The fecal shield is a double-edged sword for larvae of a leaf beetle.

Larvae of some leaf beetles carry masses of feces covering parts or all of the body, which is called a "fecal shield". In general, the shield is thought to be a defense structure against natural enemies. However, some studies have suggested that defense effectiveness varies depending on the natural enemy. In this study, we used a fecal retention leaf beetle Ophrida xanthospilota (Coleoptera: Chrysomelidae), and 2 local generalist predators (an ant, Camponotus japonicus (Hymenoptera: Formicidae) and a stinkbug, Arma custos [Hemiptera: Pentatomidae]) as a system to test the hypothesis that the fecal shield of O. xanthospilota plays different roles in predation behavior of different predators and can provide multiple chemical communication signals in predator-prey interactions. Prey bioassays showed that the fecal shield of O. xanthospilota larvae repelled the ant C. japonicus while attracting the stinkbug A. custos. The results also strongly demonstrated that hexane extracts of the fecal shield significantly repelled C. japonicus, while dichloromethane (DCM) extracts did not inhibit ant predation. Interestingly, DCM extracts attracted A. custos, but hexane extracts did not. Therefore, we suggest that the fecal shield is a double-edged sword for the larvae of O. xanthospilota. Our results also indicated that the risk-benefit tradeoff of an insect should be estimated at a community level involving multiple enemies (predators and parasites) and herbivores, rather than in a single prey-predator pair.

Parallel metatranscriptome analysis reveals degradation of plant secondary metabolites by beetles and their gut symbionts.

Switching to a new host plant is a driving force for divergence and speciation in herbivorous insects. This process of incorporating a novel host plant into the diet may require a number of adaptations in the insect herbivores that allow them to consume host plant tissue that may contain toxic secondary chemicals. As a result, herbivorous insects are predicted to have evolved efficient ways to detoxify major plant defences and increase fitness by either relying on their own genomes or by recruiting other organisms such as microbial gut symbionts. In the present study we used parallel metatranscriptomic analyses of Altica flea beetles and their gut symbionts to explore the contributions of beetle detoxification mechanisms versus detoxification by their gut consortium. We compared the gut meta-transcriptomes of two sympatric Altica species that feed exclusively on different host plant species as well as their F1 hybrids that were fed one of the two host plant species. These comparisons revealed that gene expression patterns of Altica are dependent on both beetle species identity and diet. The community structure of gut symbionts was also dependent on the identity of the beetle species, and the gene expression patterns of the gut symbionts were significantly correlated with beetle species and plant diet. Some of the enriched genes identified in the beetles and gut symbionts are involved in the degradation of secondary metabolites produced by plants, suggesting that Altica flea beetles may use their gut microbiota to help them feed on and adapt to their host plants.

Two Apriona Species Sharing a Host Niche Have Different Gut Microbiome Diversity.

The adaptability of herbivorous insects to toxic plant defense compounds is partly related to the structure of the gut microbiome. To overcome plant resistance, the insect gut microbiome should respond to a wide range of allelochemicals derived from dietary niches. Nevertheless, for sibling herbivorous insect species, whether the gut microbiome contributes to success in food niche competition is unclear. Based on 16S rDNA high-throughput sequencing, the gut microbiomes of two Apriona species that share the same food niche were investigated in this study to determine whether the gut microbiome contributes to insect success in food-niche competition. Our observations indicated that the gut microbiome tended to play a part in host niche competition between the two Apriona species. The gut microbiome of Apriona swainsoni had many enriched pathways that can help degrade plant toxic secondary compounds, including xenobiotic biodegradation and metabolism, terpenoid and polyketide metabolism, and secondary metabolite biosynthesis. Meanwhile, A. swainsoni hosted a much greater variety of microorganisms and had more viable bacteria than A. germari. We conclude that gut microbes may influence the coevolution of herbivores and host plants. Gut bacteria may not only serve to boost nutritional relationships, but may also play an important role in insect food niche competition.

