEN RNeasy Micro Kit (QIAGEN; Hilden, Germany), including QIAshredder homogenization and DNase treatment. Similarly, three independent batches of cell cultures at split 6 were thawed, re-seeded in culture, and processed for RNA extraction using the same protocol.

RNA concentration and purity were evaluated using a NanoDrop One spectrophotometer (Thermo Fisher Scientific; Wilmington, DE, USA). mRNA libraries were constructed using the TruSeq Stranded mRNA kit (Illumina, Inc.; San Diego, CA, USA) and sequenced on an Illumina NextSeq 550 platform (Illumina, Inc.; San Diego, CA, USA) at Polo GGB (Siena, Italy). Raw sequencing data were quality-checked using MultiQC v1.17 [22] and trimmed with fastp v0.23.2 [23] using default parameters.

The most recently published P. japonica transcriptome [24] was used as the reference to quantify non-normalized read counts for each group (three cultured cells libraries and three fresh tissues libraries). Transcript quantification was performed using Salmon v0.14.1 with the validateMappings flag enabled [25]. Read counts were extracted using the quantmerge command and used for differential expression analysis with the DESeq2 v1.44.0 R package [26]. Read counts were variance-stabilizing transformed (vst), and batch effects were removed using the removeBatchEffect function from the limma v3.60.2 R package [27]. Differential expression between cultured cells and fresh tissues was assessed using the ashr shrinkage method [28]. Data was graphically curated in R-studio v. 4.4.0 employing ggplot2 v. 3.5.1 [18].

3. Results

Cell cultures were successfully initiated from P. japonica larval fat bodies. Upon plating, dissociated cells exhibited mixed growth behaviors, including the rapid formation of compact cellular aggregates (spheroids) that detached from the substrate and floated in suspension (Figure 1a). This aggregative behavior was prominent during the first few days of culture. Over time, particularly after the first split, spheroid formation declined, and adherent cells became predominant (Figure 1b). By the second split (approximately two weeks after seeding), circular adherent clusters began to emerge, indicating the onset of organized monolayer formation (Figure 1c). By the third to fourth week (split 3), cultures were largely composed of adherent cells, homogeneously distributed across the culture surface (Figure 2a). Fluorescence microscopy after DAPI staining revealed well-defined nuclei throughout the culture period, confirming nuclear integrity and cellular health (Figure 2b–d). By the end of the fourth week, with the culture dominated by adherent, morphologically stable cells, the mean cell diameter was measured at 15.7 ± 1.6 μm (mean, SD; Figure 2).

At split 5, cell ultrastructure was further investigated using transmission electron microscopy. Cells displayed round to oval morphologies with dimensions averaging 21 ± 8.7 µm in diameter (mean ± SD). Cytoplasmic features were consistent with their insect fat body origin, including well-preserved nuclei, mitochondria, abundant lipid droplets of variable sizes, and numerous electron-dense granules and proteinaceous inclusions (Figure 3a–c).

To monitor cell growth, cell numbers were assessed up to split 5 (week 5), corresponding to the time point preceding the molecular and cellular biology experiments. Two days after seeding (week 0), the culture contained 675,000 ± 49,497 cells (mean ± SD; Figure 4). Across subsequent splits, cell numbers showed limited variation, with a modest increase by week 4 (28 days), reaching 960,000 ± 141,421 cells (mean ± SD; Figure 4). The final measurement at week 5 recorded 770,000 ± 42,426 cells (mean ± SD; Figure 4). At split eight, a bacterial contamination was detected despite the presence of antibiotics in the culture medium, after which cell numbers sharply declined.

Importantly, the cryopreservation and subsequent thawing of cells in liquid nitrogen did not negatively affect cell viability, suggesting potential for temporary storage and short-term reuse.

PCR amplification of the cox1 barcode gene successfully yielded a clean amplicon. Sanger sequencing followed by BLASTn analysis confirmed a 100% match to P. japonica reference sequences (GenBank accession: OP903034.1), validating the taxonomic origin of the cell culture.

RNA sequencing yielded a total of 42.4–54.8 million raw paired-end reads per sample. Quality trimming removed a maximum of 5% of reads across all libraries. Principal component analysis (PCA) of variance-stabilized counts showed a modest separation between cultured cells and fresh tissue along PC1, which accounted for 40% of the variance. Fresh tissue samples formed a tight cluster, whereas cultured samples displayed much broader dispersion. This variability was captured primarily along PC2 (34% of the variance), highlighting substantial transcriptomic heterogeneity among the in vitro cultures and a more limited heterogeneity between cultured cell and fresh tissue samples (Figure 5).

Differential expression analysis identified a total of 17,524 differentially expressed transcripts between cultured cells and fresh tissues. Nevertheless, among these, only eight transcripts were significantly upregulated in cultured cells. BLASTp v. 2.17.0 searches against the UniProt database annotated these transcripts as reported in

Table 1. Annotation of transcripts significantly upregulated in Popillia japonica cultured cells compared with fresh fat body tissues. The table reports transcript identifiers from the Trinity assembly, corresponding UniProt accession numbers, and functional descriptions based on BLASTp searches. Full nucleotide sequences of these transcripts are provided as Supplementary Material (File S1) and derive from the transcriptome assembly published by Cucini et al. [24].

