Molecular evolution of dietary shifts in ladybird beetles (Coleoptera: Coccinellidae): from fungivory to carnivory and herbivory

Abstract

Background

Dietary shifts are major evolutionary steps that shape ecological niches and biodiversity. The beetle family Coccinellidae, commonly known as ladybirds, first transitioned from a fungivorous to an insectivorous and subsequently a plant diet. However, the molecular basis of this dietary diversification remained unexplored.

Results

We investigated the molecular evolution of dietary shifts in ladybirds, focusing on the transitions from fungivory to carnivory (Coccinellidae) and from carnivory to herbivory (Epilachnini), by comparing 25 genomes and 62 transcriptomes of beetles. Our analysis shows that chemosensory gene families have undergone significant expansions at both nodes of diet change and were differentially expressed in feeding experiments, suggesting that they may be related to foraging. We found expansions of digestive and detoxifying gene families and losses of chitin-related digestive genes in the herbivorous ladybirds, and absence of most plant cell wall-degrading enzymes in the ladybirds dating from the transition to carnivory, likely indicating the effect of different digestion requirements on the gene repertoire. Immunity effector genes tend to emerge or have specific amino acid sequence compositions in carnivorous ladybirds and are downregulated under suboptimal dietary treatments, suggesting a potential function of these genes related to microbial symbionts in the sternorrhynchan prey.

Conclusions

Our study provides a comprehensive comparative genomic analysis to address evolution of chemosensory, digestive, detoxifying, and immune genes associated with dietary shifts in ladybirds. Ladybirds can be considered a ubiquitous example of dietary shifts in insects, and thus a promising model system for evolutionary and applied biology.

Background

The acquisition and specialization of specific foods has been a major factor shaping the evolution of heterotrophic organisms, such as animals. Vertebrates, especially hominids and other mammals, have been the subject of several studies focusing on the anatomical and molecular basis of dietary shifts [1,2,3,4,5]. For invertebrates such as insects, the evolution of plant-based diets and its impact on their extraordinary diversification has been the subject of countless studies, spurred in part by the seminal work of Ehrlich and Raven [6] on the coevolution of butterflies and plants. While the evolutionary implications of a transition to herbivory are still much debated [7, 8], an adequate understanding of the molecular basis and mechanism of dietary shifts in insects remains challenging due to the lack of studies on taxa with appropriate dietary breath, phylogenetic diversity, and relatively recent age. Only Drosophila, with at least three herbivorous species, has emerged as a system to study insect diet shifts [9]. Here, we present genomic data and analyses to demonstrate that ladybird beetles may be a promising group to study the evolution of dietary shifts in insects.

Ladybird beetles (Coleoptera: Coccinellidae) comprise more than 6000 species worldwide [10]. They belong to the superfamily Coccinelloidea, a conglomerate of 15 currently recognized taxonomic families [11, 12]. Although largely known as voracious predators of aphids, the diets of the thousands of species of ladybirds are highly diverse. Approximately 36% of ladybirds feed primarily on coccids (including mealybugs and other scale insects), 20% feed on aphids, and 20% feed on plant leaves, while the remainder feed on fungi, pollen, or other insects [13,14,15,16]. In general, Coccinellidae are recognized as the largest group and the only family of beetles that prey extensively on sternorrhynchan insects (coccids, aphids, whiteflies, and psyllids), in contrast to other Coccinelloidea that feed primarily on fungi [13, 17].

Ladybird ancestors expanded their dietary preferences after angiosperm diversification in the Late Cretaceous [18, 19]. Host association and phylogenetic analyses on Coccinellidae and some Coccinelloidea indicated a putative dietary shift from fungi to Coccoidea [14], with further specialization in some major clades (e.g., Coccinellini mainly on aphids, Serangiini mainly on whiteflies) or a shift to other food sources such as plant leaves (e.g., Epilachnini, Bulaea), other arthropod lineages (e.g., preying on mites by representatives of Stethorini), and fungi (e.g., some genera in Coccinellini) [14, 20,21,22]. These dietary shifts make ladybirds a promising group to study the evolution of feeding habits. Comparative anatomical and physiological studies have revealed phenotypic differences between carnivorous and herbivorous ladybirds [23,24,25,26,27,28], for example, herbivorous ladybirds have a digestive system at least twice the length of carnivorous ones.

In addition, because of their feeding habits, several ladybird species are important in agroecosystems and biosecurity. For example, Cryptolaemus montrouzieri is widely used for biocontrol of mealybugs [29], Harmonia axyridis is used for aphid control but it is also a major invasive species [30], the herbivorous Henosepilachna vigintioctopunctata is considered a major pest of Solanaceae [31], and Micraspis discolor can complete its life cycle by feeding on aphids or pollen; its impact on rice fields is still unclear [32] (Fig. 1). The prominent role of ladybirds in agricultural activities and natural ecosystems requires a deeper understanding of the mechanisms involved in their feeding habits.

[IMAGE OMITTED: SEE PDF]

Comparative genomics has become an essential tool for studying evolutionary questions, and genomic data combined with transcriptome expression profiles have been used to explain complex phenotypes in organisms, including their feeding habits. For example, such approaches have shed light on macroevolutionary signatures and genomic mechanisms of novel host plant shifts in butterflies [33], insect dietary adaptation in blind snakes [34], and herbivory and carnivory in mammals [1, 2, 35,36,37].

To investigate genomic changes associated with dietary shifts, one would need to comprehensively compare the sequenced genomes of multiple ladybirds that differ in their diets, as has been done in animal groups [2]. However, the currently available number of ladybird species with sequenced genomes precludes such a genome-based analysis, although the number of ladybird genomes and transcriptomes has increased significantly in recent years [38,39,40,41,42,43,44,45,46,47].

In other arthropods, various genome-based studies over the years have provided insights into genes related to feeding habits. These studies have shown that genes underlying dietary adaptations are often related to chemosensation, digestion, detoxification, and immunity. For example, a gustatory receptor encoded by a chemosensory gene was reported to determine the feeding preference for mulberry leaves in the silkworm [48]. Digestion-related genes encoding plant cell wall degrading enzymes (PCWDEs), acquired by horizontal gene transfer from bacteria and fungi, are used for lignocellulose degradation in at least two groups of herbivorous beetles, Buprestidae and Phytophaga [49,50,51,52]. The expansions of several detoxification-associated gene families are found to be potentially key to the polyphagous diet of spiders [53]. Overall, genes related to chemosensation, digestion, and detoxification appear to contribute to host adaptation and plant component metabolism in general in many herbivorous insects, such as beetles [54, 55], cactophilic flies [56,57,58], cotton bollworms [59], and plant hoppers [60], with a few studies on dietary adaptation in carnivorous insects (e.g., praying mantis [61], green lacewing [62], and hoverfly [63]). Furthermore, genomic and transcriptomic analyses have shown that diet affects insect immunity, and thus immune system responses may be relevant as evolutionary drivers of dietary shifts [64,65,66]. We have previously used transcriptomic data to study ladybird genes involved in adaptation to novel prey [67,68,69,70] and genomic data to study dietary adaptation to scale insects in C. montrouzieri and Novius pumilus [38, 39]. These studies also revealed the importance of genes associated with chemosensation, digestion, detoxification, and immunity.

In the present study, we report new insights into the molecular basis of ladybird dietary shifts from the analysis of a newly assembled dataset. Our dataset includes four newly sequenced and assembled genomes of ladybirds with different diets (i.e., the coccidophagous C. montrouzieri [CMONT, chromosome level], the aphidophagous M. discolor [MDISC], and the herbivorous H. vigintioctopunctata [HVIGI] and Cynegetis impunctata [CIMPU]) and transcriptomes of 25 species of Coccinelloidea. We complemented these data with six publicly available ladybird genomes, 37 species of additional Coccinelloidea transcriptomes, and the genomes of 15 beetle species as outgroups, providing the basis for comparative genomics at three diet-shift nodes: Coccinellidae, Coccinellini, and Epilachnini (Fig. 1). Given that previous research has found genes related to chemosensation, digestion, detoxification, and immunity to be of major importance in the evolutionary history of dietary shifts in animals, we hypothesized that protein-coding genes related to these functions should be relevant to dietary adaptations in the ladybirds, and that members of these gene families should be (1) involved in adaptive lineage-specific gene family dynamics or specific amino acid sequence compositions, (2) differentially expressed under different dietary treatments, and (3) highly expressed in correspondingly relevant tissues (i.e., gut for digestive and detoxifying genes, antenna and head for chemosensory genes). Focusing on these candidate gene families, we performed phylogenetic analyses coupled with diet- and tissue-specific transcriptome profiling to further explore their evolutionary history and expression patterns in relation to dietary shifts.

Results

Genomes, transcriptomes, and ortholog groups of 69 ladybird species (see Sect. 1 of the Additional file 1 for details)

To increase the number of ladybird species with available genomes, we first de novo sequenced and assembled the genomes of three species (i.e., M. discolor, H. vigintioctopunctata, C. impunctata) using a combination of short reads (Illumina) and long reads (Nanopore, Pacbio). For a fourth species, C. montrouzieri, we used data from the previously published contig-level assembly (Li, Huang [38]) to construct a chromosome-level assembly using Hi-C technology. The completeness of these new resources was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool (OrthoDB version 10, Insecta lineage dataset, n = 1367) [71, 72]. The gene space completeness scores and assembly N50 values were as follows: C. montrouzieri, 99.2%, 101.22 Mb; M. discolor, 94.3%, 2.63 Mb; H. vigintioctopunctata, 99.3%, 5.76 Mb; and C. impunctata, 98.2%, 562.46 kb (Table 1). We complemented this dataset by including published assemblies of an additional six species, resulting in a total of ten ingroup genomes for our analyses [38,39,40,41,42, 46, 47]. Note that the species whose genomes we have selected for our comparative analyses are predominantly carnivores and herbivores (Table 1, Fig. 1). Specifically, two of the species feed on scale insects (i.e., C. montrouzieri [CMONT] and Novius pumilus [NPUMI]), three feed on plant leaves (i.e., H. vigintioctopunctata [HVIGI], C. impunctata [CIMPU], and Henosepilachna vigintioctomaculata [HVIMA]), and five feed primarily on aphids (i.e., M. discolor [MDISC], H. axyridis [HAXYR], Coccinella septempunctata [CSEPT], Adalia bipunctata [ABIPU], and Propylea japonica [PJAPO]).