The draft genome of the specialist flea beetle Altica viridicyanea (Coleoptera: Chrysomelidae).

BACKGROUND: Altica (Coleoptera: Chrysomelidae) is a highly diverse and taxonomically challenging flea beetle genus that has been used to address questions related to host plant specialization, reproductive isolation, and ecological speciation. To further evolutionary studies in this interesting group, here we present a draft genome of a representative specialist, Altica viridicyanea, the first Alticinae genome reported thus far. RESULTS: The genome is 864.8 Mb and consists of 4490 scaffolds with a N50 size of 557 kb, which covered 98.6% complete and 0.4% partial insect Benchmarking Universal Single-Copy Orthologs. Repetitive sequences accounted for 62.9% of the assembly, and a total of 17,730 protein-coding gene models and 2462 non-coding RNA models were predicted. To provide insight into host plant specialization of this monophagous species, we examined the key gene families involved in chemosensation, detoxification of plant secondary chemistry, and plant cell wall-degradation. CONCLUSIONS: The genome assembled in this work provides an important resource for further studies on host plant adaptation and functionally affiliated genes. Moreover, this work also opens the way for comparative genomics studies among closely related Altica species, which may provide insight into the molecular evolutionary processes that occur during ecological speciation.

Gut bacterial communities and their contribution to performance of specialist Altica flea beetles.

Host plant shifts are a common mode of speciation in herbivorous insects. Although insects can evolve adaptations to successfully incorporate a new host plant, it is becoming increasingly recognized that the gut bacterial community may play a significant role in allowing insects to detoxify novel plant chemical defenses. Here, we examined differences in gut bacterial communities between Altica flea beetle species that feed on phylogenetically unrelated host plants in sympatry. We surveyed the gut bacterial communities of three closely related flea beetles from multiple locations using 16S rRNA amplicon sequencing. The results showed that the beetle species shared a high proportion (80.7%) of operational taxonomic units. Alpha-diversity indicators suggested that gut bacterial diversity did not differ among host species, whereas geography had a significant effect on bacterial diversity. In contrast, analyses of beta-diversity showed significant differences in gut bacterial composition among beetle species when we used species composition and relative abundance metrics, but there was no difference in composition when species presence/absence and phylogenetic distance indices were used. Within host beetle species, gut bacterial composition varied significantly among sites. A metagenomic functionality analysis predicted that the gut microbes had functions involved in xenobiotic biodegradation and metabolism as well as metabolism of terpenoids and polyketides. These predictions, however, did not differ among beetle host species. Antibiotic curing experiments showed that development time was significantly prolonged, and there was a significant decline in body weight of newly emerged adults in beetles lacking gut bacteria, suggesting the beetles may receive a potential benefit from the gut microbe-insect interaction. On the whole, our results suggest that although the gut bacterial community did not show clear host-specific patterns among Altica species, spatiotemporal variability is an important determinant of gut bacterial communities. Furthermore, the similarity of communities among these beetle species suggests that microbial facilitation may not be a determinant of host plant shifts in Altica.

The complete mitochondrial genome of the cowpea weevil, Callosobruchus maculates (Coleoptera: Chrysomelidae: Bruchinae) and a related phylogenetic analysis of Chrysomelidae.

In this study, the complete 17,809 bp mitochondrial genome of Callosobruchus maculates (F.) (Coleoptera: Chrysomelidae: Bruchinae) was sequenced using Illumina's HiSeq2000 platform. The mitogenome is a double-stranded circular molecule of 17,809 bp in length with 21 transfer RNA genes, 13 protein-coding genes, and two ribosomal RNA genes as in other insects. Specially, there is a 2008 bp-inserted segment between ND2 and tRNA-Trp from 1180 to 3187, which cannot be aligned to any known gene of mitogenomes. To estimate the taxonomic status of Bruchinae, total 17 species from eight subfamilies of Chrysomelidae were selected as ingroups and three species of Lamiinae as outgroups for phylogenetic analysis based on mitogenome. The results showed that three major lineages were formed, including a basal 'Eumolpine' clade (Cassidinae, Eumolpinae, Cryptocephalinae, Clytrinae), ''Criocerine' clade (Criocerinae, Bruchinae) and 'Chrysomeline' clade (Chrysomelinae, Galerucinae s. l.). Bruchinae showed more closed relationship with Criocerinae than other subfamilies. More thorough taxon sampling will be needed to well understand the relationship in Chrysomelidae.