Transcript ID	Uniprot Entry	Uniprot Description
TRINITY_DN1956_c0_g1_i13	A0AAW1KPW4	Ferritin
TRINITY_DN1923_c0_g1_i2	A0A7 × 3FWF4	Tubulin alpha chain
TRINITY_DN157_c0_g2_i2	A0AAW1MH71	Gelsolin repeat
TRINITY_DN3867_c0_g1_i3	A0AAW1IX89	Insect allergen related repeat, nitrile-specifier detoxification
TRINITY_DN4053_c0_g1_i3	A0AAW1IYE1	Zinc carboxypeptidase A 1
TRINITY_DN4191_c3_g1_i4	A0ABM2A4K7	Uncharacterized protein
TRINITY_DN11913_c0_g1_i3	A0AAW1LPJ0	Cold-shock DNA-binding domain
TRINITY_DN10907_c0_g2_i2	A0AAW1I9E2	60S ribosomal protein L4 C-terminal domain

.

4. Discussion

The rapid spread of P. japonica in non-native regions poses a substantial threat to agriculture, natural ecosystems, and local economies due to its broad host range and high reproductive potential. As invasions intensify, countries are actively seeking effective containment strategies to reduce its impact. However, current management efforts and ongoing research—such as the use of insecticides [24]—are limited to already invaded areas.

In this context, the development of a cell line derived from P. japonica represents a useful experimental resource, toward which the present study constitutes a significant initial step. Insect cell lines have proven instrumental across a range of disciplines, from virology and immunology to toxicology and biotechnology. To contribute to this growing research infrastructure, we provide the first attempt to establish a cell culture from P. japonica, derived from larval fat bodies—a tissue widely used for insect cell line development due to its metabolic plasticity and proliferative capacity [29,30,31]. Insect fat bodies are mainly composed of trophocytes (also called adipocytes) and associated cells known as oenocytes [32], primarily related to the detoxification of toxic compounds [33]. Interestingly, our P. japonica cell culture consisted predominantly of vacuolated cells rich in lipid droplets and dense inclusions, likely corresponding to trophocytes (Figure 3). While previous studies have demonstrated the possibility of a successful establishment of continuous insect cell lines from single tissues (e.g., [31]) or mixed organ sources [34,35], our P. japonica cell culture exhibited limited longevity, with stable proliferation maintained for up to five splits. The subsequent decline in cell numbers and contamination observed at later splits indicate that the system did not achieve long-term stability or continuous passaging capability. This instability likely reflects the non-transformed nature of the starting cells, which retained differentiation features incompatible with sustained, immortalized growth.

Moreover, we investigated gene expression differences between fresh fat bodies and cultured cells. Overall, the transcriptomic divergence between the two conditions was present but not substantial. Although PCA revealed a separation between cultured cells and fresh tissues (Figure 5), this pattern was primarily driven by a broad dispersion among cultured replicates, indicating a high degree of intra-culture transcriptomic heterogeneity rather than a uniform transcriptional shift. In line with this pattern, only a small subset of transcripts was significantly upregulated in cultured cells compared with fresh fat bodies. Together, these results suggest that the observed transcriptional differences primarily reflect adaptive responses to in vitro culture conditions rather than a clear functional reprogramming or mechanistic transition associated with stable cell line establishment. This limited differential expression likely reflects both the reduced cellular complexity of the in vitro system and the selective activation of genes required for survival and modest proliferation under culture conditions.

Among the upregulated transcripts (Table 1), ferritin has previously been identified as a key regulator of iron homeostasis and an essential mitogenic factor in insect cell cultures, where iron-loaded ferritin promotes cell growth and proliferation. Its upregulation in P. japonica cultured cells is therefore consistent with the observed capacity of these cells to proliferate under in vitro conditions, albeit for a limited number of splits [36]. Other upregulated transcripts encode proteins involved in stress response and metabolic plasticity, including insect allergen-related repeats, nitrile-specifier detoxification-related proteins, and cold-shock domain-containing proteins. The induction of these genes likely represents a generalized adaptive response to in vitro culture conditions and repeated passaging, highlighting transcriptional adjustments associated with cell maintenance and survival rather than fat body-specific functional specialization.

Although the study has some limitations—such as contamination in later splits and the failure to immortalize cells—it provides a solid foundation for future research on the cellular and molecular biology of the Japanese beetle. In its current form, this cell culture should therefore be regarded as a short-term model, suitable for preliminary molecular analyses and early in vitro observations rather than as a stable or continuous experimental platform. Future work can refine culture conditions and explore strategies for immortalization, a challenging objective given the low success rate and substantial effort required to establish continuous cell lines. Importantly, this species-specific short-term culture model represents a valuable first step toward exploratory in vitro experimentation in P. japonica, enabling preliminary studies on physiology, pathogen interactions, insecticide responses, and the identification of molecular targets for potential genetic control strategies.