[IMAGE OMITTED: SEE PDF]

To study the evolution of protein-coding genes in ladybird beetles, we annotated the complete predicted protein sets in all ten ladybird genomes and collected the annotated protein sets of fifteen published beetle genomes [79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94] belonging to different families as outgroups (Fig. 1, Additional file 2: Table SE1). The BUSCO completeness scores of the annotated protein-coding genes from the resulting 25 beetle genomes (hereafter referred to as the genome dataset) ranged from 90.2 to 99.6%.

We also inferred protein-coding gene sets from 62 Coccinelloidea transcriptomes, consisting of 37 published and 25 newly sequenced ones (BUSCO completeness scores: 82.0 to 97.4%). This resulted in a total of 87 gene sets (69 from ladybirds and 18 from outgroup beetles) for which we identified orthologous genes (ortholog groups; OGs). Protein-coding genes were assigned to a total of 148,089 OGs, including 1074 common to all 87 species, which formed the basis of our downstream phylogenomic analyses.

Time-calibrated phylogeny and ancestral state reconstruction (see Sect. 2 of Additional file 1 for details)

We inferred the phylogeny of the 87 beetle species studied by analyzing 770 OGs of nearly single-copy protein-coding genes. Concatenation of the corresponding amino acid alignments resulted in a supermatrix of 295,332 amino acid (aa) positions. Phylogenetic analysis of this supermatrix using the maximum likelihood optimality criterion was performed with IQ-TREE [95], specifying a gene-partitioned matrix and selecting the most appropriate substitution model for each partition separately. The resulting reconstructed phylogenetic tree confirmed all major taxonomic lineages of interest (i.e., Coccinellidae, Coccinellini, Epilachnini) as monophyletic entities with high statistical support (ultrafast bootstrap values = 100%).

We estimated divergence times with a Bayesian approach using MCMCTREE [96] and eleven fossil calibration points. The time-calibrated phylogeny suggests that Coccinellidae originated in the Early Cretaceous, 142 million years ago (= Ma; 95% confidence intervals [CI]: 156–131 Ma). The crown groups Coccinellini and Epilachnini originated in the Paleogene, 54 Ma (95% CI 60–50 Ma) and 48 Ma (95% CI 53–44 Ma), respectively.

Using this time-calibrated phylogeny, we reconstructed the ancestral character states of feeding habits at all nodes within Coccinelloidea using published data on feeding habits across the lineage. Species for which no feeding information was available were discarded from the analysis, leaving a total of 61 species for stochastic character mapping using the phytools package [97]. Our results indicate that the diet of the most recent common ancestor of Coccinellidae was most likely scale insects (state probability (SP) = 81%) and that its diet was most likely derived from an ancestral fungivorous diet (Fig. 1), in agreement with previous analyses [14, 17, 22, 98]. Dietary shifts from coccidophagy to aphidophagy and to herbivory likely occurred in the lineages leading to Coccinellini and Epilachnini, respectively (SP ≥ 99%). The identified shifts provide the basis for our comparative genomic and transcriptomic analyses to explore the evolutionary history and expression patterns of candidate gene families in ladybirds.

Evolutionary histories of gene families putatively associated with dietary shifts (see Sects. 3 and 4 of Additional file 1 for details)

Focusing on our genome dataset, we searched for signatures of selection in the subset of single-copy orthologs using PAML [96], and we quantified gene family dynamics (expansions and contractions) using CAFE [99] across all OGs. In addition, we quantified gene losses from phylogenetic lineages and de novo lineage-specific gene emergence by comparing gene counts across the phylogeny. The selection analyses identified a small number of genes with sites showing signatures of positive, relaxed, or enhanced selection along branches (Sect. 3 of Additional file 1). For convenience, we hereafter refer to OGs with significant expansions and contractions, or with clade-specific gene loss, or with lineage-specific de novo gene emergence, as lineage-specific evolving gene families (LEGFs). We focused our analysis of LEGFs on the three nodes of the ladybird phylogeny associated with major dietary shifts (Fig. 1), viz: Coccinellidae, Coccinellini, and Epilachnini.

We identified overrepresented functional gene categories using clusterProfiler [100]. LEGFs at the Coccinellidae, Coccinellini, and Epilachnini nodes were enriched with gene families functionally related mainly to chemosensation (CSP, OBP, OR), digestion (CAT, CBPD, CP, FABP, GLC, GLUT, LIP, NAT, SP), detoxification (AKR, COE, GDH, GST, P450, UGT), and immunity (SPI) (Fig. 2A; candidate gene family abbreviations are listed in the Abbreviations section).

[IMAGE OMITTED: SEE PDF]

Diet-specific differentially expressed genes and tissue-specific gene expression (see Sects. 5 and 6 of Additional file 1 for details)

Six ladybird species (five carnivorous, one herbivorous) were subjected to diet-specific experiments and transcriptome profiling (Additional file 1: Table S5.1). Transcriptome profiling of different tissues (i.e., abdomen without gut, antennae, gut, head, legs, thorax) was performed on C. montrouzieri and C. septempunctata, and transcriptome profiling of gut and body without gut was performed on C. impunctata. We found that gene families related to chemosensation (CSP, OBP), digestion (CAT, GLC, GLUT), and detoxification (ABC, COE, GST, P450, UGT) were significantly enriched in diet-specific comparisons in at least three ladybird species (e.g., aphid vs. mealybug diet treatments in female adults of C. montrouzieri) (Figs. 2B, C). Digestion- and detoxification-related genes were mainly highly expressed in the gut, while chemosensation-related genes were found to be highly expressed in the antennae or head (Fig. 2B). In addition, OG enrichment analysis of diet-specific differentially expressed genes (= DEGs) revealed that specific OGs containing large numbers of DEGs mostly belong to gene families related to chemosensation, digestion, detoxification, and immunity (Additional file 1: Tables S5.2 and S5.3). The LEGFs at the Coccinellidae, Coccinellini, and Epilachnini nodes also tended to be differentially expressed in the corresponding diet comparisons (Additional file 1: Figures S5.2, S5.3 and S5.7).

Evolutionary dynamics of the candidate gene families

Our hypothesis that genes related to chemosensation, digestion, detoxification, and immunity are likely relevant to ladybird dietary adaptation are supported by our statistical enrichment analyses. We subsequently present additional information (e.g., OG size, clade-level gene dynamics and sequence composition, diet- and tissue-specific expression patterns) on gene families related to these functions, with the main results summarized in Table 2. The gene dynamics (e.g., expansion, de novo gene emergence and gene loss) were detected at not only OG level but also clade level (see methods for details).

[IMAGE OMITTED: SEE PDF]

Chemosensory gene families (see Sect. 7 of Additional file 1 for details)

We found evolutionary dynamics in the chemosensation-related OGs, with evidence of expansion (OBP, SNMP) and contraction (OR) events (Fig. 3A). The chemosensation-related OGs also contained diet-specific DEGs (mainly OBP, CSP and SNMP, Fig. 3B). Compared to other chemosensation-related OGs, the largest OG of OBP (OG0000120) was notably expanded in Coccinellidae and Epilachnini (Figs. 1 and 3A). Clade C5 of OG0000120 contributes the most to the gene repertoire expansion in Coccinellidae (Fig. 3C), with 3/5 genes being highly expressed in the antennae in C. montrouzieri, whereas half of genes in C. montrouzieri and C. septempunctata of clades C2 and C3 are clearly expressed in the head (Additional file 1: Figure S7.3). The gene expansion in OG0000120 of the tribe Epilachnini is largely due to a single gene duplication in clade C5 (Fig. 3C). Overall, approximately 50% of the genes in OG0000120 were found to have diet-specific expression in ladybirds (Fig. 3B).

[IMAGE OMITTED: SEE PDF]

Digestive gene families (see Sect. 8 of Additional file 1 for details)

We examined the presence of genes encoding PCWDEs of interest (i.e., cellulases and pectinases) in Coccinellidae and outgroup beetles. We found that Coccinellidae encode only GLC (glycosyl hydrolase family 1 (GH1) domain-contained) genes, while most outgroup beetles encode not only genes of this family but also glycosyl hydrolase family 9 (GH9) and carbohydrate esterase family 13 (CE13) genes (Figs. 1 and 3A). Among the candidate digestive OGs, those belonging to at least seven gene families exhibit expansions (ASP, CBPD, CP, FABP, FATP, GLC, GLUT) in Epilachnini (Figs. 1 and 3A). These OGs also contain a relatively high proportion (13–100%) of genes that are differentially expressed under different dietary treatments (Fig. 3B) as well as highly expressed in the gut (Additional file 1: Figures S8.3, S8.9 and S8.13). In Coccinellidae, only the OGs of GLUT and FABP were expanded. We found that genes of these two gene families are highly expressed in the gut and show large numbers of diet-specific DEGs (25–67%) in the carnivorous ladybirds. Among the candidate chitin-degrading enzymes, we found genes of the chitinase OG0000184 and of the lytic polysaccharide mono-oxygenase (LPMO) OG0000406 to be often highly expressed in the gut. Epilachnini lost genes of the OG0000184 clades C3 and C5 (Figs. 3C) as well as all genes of OG0000406 (Fig. 3A). In M. discolor, the genes of both clades (C3: 2/2, C5: 1/1) in OG0000184 and 2/4 genes of OG0000406 were downregulated when individuals were fed on pollen instead of insects. In Coccinellini, the CAT OG0000065 is significantly contracted in size (Figs. 1 and 3A). We found that most genes in this OG are highly expressed in the gut, and we found that expression of many genes in this OG was upregulated in C. montrouzieri (50%) and downregulated in Coccinellini (17–30%) when individuals of each taxon were fed on suboptimal diets (e.g., aphids for coccidophagous C. montrouzieri, and mealybugs for aphidophagous Coccinellini species).

Detoxifying gene families (see Sect. 9 in Additional file 1 for details)

We found two UGT OGs that were significantly expanded in Coccinellidae (Fig. 3A). In the carnivorous Coccinellidae, most of the underlying genes were highly expressed in the gut (50–100%; Additional file 1: Figure S9.7) and exhibited diet-specific DEGs (29–50%; Fig. 3B). OGs of the following families are significantly expanded in Epilachnini (Fig. 3A): two of GDH, two of P450, two of UGT, and one each of ABC, AKR, COE, and GST. Genes from these OGs were highly expressed in the gut (Additional file 1: Figures S9.3, S9.7, S9.11 and S9.15). Experiments on H. vigintioctopunctata showed that the expression of many genes from these OGs is diet-dependent (5–29%; Fig. 3B). Such diet-dependent gene expression differences were also found in the carnivorous Coccinellidae (15–85%; Fig. 3B).

Immune gene families (see Sect. 10 of Additional file 1 for details)

We found that OG0000225 of a SPI was significantly expanded at the Coccinellidae and Epilachnini nodes and to be contracted at the Coccinellini node (Fig. 3A). Most immune genes (i.e., ATTs, COLs, CLYSs, CWHs DEFs, GNBPs, ILYSs, and PGRPs) were downregulated when the carnivorous ladybirds were fed a suboptimal diet (e.g., moth eggs) (Fig. 3B). Five of 19 genes of the SPI OG0000225 were downregulated in H. vigintioctopunctata when fed with a suboptimal diet (sugar water) and five out of nine genes were upregulated in C. montrouzieri when fed with a suboptimal diet (i.e., moth eggs, and aphids). The following gene families are unique to ladybirds (Fig. 3A) and show a diet-specific DEG in the carnivorous species (Fig. 3B): CWH (OG0001441), DEF (OG0009673), and ILYS (OG0009100). Among the antimicrobial peptides, the clade C5 of the ATT OG0000273 lost genes in Coccinellini but included genes in most other Coccinellidae (Additional file 1: Figure S10.7). In our experiments with C. montrouzieri, we found that three of the four genes of OG0000273 were downregulated when the beetles were fed with a suboptimal diet. Clade C4 of the COL OG0000458 (Fig. 3C) shows unique arrangements at the motif level, with relatively extensive lengths in Coccinellini and other Coccinellidae (33–40 aa, totally 136 aa; Additional file 1: Figure S10.8). Another specific motif was found in genes of carnivorous ladybirds in the DEF OG0009673, which is absent in Epilachnini, resulting in the loss of the functional domain of the defensin (Additional file 1: Figure S10.6).

Discussion

Comparison of the genomes and transcriptomes of ladybirds sampled from across their phylogeny and of outgroup beetles, combined with gene expression experiments, provided us with a list of promising candidate genes that are likely to enable the use of different diets by different species. These candidate genes are predicted to be functionally involved in chemosensation, digestion, detoxification, and immunity (Fig. 4) and are discussed in detail below.

[IMAGE OMITTED: SEE PDF]

Using genomes and transcriptomes across major ladybird lineages, our comparative analysis revealed several genomic signatures of genes related to chemosensation, digestion, detoxification, and immunity, consistent with the molecular adaptation to diet shifts at the nodes of Coccinellidae (fungivorous to an insectivorous diet), Coccinellini (aphidophagous diet) or Epilachnini (herbivorous diet) (Fig. 4).

Evolution of chemosensory genes in ladybirds possibly related to food searching

We analyzed the gene evolutionary dynamics and transcriptome profiling of the chemosensory genes, including those encoding the chemosensory receptors and soluble binding proteins. OBPs and SNMPs expanded at the node of Coccinellidae, thus possibly in the context of a dietary shift from fungivory to carnivory. OBPs also expanded at the node of Epilachnini and thus possibly in the context with a dietary shift from carnivory to herbivory. In insects, OBPs play a key role in the perception of sex and alarm pheromones as well as of host plant volatiles [101, 102]. SNMPs have been reported to be essential for the perception of fatty acid-derived odorants in Drosophila [103]. Carnivorous ladybirds are known to use such chemical cues, for example the aphid alarm pheromone (E)-β-farnesene [104], the mealybug sex pheromone chrysanthemyl 2-acetoxy-3-methylbutanoate [105], and methyl salicylate, which indicates plant damage [106]. OBPs have also been associated with the perception of functionally similar volatiles in carnivorous ladybirds [107,108,109]. Herbivorous ladybirds are able to locate their host plants through plant volatiles [110]. These adaptative requirements in ladybirds may drive the expansions of the chemosensory gene families, especially OBPs. Interestingly, some of the volatiles can be used by ladybirds to locate both sternorrhynchan insect prey and host plants, and even host fungi, such as (E)-β-farnesene, methyl salicylate, (-)-α pinene, and 1-octen-3-ol [106, 110,111,112,113]. It provides a reasonable explanation how diet shift could have evolved without temporary fitness reduction and why expansions at Coccinellidae and Epilachnini nodes could occur in the same OG.

We found that the expression of genes of the soluble binding protein family (i.e., CSPs, OBPs, and SNMPs) was affected by the type of food the beetles were fed. A possible explanation could be an autoregulation of protein abundance in response to the semiochemical environment. Such a mechanism could increase the sensitivity not only to detect volatiles of the preferred diet—and in consequence a better ability to track it [114]—but also to avoid suboptimal diets [114,115,116].

Evolution of digestion and detoxification genes in ladybirds likely reflects adaptation to plant components

In the herbivorous tribe Epilachnini, digestion-related OGs (i.e., ASPs, CBPDs, CPs, FABPs, FATPs, GLCs, and GLUTs) have expanded. These genes tend to be highly expressed in the gut and are downregulated in dietary experiments when H. vigintioctopunctata is fed sugar water instead of plant leaves. We also found that the digestion-related OGs GLUTs and FABPs are expanded at the node of Coccinellidae, with diet-specific DEGs and high expression in gut in the carnivorous ladybirds. The observed expansions and gene expression likely reflect adaptation to metabolize plant components. For example, the expansion of a GLC has been hypothesized to have been critical for the evolution of herbivory by enabling digestion, or detoxification, of plant cell components [49, 117]. We found that GLUTs and FABPs are expanded at both the Coccinellidae and Epilachnini nodes, which is associated with the switches from fungivory to carnivory and from carnivory to herbivory. This at first glance counterintuitive result may be explained by the specific prey of these ladybirds: sternorrhynchan insects. Their gut may contain plant components (e.g., phytoene, carotenoids) that ladybirds with specific GLUTs and FABPs may be able to digest or detoxify [118]. CBPD, whose expansion we found to be associated with a switch to herbivory, is thought to be required to ensure the structural and functional integrity of the peritrophic membrane in the gut and to influence the digestibility of plant tissues [119,120,121]. CBPD could thus help herbivorous ladybirds, which have a longer gut than carnivorous ladybirds [27, 28, 73], to digest plant components.

Several of the OGs related to detoxification (i.e., ABC, AKR, COE, GDH, GST, P450, and UGT) have expanded in the tribe Epilachnini. We found that the genes of these OGs were predominantly upregulated in dietary experiments on H. vigintioctopunctata, in which individuals were fed plant leaves instead of sugar water, and highly expressed in the gut of the ladybirds. These genes cover all the detoxifying gene families we considered and all the three phases in the insect detoxification enzyme system [122]. Similar results have been reported in studies of other herbivorous beetles [55, 81]. It has been reported that H. vigintioctomaculata is not deterred by host plant alkaloid toxins such as α-solanine, α-chaconine, and tomatine [123, 124]. Thus, it appears that Epilachnini can neutralize plant secondary compounds, probably with the help of proteins from the detoxification-related protein families listed above, as has been reported in other herbivorous insects [125, 126].

Although typically associated with benefits for herbivorous species, detoxification-related genes are also important for carnivorous species. Previous studies on ladybirds have already reported major changes in the expression of genes encoding proteins related to detoxification in carnivorous ladybirds in response to different dietary treatments [38, 67, 69]. In our study, we found two UGT OGs to have significantly expanded in Coccinellidae. These two protein families are likely involved in dietary toxin process, as we found them to be expressed primarily in the gut and their expression to respond to dietary treatments. Some toxins (e.g., glycosides, glucosinolates, isothiocyanates, and alkaloids) have been reported from the prey of carnivorous ladybirds, such as aphids and coccids, which are originally acquired from the plant or synthesized using plant components [13, 104, 127], suggesting a need to cope with toxins also in carnivorous ladybirds. Expansion of detoxification-related genes has been previously reported in other carnivorous insects, such as praying mantises [61] and green lacewings [62], indicating a convergent molecular adaptation to toxic prey.

The large numbers of digestive and detoxifying OGs exhibiting expansions in Epilachnini is similar to the pattern found in other herbivorous beetles and insects [48, 54, 56,57,58,59,60, 81, 128,129,130]. Compared with the carnivorous ladybirds, we found more genes associated with digestion or detoxification in herbivorous ladybirds, suggesting the need for a more sophisticated system of digestion and detoxification to digest plant leaves.

Loss and contraction of genes related to carnivory and herbivory in ladybirds

We found that most of the PCWDE gene families known from beetles [49, 131, 132], especially the widely existing endocellulase GH9 and pectinase CE13, are absent in Coccinellidae, with the exception of GLC (GH1 domain-contained). The absence of GH9 in Coccinellidae has been reported previously [49]. The different enzymatic requirements for digestion of fungi versus arthropods may account for this widespread loss of PCWDE genes at the base of Coccinellidae.

Two genes related to chitin metabolism (a CHT and an LMPO) are highly expressed in the gut of carnivorous species of the Coccinellidae and are partially lost in Epilachnini. Although LMPOs have been reported to be possibly involved in the digestion of cellulose [133], in the ladybirds it seems possible that these genes play roles in chitin degradation [134,135,136,137] based on the above results. Consistent with this idea, we found an upregulation of these two genes in M. discolor when fed with an insect diet instead of a pollen diet. Woodring [138] reported the absence of PCWDEs in carnivorous praying mantids and the absence of chitinases in herbivorous stick insects, a pattern reminiscent of that found in ladybirds. This suggests that carnivory and herbivory are associated with predictive enzyme requirements in different insects. However, the lack of PCWDEs, particularly endocellulases and pectinases, has been passed on to the Epilachnini lineage, resulting in an abnormal condition. The PCWDEs in other herbivorous beetles, such as weevils and leaf beetles, could be acquired from fungi through horizontal gene transfer [49]. This suggests that reduced opportunities to horizontally acquire the fungi PCWDEs due to their carnivorous ancestry may be one of the reasons of the absence of most PCWDEs in Epilachnini. The only PCWDE present in Epilachnini, GH1, is thought to act as a hemicellulose. This raises the question of how Epilachnini cope with the lack of PCWDEs, such as the endocellulase GH9, which has synergism with GH1 and is found in most other beetles [49, 139]. These findings suggest that Epilachnini may have adopted alternative strategies to deal with plant cell wall components in their diet compared to other herbivorous beetles. For example, some Epilachnini are known to primarily scrape the soft tissues of plant leaves, chew them, and suck the exposed sap. Thus, Epilachnini leave most of the cellulose on the leaves compared to other leaf-feeding beetles that swallow leaf fragments [16, 140, 141]. In addition, the omnivorous ladybird M. discolor was found to harbor putative cellulolytic bacteria [70], and some isolated bacteria from Epilachnini species have the ability to hydrolyze the cellulose, though they are relatively low in abundance [142]. These support a potential role of symbiotic microbes to digest plant cell walls in the ladybirds. We hypothesize that similar mechanical and/or microbial strategies exist in all Epilachnini species.

We found that genes of the family CAT are highly expressed in the gut and exhibit diet-specific expression patterns. These results are in line with the idea that proteins of this family serve critical digestive functions in both carnivorous and herbivorous ladybirds [143,144,145,146,147]. However, we found that this family is significantly contracted in the tribe Coccinellini, whose species feed on aphids instead of coccids. Since research on the coccidophagous ladybird C. montrouzieri has shown that the enzymatic activity of CAT is reduced when individuals of this species are fed with aphids [144], we hypothesize that CAT is required to digest a specific component present in coccids or mealybugs.

Evolution of immune effector genes in carnivorous ladybirds in relation to prey symbionts

Reports linking immunity-related genes to insect feeding habits are rare [64,65,66]. We have previously found that immune effector genes are downregulated when ladybirds are fed with a suboptimal diet, and that immunity-related gene families are expanded in size in the mealybug predator C. montrouzieri [38, 67]. In this study, we also found that several immune effector OGs, such as CWHs, COLs, DEFs, and ILYSs, appear to have evolved de novo in Coccinellidae. In at least one case, CWHs, the genes were obtained by ladybird ancestors from bacteria via horizontal gene transfer [19]. We found that many genes of immunity-related families (i.e., ATTs, CLYSs, COLs, CWHs DEFs, GNBPs, ILYSs, and PGRPs) are downregulated when the ladybirds are fed on diets other than sternorrhynchan insects (e.g., moth eggs). We also found that the SPI immunoregulator gene family is expanded at the Coccinellidae and Epilachnini nodes and includes several genes whose expression is diet-specific in the ladybirds. However, whether serpins play a role in immunity or have other functions (e.g., regulation of secretion, digestion [148]) in the ladybirds remains to be explored.

Ladybirds primarily prey on sternorrhynchan insects, which are known to harbor a diverse set of symbiont bacteria in their bacteriome or in their tissues (e.g., Tremblaya and Moranella in mealybugs, Buchnera and Serratia in aphids [149, 150]). These symbionts can protect their hosts from predators [151, 152] or colonize and thereby harm predators [150]. It is therefore reasonable to assume that ladybirds have evolved strategies to cope with these bacteria. We interpret the higher expression of genes from several immunity-related families when ladybirds are fed sternorrhynchan insects compared to alternative diets as a possible indication of an immune response against bacteria.

Compared to genes in families related to chemosensation, digestion, and detoxification, we found few lineage-specific changes in family size via expansion or loss and diet-specific changes in expression of genes related to immunity (Fig. 2). We found that dietary shifts were associated primarily by de novo emergence and amino acid changes in immunity effector genes. This is similar to the evolutionary mechanisms of antimicrobial peptides in Drosophila that are driven by the bacteria in the flies’ diet [66].

Conclusions

Ladybirds serve as a promising model group to study of the evolution of dietary shifts in insects, with relevance for agricultural activities and ecosystems in general. In this study, we shed light on the molecular mechanisms underlying dietary shifts in ladybirds, in particular the transitions from fungivory to carnivory and from carnivory to herbivory. This was achieved by applying a combination of comparative genomic analyses using the new genomes of four ladybird species and 21 published ladybird and outgroup beetle genomes, as well as the transcriptomes of 62 related species, together with diet- and tissue-specific transcriptome profiling representing the major feeding guilds: carnivores versus herbivores. Our study revealed diet-shift-specific patterns in the evolution of multigene families related to chemosensation, digestion, detoxification, and immunity. Specifically, we found notable changes in the gene repertoires at phylogenetic nodes with diet shifts from fungi to sternorrhynchan insects (Coccinellidae), from sternorrhynchan insects to plants (Epilachinini), and from coccids to aphids (Coccinellini).

In Coccinellidae, the evolutionary dynamics of gene families related to chemosensation (expansion of OBPs and SNMPs), digestion (loss of PCWDEs and expansion of GLUTs and FABPs), detoxification (expansion of UGTs), and immunity (emergence or specific sequence composition of immune effector genes) can be seen as possible adaptations to foraging, digestion, and the management of toxins and symbiotic bacteria during the dietary transition from fungi to sternorrhynchan insects. In Coccinellini, the reduction in size of the CAT digestive gene family could be a consequence of significant differences in protein components between aphids and coccids, while the loss of a specific clade of attacins and the specific sequence composition of coleoptericin could have been driven by symbiotic bacteria in aphids serving as prey. In Epilachnini, changes in gene families related to chemosensation (expansion of OBPs), digestion (loss of chitin-related genes, expansion of ASPs, CBPDs, CPs, FABPs, FATPs, GLCs, GLUTs), and detoxification (expansion of almost all gene families) could represent adaptations to perceive volatiles of, digest, and cope with toxins in plants.

However, to make the model system of dietary shifts in the ladybirds more complete and precise, our hypotheses on the function of specific gene families should be further supported by experimental verifications. In addition, the genomes of the ladybirds with other feeding habits not covered in our research are worthy to be studied in the future. For example, the genomes of the fungivorous ladybirds (e.g., Illeis and Halyzia) would provide insights into the dynamics during the back transition from carnivory to fungivory, and the genomes of non-sternorrhyncha carnivores (e.g., Stethorus) could be used to investigate the adaptive mechanisms to sternorrhynchan prey in the ladybirds in comparison with those of sternorrhyncha-feeding carnivores. Molecular evolution of metabolizing specific diet components with different contents (e.g., different chitins in insects and fungi, cellulose and toxins in different host plants, wax covered on prey and used as camouflage by ladybird larvae) during the dietary shifts of ladybirds could be explored more deeply, considering the global gene pathways and even the symbiotic microbiomes.

Methods

We generated a chromosome-level assembly from an already existing C. montrouzieri contig-level genome assembly [38] using Hi-C technology. We de novo sequenced the genomes of M. discolor and H. vigintioctopunctata using a combination of Oxford Nanopore long read and Illumina short-read DNA sequencing technologies and the C. impunctata genome using a combination of PacBio, 10X Genomics, and Illumina DNA sequencing technologies. We used the following software packages to assemble the read data from these genomes: ARCS v. 1.2.4 [153], Canu v. 1.5 [154], LACHESIS [155], NextDenovo v. 2.5.0 [156], NextPolish v. 1.4.1 [157], Pilon v. 1.21 [158], Racon v. 1.32 [159], and wtdbg v. 2.5 [160]. Other published genomes of the ladybirds before our analyses were used in our research (Table 1). Our research included the genome of H. vigintioctomaculata close to H. vigintioctopunctata, because it has higher assembly level and potentially more complete gene contents related to herbivory. The completeness of the protein-coding gene space of the resulting assemblies was assessed using the BUSCO v. 5.2.2 pipeline [71] and specifying the Insecta ortholog set of OrthoDB v. 10 [72]. Structural and functional annotation of protein-coding genes in the genome assemblies was performed using the FunAnnotate v. 1.8.1 pipeline [161] and applying the procedures described by Tang, Huang [39]. Coccinelloidea transcriptomes of whole individuals were sequenced using Illumina short-read DNA sequencing technology, and non-redundant transcriptomic protein sets were inferred using EvidentialGene v. 2018.06.18 [162]. Ortholog group assignment and annotation were performed by OrthoFinder v2.5.4 [163] and KinFin v1.0.3 [164].

We inferred a species tree of the investigated taxa from the phylogenetic signal contained in 770 near single-copy protein-coding genes (including the single-copy OGs in the genome dataset and the OGs selected for phylogenetic reconstruction by OrthoFinder in 87 species) using IQ-TREE v. 2.1.4-beta [95]. The best models and partitions were selected using ModelFinder [165] in IQ-TREE with minimum Bayesian information criterion (BIC) score. The adephagan beetle Nebria ingens was selected to root the tree. Divergence times were estimated using MCMCTREE in PAML v. 4.8a [96] and the age of eleven fossils for calibration (Additional file 1: Table S2.1). Dietary evolution was traced on the inferred phylogeny using the fitMk function in the R package phytools [97].

CAFE v. 5.0 [99] was used to identify contractions and expansions of OGs of species with sequenced genomes, using the genome dataset except P. japonica due to its species-specific high gene duplication level of 20.0%, which directly affects gene count data. The lineage-specific OGs and linage-absent OGs were also identified as de novo emergences and losses from the whole subgroups. For the OGs related to chemosensation, digestion, detoxification, and immunity of interest, we also considered clade-level dynamics, and thought clade-specific gene duplications, de novo gene emergence and loss as supplementary of expansion, and de novo gene emergence and loss in OG size. Phylogenies of OGs were inferred using IQ-TREE [95]. The inferred phylogenies served as the basis for identifying ladybird clades, as well as taxonomic clade-specific gene duplications and de novo gene emergence or loss using the OG tree reconciliation implemented in OrthoFinder [163]. Motif analysis of the amino acid sequences of the 25 beetle genomes was performed using MEME v. 5.4.1 [166]. Pfam domains reported in previous studies were used to identify PCWDEs [49, 131, 132].

Ladybird samples of different diet treatments or different tissues were prepared and sequenced using Illumina platform. Abundance estimation and DEG detection were performed by HISAT2 v2.2.0 [167], StringTie v2.1.4 [168], and DESeq2 [169]. For tissue-specific expression, an upregulated DEG detected in a specific tissue group compared with other tissue groups were considered as a gene highly expressed in this tissue. Enrichment was performed using clusterProfiler package [100] and a customized annotation based on our gene family identification. In order to avoid biases of the enrichment results, we also added the Pfam annotations of other OGs or genes into the enrichments.

More detailed information on the procedures used, as well as additional results and discussion, can be found in Additional file 1.

Data availability

Raw reads from the genome and transcriptome sequencing were deposited at NCBI (BioProject accessions: PRJNA626074, PRJNA509782, PRJNA549114, PRJNA776094, PRJNA956151, PRJNA956140, PRJNA956138, PRJNA956078, PRJNA955835, and PRJNA967842).

Abbreviations

ABC:

ATP-binding cassette transporter (detoxification)

AKR:

Aldo-keto reductase (detoxification)

AMY:

α-Amylase (digestion)

AP:

Aminopeptidase (digestion)

ASP:

Aspartic proteinases (digestion)

ATT:

Attacin (immunity)

CAT:

Cysteine proteinase (digestion)

CBPD:

Chitin binding Peritrophin-A domain containing protein (digestion)

CLYS:

C-type lysozyme (immunity)

COE:

Carboxylesterase (detoxification)

COL:

Coleoptericin (immunity)

CP:

Carboxypeptidase (digestion)

CSP:

Chemosensory protein (chemosensation)

CTL:

C-type lectin (immunity)

CWH:

Cell wall hydrolase (immunity)

DEF:

Defensin (immunity)

FABP:

Fatty acid binding protein (digestion)

FATP:

Fatty acid transport protein (digestion)

FREP:

Fibrinogen-related protein (immunity)

GDH:

Glucose dehydrogenase (detoxification)

GLC:

α/β-Glucosidase (digestion)

GLUT:

Glucose transporter protein (digestion)

GNBP:

Gram-negative binding protein (immunity)

GR:

Gustatory receptor (chemosensation)

GST:

Glutathione S-transferase (detoxification)

ILYS:

I-type lysozyme (immunity)

IR:

Ionotropic receptor (chemosensation)

LIP:

Lipase (digestion)

LPMO:

Lytic polysaccharide mono-oxygenase (digestion)

MMP:

Metalloproteinase (digestion)

NAT:

Nutrient amino acid transporter (digestion)

NPC:

Niemann-Pick C1 (digestion)

OBP:

Odorant-binding protein (chemosensation)

OR:

Odorant receptor (chemosensation)

P450:

Cytochrome P450 (detoxification)

PGRP:

Peptidoglycan recognition protein (immunity)

SCP:

Sterol carrier protein (digestion)

SGLT:

Sodium-driven glucose symporter (digestion)

SNMP:

Sensory neuron membrane protein (chemosensation)

SP:

Serine proteinase (digestion)

SPI:

Serine protease inhibitor, serpin (immunity)

UGT:

UDP-glucuronosyltransferase (detoxification)

References

Pajic P, Pavlidis P, Dean K, Neznanova L, Romano R-A, Garneau D, et al. Independent amylase gene copy number bursts correlate with dietary preferences in mammals. eLife. 2019;8:e44628.

Hecker N, Sharma V, Hiller M. Convergent gene losses illuminate metabolic and physiological changes in herbivores and carnivores. P Natl Acad Sci USA. 2019;116(8):3036–41.

Kim S, Cho YS, Kim H-M, Chung O, Kim H, Jho S, et al. Comparison of carnivore, omnivore, and herbivore mammalian genomes with a new leopard assembly. Genome Biol. 2016;17:211.

Luca F, Perry GH, Di Rienzo A. Evolutionary Adaptations to Dietary Changes. Annu Rev Nutr. 2010;30:291–314.

Potter JHT, Davies KTJ, Yohe LR, Sanchez MKR, Rengifo EM, Struebig M, et al. Dietary Diversification and Specialization in Neotropical Bats Facilitated by Early Molecular Evolution. Mol Biol Evol. 2021;38(9):3864–83.

Ehrlich PR, Raven PH. Butterflies and Plants: A Study in Coevolution. Evolution. 1964;18(4):586–608.

Mayhew PJ. Why are there so many insect species? Perspectives from fossils and phylogenies. Biol Rev. 2007;82(3):425–54.

Wiens JJ, Lapoint RT, Whiteman NK. Herbivory increases diversification across insect clades. Nat Commun. 2015;6:8370.

Groen SC, Whiteman NK. Using Drosophila to study the evolution of herbivory and diet specialization. Curr Opin Insect Sci. 2016;14:66–72.

Ślipiński A. Australian Ladybird Beetles (Coleoptera: Coccinellidae): Their biology and classification. Collingwood: CSIRO Publishing; 2013.

Robertson JA, Ślipiński A, Moulton M, Shockley FW, Giorgi A, Lord NP, et al. Phylogeny and classification of Cucujoidea and the recognition of a new superfamily Coccinelloidea (Coleoptera: Cucujiformia). Syst Entomol. 2015;40(4):745–78.

Arriaga-Varela E, Tomaszewska W, Szawaryn K, Robertson J, Seedel M, Ślipiński A, et al. The resurrection of Cerasommatidiidae, an enigmatic group of coccinelloid beetles (Coleoptera: Coccinelloidea) based on molecular and morphological evidence. Zool J Linn Soc-Lond. 2023;197(4):1078–115.

Hodek I, Honěk A. Scale insects, mealybugs, whiteflies and psyllids (Hemiptera, Sternorrhyncha) as prey of ladybirds. Biol Control. 2009;51(2):232–43.

Giorgi JA, Vandenberg NJ, McHugh JV, Forrester JA, Ślipiński SA, Miller KB, et al. The evolution of food preferences in Coccinellidae. Biol Control. 2009;51(2):215–31.

Magro A, Lecompte E, Magné F, Hemptinne J-L, Crouau-Roy B. Phylogeny of ladybirds (Coleoptera: Coccinellidae): Are the subfamilies monophyletic? Mol Phylogenet Evol. 2010;54(3):833–48.

Tomaszewska W, Szawaryn K. Epilachnini (Coleoptera: Coccinellidae)—A Revision of the World Genera. J Insect Sci. 2016;16(1):101.

Leschen RAB. Beetles feeding on bugs (Coleoptera, Hemiptera): repeated shifts from mycophagous ancestors. Invertebr Taxon. 2000;14(6):917–29.

Che LH, Zhang P, Deng SH, Escalona HE, Wang XM, Li Y, et al. New insights into the phylogeny and evolution of lady beetles (Coleoptera: Coccinellidae) by extensive sampling of genes and species. Mol Phylogenet Evol. 2021;156: 107045.

Li H-S, Tang X-F, Huang Y-H, Xu Z-Y, Chen M-L, Du X-Y, et al. Horizontally acquired antibacterial genes associated with adaptive radiation of ladybird beetles. BMC Biol. 2021;19(1):7.

Katoh T, Koji S, Ishida TA, Matsubayashi KW, Kahono S, Kobayashi N, et al. Phylogeny of Epilachna, Henosepilachna, and Some Minor Genera of Phytophagous Ladybird Beetles (Coleoptera: Coccinellidae: Coccinellinae: Epilachnini), with an Analysis of Ancestral Biogeography and Host-Plant Utilization. Zool Sci. 2014;31(12):820–30.

Nattier R, Michel-Salzat A, Almeida LM, Chifflet-Belle P, Magro A, Salazar K, et al. Phylogeny and divergence dating of the ladybird beetle tribe Coccinellini Latreille (Coleoptera: Coccinellidae: Coccinellinae). Syst Entomol. 2021;46(3):632–48.

Escalona HE, Zwick A, Li H-S, Li JH, Wang XM, Pang H, et al. Molecular phylogeny reveals food plasticity in the evolution of true ladybird beetles (Coleoptera: Coccinellidae: Coccinellini). BMC Evol Biol. 2017;17:151.

Potts SF. The alimentary canal of the Mexican bean beetle. Ohio J Sci. 1927;37(3):127–37.

Pradhan S. Neuro-Muscular Study of the Mouth-Parts of Coccinella septempunctata, with a Comparison of the Mouth-Parts in Carnivorous and Herbivorous Coccinelids. Rec Zool Surv India. 1938;40:341–58.

Pradhan S. The Alimentary Canal and Pro-epithelial Re-generation in Coccinella septempunctata with a comparison of Carnivorous and Herbivorous Coccinellids. Quarterly Journal of Microscopical Science. 1939;81:451–78.

Pradhan S. The alimentary canal of Epilachna indica (Coccinellidae: Coleoptera) with a discussion on the activity of the midgut epithelium. Journal of the Royal Asiatic Society of Bengal Science. 1936;2:127–56.

Aldigail SA, Alsaggaff AI, Al-Azab AM. Anatomical and histological study on the digestive canal of Epilachna chrysomelina (Coleoptera: Coccinellidae). Biosci, Biotechnol Res Asia. 2013;10(1):183–92.

Sakurai H. Physiological Studies on the Digestion of Coccinellid Beetles (Coleoptera : Coccinellidae), with Special Reference to their Food Habits. Appl Entomol Zool. 1968;3(3):130–8.

Kairo MTK, Paraiso O, Gautam RD, Peterkin DD. Cryptolaemus montrouzieri (Mulsant) (Coccinellidae: Scymninae): A Review of Biology, Ecology, and Use in Biological Control with Particular Reference to Potential Impact on Non-Target Organisms. CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources. 2013;8(5):1–20.

Koch RL. The multicolored Asian lady beetle, Harmonia axyridis: A review of its biology, uses in biological control, and non-target impacts. J Insect Sci. 2003;3:32.

Sharma A, Thakur A, Kaur S, Pati PK. Effect of Alternaria alternata on the coccinellid pest Henosepilachna vigintioctopunctata and its implications for biological pest management. J Pest Sci. 2012;85(4):513–8.

Shanker C, Mohan M, Sampathkumar M, Lydia C, Katti G. Functional significance of Micraspis discolor (F.) (Coccinellidae: Coleoptera) in rice ecosystem. J Appl Entomol. 2013;137(8):601–9.

Allio R, Nabholz B, Wanke S, Chomicki G, Pérez-Escobar OA, Cotton AM, et al. Genome-wide macroevolutionary signatures of key innovations in butterflies colonizing new host plants. Nat Commun. 2021;12(1):354.

Peng CJ, Wu D-D, Ren J-L, Peng Z-L, Ma ZF, Wu W, et al. Large-scale snake genome analyses provide insights into vertebrate development. Cell. 2023;186(14):2959–76.

Li GT, Wei HY, Bi JJ, Ding XY, Li LL, Xu SX, et al. Insights into Dietary Switch in Cetaceans: Evidence from Molecular Evolution of Proteinases and Lipases. J Mol Evol. 2020;88(6):521–35.

Zhou XM, Wang BS, Pan Q, Zhang JB, Kumar S, Sun XQ, et al. Whole-genome sequencing of the snub-nosed monkey provides insights into folivory and evolutionary history. Nat Genet. 2014;46(12):1303–10.

Wang ZF, Xu SX, Du KX, Huang F, Chen Z, Zhou KY, et al. Evolution of Digestive Enzymes and RNASE1 Provides Insights into Dietary Switch of Cetaceans. Mol Biol Evol. 2016;33(12):3144–57.

Li H-S, Huang Y-H, Chen M-L, Ren Z, Qiu B-Y, De Clercq P, et al. Genomic insight into diet adaptation in the biological control agent Cryptolaemus montrouzieri. BMC Genomics. 2021;22(1):135.

Tang X-F, Huang Y-H, Li H-S, Chen P-T, Yang H-Y, Liang Y-S, et al. Genomic insight into the scale specialization of the biological control agent Novius pumilus (Weise, 1892). BMC Genomics. 2022;23(1):90.

Zhang LJ, Li S, Luo JY, Du P, Wu LK, Li YR, et al. Chromosome-level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures. Mol Ecol Resour. 2020;20(1):292–307.

Boyes D, Crowley LM, University of Oxford and Wytham Woods Genome Acquisition Lab, Darwin Tree of Life Barcoding collective, Wellcome Sanger Institute Tree of Life programme, Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective, et al. The genome sequence of the harlequin ladybird, Harmonia axyridis (Pallas, 1773) [version 1; peer review: 1 approved with reservations]. Wellcome Open Res. 2021:6:300.

Crowley LM, University of Oxford and Wytham Woods Genome Acquisition Lab, Darwin Tree of Life Barcoding collective, Wellcome Sanger Institute Tree of Life programme, Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective, Tree of Life Core Informatics collective, et al. The genome sequence of the seven-spotted ladybird, Coccinella septempunctata Linnaeus, 1758 [version 1; peer review: 2 approved]. Wellcome Open Res. 2021:6:319.

Ando T, Matsuda T, Goto K, Hara K, Ito A, Hirata J, et al. Repeated inversions within a pannier intron drive diversification of intraspecific colour patterns of ladybird beetles. Nat Commun. 2018;9:3843.

Chen MY, Mei Y, Chen X, Chen X, Xiao D, He K, et al. A chromosome-level assembly of the harlequin ladybird Harmonia axyridis as a genomic resource to study beetle and invasion biology. Mol Ecol Resour. 2021;21(4):1318–32.

Gautier M, Yamaguchi J, Foucaud J, Loiseau A, Ausset A, Facon B, et al. The Genomic Basis of Color Pattern Polymorphism in the Harlequin Ladybird. Curr Biol. 2018;28(20):3296–302.

Wellcome Sanger Institute Tree of Life programme, Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective, Tree of Life Core Informatics collective, Goate Z, Darwin Tree of Life Consortium. The genome sequence of the two-spot ladybird, Adalia bipunctata (Linnaeus, 1758) [version 1; peer review: 2 approved, 3 approved with reservations]. Wellcome Open Res. 2022:7:288.

Zhu WB, Chi SQ, Wang YC, Li HR, Wang ZK, Gu SD, et al. A chromosome-level genome assembly of the Henosepilachna vigintioctomaculata provides insights into the evolution of ladybird beetles. DNA Res. 2023;30(1):dsad001.

Zhang Z-J, Zhang S-S, Niu B-L, Ji D-F, Liu X-J, Li M-W, et al. A determining factor for insect feeding preference in the silkworm, Bombyx mori. Plos Biol. 2019;17(2): e3000162.

McKenna DD, Shin S, Ahrens D, Balke M, Beza-Beza C, Clarke DJ, et al. The evolution and genomic basis of beetle diversity. P Natl Acad Sci USA. 2019;116(49):24729–37.

Wybouw N, Pauchet Y, Heckel DG, Van Leeuwen T. Horizontal gene transfer contributes to the evolution of arthropod herbivory. Genome Biol Evol. 2016;8(6):1785–801.

Kirsch R, Gramzow L, Theißen G, Siegfried BD, ffrench-Constant RH, Heckel DG, et al. Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: Key events in the evolution of herbivory in beetles. Insect Biochem Molec. 2014:52:33–50.

Kirsch R, Okamura Y, Haeger W, Vogel H, Kunert G, Pauchet Y. Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival. P Natl Acad Sci USA. 2022;119(40): e2205857119.

Fan Z, Yuan T, Liu P, Wang LY, Jin JF, Zhang F, et al. A chromosome-level genome of the spider Trichonephila antipodiana reveals the genetic basis of its polyphagy and evidence of an ancient whole-genome duplication event. GigaScience. 2021;10(3):giab016.

Muller C, Vogel H, Heckel DG. Transcriptional responses to short-term and long-term host plant experience and parasite load in an oligophagous beetle. Mol Ecol. 2017;26(22):6370–83.

Seppey M, Ioannidis P, Emerson BC, Pitteloud C, Robinson-Rechavi M, Roux J, et al. Genomic signatures accompanying the dietary shift to phytophagy in polyphagan beetles. Genome Biol. 2019;20:98.

Matzkin LM. Population transcriptomics of cactus host shifts in Drosophila mojavensis. Mol Ecol. 2012;21(10):2428–39.

Diaz F, Allan CW, Matzkin LM. Positive selection at sites of chemosensory genes is associated with the recent divergence and local ecological adaptation in cactophilic Drosophila. BMC Evol Biol. 2018;18:144.

Rane RV, Pearce SL, Li F, Coppin C, Schiffer M, Shirriffs J, et al. Genomic changes associated with adaptation to arid environments in cactophilic Drosophila species. BMC Genomics. 2019;20:52.

Pearce SL, Clarke DF, East PD, Elfekih S, Gordon KHJ, Jermiin LS, et al. Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species. BMC Biol. 2017;15:63.

He P, Engsontia P, Chen GL, Yin Q, Wang J, Lu X, et al. Molecular characterization and evolution of a chemosensory receptor gene family in three notorious rice planthoppers, Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus, based on genome and transcriptome analyses. Pest Manag Sci. 2018;74(9):2156–67.

Huang GP, Song LY, Du X, Huang X, Wei FW. Evolutionary genomics of camouflage innovation in the orchid mantis. Nat Commun. 2023;14(1):4821.

Wang YY, Zhang RY, Wang MQ, Zhang LS, Shi C-M, Li J, et al. The first chromosome-level genome assembly of a green lacewing Chrysopa pallens and its implication for biological control. Mol Ecol Resour. 2022;22(2):755–67.

Yuan H, Gao BJ, Wu C, Zhang L, Li H, Xiao YT, et al. Genome of the hoverfly Eupeodes corollae provides insights into the evolution of predation and pollination in insects. BMC Biol. 2022;20(1):157.

Vilcinskas A. Evolutionary plasticity of insect immunity. J Insect Physiol. 2013;59(2):123–9.

Vogel H, Muller A, Heckel DG, Gutzeit H, Vilcinskas A. Nutritional immunology: Diversification and diet-dependent expression of antimicrobial peptides in the black soldier fly Hermetia illucens. Dev Comp Immunol. 2018;78:141–8.

Hanson MA, Grollmus L, Lemaitre B. Ecology-relevant bacteria drive the evolution of host antimicrobial peptides in Drosophila. Science. 2023;381(6655):eadg5725.

Chen M-L, Huang Y-H, Qiu B-Y, Chen P-T, Du X-Y, Li H-S, et al. Changes in life history traits and transcriptional regulation of Coccinellini ladybirds in using alternative prey. BMC Genomics. 2020;21(1):44.

Chen M-L, Wang T, Huang Y-H, Qiu B-Y, Li H-S, Pang H. Physiological and Evolutionary Changes in a Biological Control Agent During Prey Shifts Over Several Generations. Front Physiol. 2018;9:971.

Li H-S, Pan C, De Clercq P, Ślipiński A, Pang H. Variation in life history traits and transcriptome associated with adaptation to diet shifts in the ladybird Cryptolaemus montrouzieri. BMC Genomics. 2016;17:281.

Huang Y-H, Du X-Y, Chen P-T, Tang X-F, Gong S-R, Zhang P-F, et al. Is pollinivory in the omnivorous ladybird beetle Micraspis discolor (Coleoptera: Coccinellidae) symbiosis-dependent? Biol Control. 2022;169: 104867.

Manni M, Berkeley MR, Seppey M, Simao FA, Zdobnov EM. BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Mol Biol Evol. 2021;38(10):4647–54.

Kriventseva EV, Kuznetsov D, Tegenfeldt F, Manni M, Dias R, Simao FA, et al. OrthoDB v10: sampling the diversity of animal, plant, fungal, protist, bacterial and viral genomes for evolutionary and functional annotations of orthologs. Nucleic Acids Res. 2019;47(D1):D807–11.

Hodek I. Biology of Coccinellidae. Dordrecht: Springer Science & Business Media; 2013.

Hodek I, Evans EW. Food relationships. In: Hodek I, van Emden HF, Honěk A, editors. Ecology and Behaviour of the Ladybird Beetles (Coccinellidae). West Sussex: John Wiley & Sons; 2012. p. 141-274.

Lundgren JG. Relationships of Natural Enemies and Non-Prey Foods. Dordrecht: Springer International; 2009.

Ren SX, Wang XM, Pang H, Peng ZQ, Zeng T. Colored Pictorial Handbook of Ladybird Beetles in China. Beijing: Science Press; 2009.

Ślipiński A, Li JH, Pang H. Ladybird Beetles of the Australo-Pacific Region: Coleoptera: Coccinellidae: Coccinellini. Melbourne: CSIRO Publishing; 2020.

Kalushkov P, Chehlarov E, Nedvěd O. Suitability of Gramineous Plants as Food for the Phytophagous Ladybird Cynegetis impunctata L. (Coleoptera: Coccinellidae). Acta Zool Bulgar. 2013;65(4):561–4.

King R, Buer B, Davies TGE, Ganko E, Guest M, Hassani-Pak K, et al. The complete genome assemblies of 19 insect pests of worldwide importance to agriculture. Pestic Biochem Phys. 2023;191: 105339.

Schoville SD, Chen YH, Andersson MN, Benoit JB, Bhandari A, Bowsher JH, et al. A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Sci Rep-Uk. 2018;8:1931.

McKenna DD, Scully ED, Pauchet Y, Hoover K, Kirsch R, Geib SM, et al. Genome of the Asian longhorned beetle (Anoplophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle-plant interface. Genome Biol. 2016;17:227.

Parisot N, Vargas-Chávez C, Goubert C, Baa-Puyoulet P, Balmand S, Beranger L, et al. The transposable element-rich genome of the cereal pest Sitophilus oryzae. BMC Biol. 2021;19(1):241.

Keeling CI, Yuen MMS, Liao NY, Docking TR, Chan SK, Taylor GA, et al. Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest. Genome Biol. 2013;14(3):R27.

Keeling CI, Campbell EO, Batista PD, Shegelski VA, Trevoy SAL, Huber DPW, et al. Chromosome-level genome assembly reveals genomic architecture of northern range expansion in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Mol Ecol Resour. 2022;22(3):1149–67.

Fallon TR, Lower SE, Chang C-H, Bessho-Uehara M, Martin GJ, Bewick AJ, et al. Firefly genomes illuminate parallel origins of bioluminescence in beetles. eLife. 2018;7:e36495.

Evans JD, McKenna D, Scully E, Cook SC, Dainat B, Egekwu N, et al. Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory. GigaScience. 2018;7(12):giy138.

Cunningham CB, Ji LX, Wiberg RAW, Shelton J, McKinney EC, Parker DJ, et al. The Genome and Methylome of a Beetle with Complex Social Behavior, Nicrophorus vespilloides (Coleoptera: Silphidae). Genome Biol Evol. 2015;7(12):3383–96.

Richards S, Gibbs RA, Weinstock GM, Brown SJ, Denell R, Beeman RW, et al. The genome of the model beetle and pest Tribolium castaneum. Nature. 2008;452(7190):949–55.

Herndon N, Shelton JM, Gerischer L, Ioannidis P, Ninova M, Dönitz J, et al. Enhanced genome assembly and a new official gene set for Tribolium castaneum. BMC Genomics. 2020;21(1):47.

Thomas GWC, Dohmen E, Hughes DST, Murali SC, Poelchau M, Glastad K, et al. Gene content evolution in the arthropods. Genome Biol. 2020;21(1):15.

Zhang ZQ, Pei P, Zhang M, Li FF, Tang GH. Chromosome-level genome assembly of Dastarcus helophoroides provides insights into CYP450 genes expression upon insecticide exposure. Pest Manag Sci. 2023;79:1467–82.

Crowley LM, University of Oxford and Wytham Woods Genome Acquisition Lab, Darwin Tree of Life Barcoding collective, Wellcome Sanger Institute Tree of Life programme, Wellcome Sanger Institute Scientific Operations: DNA Pipelines collective, Tree of Life Core Informatics collective, et al. The genome sequence of the common malachite beetle, Malachius bipustulatus (Linnaeus, 1758) [version 1; peer review: 2 approved, 1 approved with reservations]. Wellcome Open Res. 2021:6:322.

Wang QY, Liu LW, Zhang SJ, Wu H, Huang JH. A chromosome-level genome assembly and intestinal transcriptome of Trypoxylus dichotomus (Coleoptera: Scarabaeidae) to understand its lignocellulose digestion ability. GigaScience. 2022;11:giac0591.

Weng Y-M, Francoeur CB, Currie CR, Kavanaugh DH, Schoville SD. A high-quality carabid genome assembly provides insights into beetle genome evolution and cold adaptation. Mol Ecol Resour. 2021;21(6):2145–65.

Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol. 2020;37(5):1530–4.

Yang ZH. PAML 4: Phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007;24(8):1586–91.

Revell LJ. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol. 2012;3(2):217–23.

Seago AE, Giorgi JA, Li JH, Ślipiński A. Phylogeny, classification and evolution of ladybird beetles (Coleoptera: Coccinellidae) based on simultaneous analysis of molecular and morphological data. Mol Phylogenet Evol. 2011;60(1):137–51.

Mendes FK, Vanderpool D, Fulton B, Hahn MW. CAFE 5 models variation in evolutionary rates among gene families. Bioinformatics. 2020;36(22–23):5516–8.

Yu GC, Wang L-G, Han YY, He Q-Y. clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters. OMICS: J Integrative Biol. 2012:16(5):284–7.

Pelosi P, Iovinella I, Felicioli A, Dani FR. Soluble proteins of chemical communication: an overview across arthropods. Front Physiol. 2014;5:320.

Pelosi P, Zhou J-J, Ban LP, Calvello M. Soluble proteins in insect chemical communication. Cell Mol Life Sci. 2006;63(14):1658–76.

Leal WS. Odorant Reception in Insects: Roles of Receptors, Binding Proteins, and Degrading Enzymes. Annu Rev Entomol. 2013;58:373–91.

Pervez A, Yadav M. Foraging Behaviour of Predaceous Ladybird Beetles: A Review. Eur J Environ Sci. 2018;8(2):102–8.

Urbina A, Verdugo JA, López E, Bergmann J, Zaviezo T, Flores MF. Searching Behavior of Cryptolaemus montrouzieri (Coleoptera: Coccinellidae) in Response to Mealybug Sex Pheromones. J Econ Entomol. 2018;111(4):1996–9.

Yang Z-K, Qu C, Pan S-X, Liu Y, Shi Z, Luo C, et al. Aphid-repellent, ladybug-attraction activities, and binding mechanism of methyl salicylate derivatives containing geraniol moiety. Pest Manag Sci. 2023;79:760–70.

Tang H-Y, Liu J-T, Xie J-X, Yi C-Q, Liu X-X, Zhang Y-J, et al. Gene cloning and ligand binding characterization of the odorant-binding protein HvarOBP2 in Hippodamia variegata (Coleoptera: Coccinellidae). Acta Entomol Sin. 2022;65(8):977–85.

Tang HY, Xie JX, Liu JT, Khashaveh A, Liu XX, Yi CQ, et al. Odorant-Binding Protein HvarOBP5 in Ladybird Hippodamia variegata Regulates the Perception of Semiochemicals from Preys and Habitat Plants. J Agr Food Chem. 2023;71(2):1067–76.

Qu C, Yang Z-K, Wang S, Zhao H-P, Li F-Q, Yang X-L, et al. Binding Affinity Characterization of Four Antennae-Enriched Odorant-Binding Proteins From Harmonia axyridis (Coleoptera: Coccinellidae). Front Physiol. 2022;13: 829766.

Piersanti S, Saitta V, Rebora M, Salerno G. Olfaction in phytophagous ladybird beetles: antennal sensilla and sensitivity to volatiles from host plants in Chnootriba elaterii. Arthropod-Plant Inte. 2022;16(6):617–30.

Li JJ, Hu H, Mao J, Yu L, Stoopen G, Wang MQ, et al. Defense of pyrethrum flowers: repelling herbivores and recruiting carnivores by producing aphid alarm pheromone. New Phytol. 2019;223(3):1607–20.

Li W, Zhang Y-F, Xie Y-P, Niu X-P. Selection response of Harmonia axyridis (Pallas) to body volatile of Ceroplastes japonicus Green. Journal of Environmental Entomology. 2016;38(2):329–36.

Tabata J, De Moraes CM, Mescher MC. Olfactory Cues from Plants Infected by Powdery Mildew Guide Foraging by a Mycophagous Ladybird Beetle. PLoS ONE. 2011;6(8): e23799.

Seagraves MP. Lady beetle oviposition behavior in response to the trophic environment. Biol Control. 2009;51(2):313–22.

Montell C. A taste of the Drosophila gustatory receptors. Curr Opin Neurobiol. 2009;19(4):345–53.

Sánchez-Gracia A, Vieira FG, Rozas J. Molecular evolution of the major chemosensory gene families in insects. Heredity. 2009;103(3):208–16.

Beran F, Pauchet Y, Kunert G, Reichelt M, Wielsch N, Vogel H, et al. Phyllotreta striolata flea beetles use host plant defense compounds to create their own glucosinolate-myrosinase system. P Natl Acad Sci USA. 2014;111(20):7349–54.

Takemura M, Maoka T, Koyanagi T, Kawase N, Nishida R, Tsuchida T, et al. Elucidation of the whole carotenoid biosynthetic pathway of aphids at the gene level and arthropodal food chain involving aphids and the red dragonfly. BMC Zool. 2021;6(1):19.

Jasrapuria S, Arakane Y, Osman G, Kramer KJ, Beeman RW, Muthukrishnan S. Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function. Insect Biochem Molec. 2010;40(3):214–27.

Rodriguez-de la Noval C, Rodriguez-Cabrera L, Izquierdo L, Espinosa LA, Hernandez D, Ponce M, et al. Functional expression of a peritrophin A-like SfPER protein is required for larval development in Spodoptera frugiperda (Lepidoptera: Noctuidae). Sci Rep-Uk. 2019;9:2630.

Tetreau G, Dittmer NT, Cao XL, Agrawal S, Chen YR, Muthukrishnan S, et al. Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects. Insect Biochem Molec. 2015;62:127–41.

Chen C-H. Activation and Detoxification Enzymes: Functions and Implications. New York: Springer; 2012.

Hori M, Nakamura H, Fujii Y, Suzuki Y, Matsuda K. Chemicals affecting the feeding preference of the Solanaceae-feeding lady beetle Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae). J Appl Entomol. 2011;135(1–2):121–31.

Matsishina NV, Ermak MV, Kim IV, Fisenko PV, Sobko OA, Klykov AG, et al. Allelochemical Interactions in the Trophic System << Henosepilachna vigintioctomaculata Motschulsky-Solanum tuberosum Linneus >>. Insects. 2023;14(5):459.

Heidel-Fischer HM, Vogel H. Molecular mechanisms of insect adaptation to plant secondary compounds. Curr Opin Insect Sci. 2015;8:8–14.

Li XC, Schuler MA, Berenbaum MR. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu Rev Entomol. 2007;52:231–53.

Francis F, Lognay G, Wathelet J-P, Haubruge E. Effects of allelochemicals from first (Brassicaceae) and second (Myzus persicae and Brevicoryne brassicae) trophic levels on Adalia bipunctata. J Chem Ecol. 2001;27(2):243–56.

Aguirre-Rojas LM, Scully ED, Trick HN, Zhu KY, Smith CM. Comparative analyses of transcriptional responses of Dectes texanus LeConte (Coleoptera: Cerambycidae) larvae fed on three different host plants and artificial diet. Sci Rep-Uk. 2021;11(1):11448.

Simon J-C, d’Alencon E, Guy E, Jacquin-Joly E, Jaquiéry J, Nouhaud P, et al. Genomics of adaptation to host-plants in herbivorous insects. Brief Funct Genomics. 2015;14(6):413–23.

Gloss AD, Abbot P, Whiteman NK. How interactions with plant chemicals shape insect genomes. Curr Opin Insect Sci. 2019;36:149–56.

Calderón-Cortés N, Quesada M, Watanabe H, Cano-Camacho H, Oyama K. Endogenous plant cell wall digestion: A key mechanism in insect evolution. Annu Rev Ecol Evol Syst. 2012;43:45–71.

Watanabe H, Tokuda G. Cellulolytic Systems in Insects. Annu Rev Entomol. 2010;55:609–32.

Sabbadin F, Hemsworth GR, Ciano L, Henrissat B, Dupree P, Tryfona T, et al. An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion. Nat Commun. 2018;9:756.

Qu M-B, Guo X-X, Kong L, Hou L-J, Yang Q. A midgut-specific lytic polysaccharide monooxygenase of Locusta migratoria is indispensable for the deconstruction of the peritrophic matrix. Insect Sci. 2022;29(5):1287–98.

Qu MB, Guo XX, Tian S, Yang Q, Kim M, Mun S, et al. AA15 lytic polysaccharide monooxygenase is required for efficient chitinous cuticle turnover during insect molting. Commun Biol. 2022;5(1):518.

Arakane Y, Muthukrishnan S. Insect chitinase and chitinase-like proteins. Cell Mol Life Sci. 2010;67(2):201–16.

Zhu QS, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S. Functional specialization among insect chitinase family genes revealed by RNA interference. P Natl Acad Sci USA. 2008;105(18):6650–5.

Woodring J. A comparison of ten digestive enzymes reveals a lack of chitinase in a phasmid and the loss of two β-glucanases in a mantid. Physiol Entomol. 2020;45(1):89–94.

Cairo JPLF, Oliveira LC, Uchima CA, Alvarez TM, Citadini APD, Cota J, et al. Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi. Insect Biochem Molec. 2013;43(10):970–81.

Howard NF. Feeding of the Mexican Bean Beetle Larva. Ann Entomol Soc Am. 1941;34(4):766–9.

Kogan M. Automatic Recordings of Masticatory Motions of Leaf-Chewing Insects. Ann Entomol Soc Am. 1973;66(1):66–9.

Taylor EC. Cellulose digestion in a leaf eating insect, the Mexican bean beetle, Epilachna varivestis. Insect Biochemistry. 1985;15(2):315–20.

Walker AJ, Ford L, Majerus MEN, Geoghegan IE, Birch N, Gatehouse JA, et al. Characterisation of the mid-gut digestive proteinase activity of the two-spot ladybird (Adalia bipunctata L.) and its sensitivity to proteinase inhibitors. Insect Biochem Molec. 1998;28(3):173–80.

Zibaee A. A Biochemical study on the types of digestive specific proteases in the ladybird, Cryptolaemus montrouzieri Mulsant (Col.: Coccinellidae) and the effects of different preys on their activit. Plant Pest Research. 2020;10(2):75–91.

Álvarez-Alfageme F, Ferry N, Castañera P, Ortego F, Gatehouse AMR. Prey mediated effects of Bt maize on fitness and digestive physiology of the red spider mite predator Stethorus punctillum Weise (Coleoptera : Coccinellidae). Transgenic Res. 2008;17(5):943–54.

Gholamzadeh-Chitgar M, Ghadamyari M, Ghanbarinezhad R. Biochemical properties of digestive proteases of melon ladybird, Epilachna chrysomelina (Fabricius) (Col.: Coccinellidae). J Entomol Soc Iran. 2017;37(3):293–304.

Koo MS, Park YC. Gut Luminal Digestive Proteinases of Adult Lady Beetle, Harmonia axyridis (Coccinellidae: Coleoptera), Fed an Artificial Diet. J Asia-Pac Entomol. 2002;5(2):167–73.

Meekins DA, Kanost MR, Michel K. Serpins in arthropod biology. Semin Cell Dev Biol. 2017;62:105–19.

Baumann P. Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annu Rev Microbiol. 2005;59:155–89.

Du X-Y, Yang H-Y, Gong S-R, Zhang P-F, Chen P-T, Liang Y-S, et al. Aphidophagous ladybird beetles adapt to an aphid symbiont. Funct Ecol. 2022;36(10):2593–604.

Brownlie JC, Johnson KN. Symbiont-mediated protection in insect hosts. Trends Microbiol. 2009;17(8):348–54.

McLean AHC. Cascading effects of defensive endosymbionts. Curr Opin Insect Sci. 2019;32:42–6.

Yeo S, Coombe L, Warren RL, Chu J, Birol I. ARCS: scaffolding genome drafts with linked reads. Bioinformatics. 2018;34(5):725–31.

Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722–36.

Burton JN, Adey A, Patwardhan RP, Qiu RL, Kitzman JO, Shendure J. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol. 2013;31(12):1119–25.

Hu J, Wang Z, Sun ZY, Hu BX, Ayoola AO, Liang F, et al. NextDenovo: an efficient error correction and accurate assembly tool for noisy long reads. Genome Biology. 2024;25(1):107.

Hu J, Fan JP, Sun ZY, Liu SL. NextPolish: a fast and efficient genome polishing tool for long-read assembly. Bioinformatics. 2020;36(7):2253–5.

Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS ONE. 2014;9(11): e112963.

Vaser R, Sović I, Nagarajan N, Šikić M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 2017;27(5):737–46.

Ruan J, Li H. Fast and accurate long-read assembly with wtdbg2. Nat Methods. 2020;17(2):155–8.

Palmer J. Funannotate: Eukaryotic Genome Annotation Pipeline. https://github.com/nextgenusfs/funannotate. Accessed 3 Oct 2021.

Gilbert D. Gene‑omes built from mRNA-seq not genome DNA. 7th annual arthropod genomics symposium. 2013.

Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20(1):238.

Laetsch DR, Blaxter ML. KinFin: Software for Taxon-Aware Analysis of Clustered Protein Sequences. G3 Genes|Genomes|Genetics. 2017:7(10):3349–57.

Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods. 2017;14(6):587–9.

Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proceed Second Int Conference Intelligent Syst Molecular Biol. 1994;2:28–36.

Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907–15.

Kovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M. Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol. 2019;20(1):278.

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.