The phylogeny of Galerucinae (Coleoptera: Chrysomelidae) and the performance of mitochondrial genomes in phylogenetic inference compared to nuclear rRNA genes.

With efficient sequencing techniques, full mitochondrial genomes are rapidly replacing other widely used markers, such as the nuclear rRNA genes, for phylogenetic analysis but their power to resolve deep levels of the tree remains controversial. We studied phylogenetic relationships of leaf beetles (Chrysomelidae) in the tribes Galerucini and Alticini (root worms and flea beetles) based on full mitochondrial genomes (103 newly sequenced), and compared their performance to the widely sequenced nuclear rRNA genes (full 18S, partial 28S). Our results show that: (i) the mitogenome is phylogenetically informative from subtribe to family level, and the per-nucleotide contribution to nodal support is higher than that of rRNA genes, (ii) the Galerucini and Alticini are reciprocally monophyletic sister groups, if the classification is adjusted to accommodate several 'problematic genera' that do not fit the dichotomy of lineages based on the presence (Alticini) or absence (Galerucini) of the jumping apparatus, and (iii) the phylogenetic results suggest a new classification system of Galerucini with eight subtribes: Oidina, Galerucina, Hylaspina, Metacyclina, Luperina, Aulacophorina, Diabroticina and Monoleptina.

Comparative transcriptome analysis of chemosensory genes in two sister leaf beetles provides insights into chemosensory speciation.

Divergence in chemosensory traits has been posited as an important component of chemosensory speciation in insects. In particular, chemosensory genes expressed in the peripheral sensory neurons are likely to influence insect behaviors such as preference for food, oviposition sites, and mates. Despite their key role in insect behavior and potentially speciation, the underlying genetic basis for divergence in chemosensory traits remains largely unexplored. One way to ascertain the role of chemosensory genes in speciation is to make comparisons of these genes across closely related species to detect the genetic signatures of divergence. Here, we used high throughput transcriptome analysis to compare chemosensory genes of the sister leaf beetles species Pyrrhalta maculicollis and P. aenescens, whose sexual isolation and host plant preference are mediated by divergent chemical signals. Although there was low overall divergence between transcriptome profiles, there were a number of genes that were differentially expressed between the species. Furthermore, we also detected two chemosensory genes under positive selection, one of which that was also differentially expressed between the species, suggesting a possible role for these genes in chemical-based premating reproductive isolation and host use. Combined with the available chemical and ecological work in this system, further studies of the divergent chemosensory genes presented here will provide insight into the process of chemosensory speciation among Pyrrhalta beetles.

The status of Bactrocera invadens Drew, Tsuruta & White (Diptera: tephritidae) inferred from complete mitochondrial genome analysis.

In this study, the complete 15,911 bp mitochondrial genome (mitogenome) of Bactrocera invadens was sequenced. To estimate the status of B. invadens, all available mitogenomes of Bactrocera were downloaded from GenBank for phylogenetic analysis. Phylogenetic analyses showed that B. invadens, B. philippinensis, B. papayae, and three B. dorsalis sequences formed a well-supported clade with very short terminal branch lengths, indicting the relatively close evolutionary relationships of these taxa. The results further supported that B. invadens, the same as B. philippinensis and B. papayae, belongs to the same species as B. dorsalis.

Adaptation to different host plant ages facilitates insect divergence without a host shift.

Host shifts and subsequent adaption to novel host plants are important drivers of speciation among phytophagous insects. However, there is considerably less evidence for host plant-mediated speciation in the absence of a host shift. Here, we investigated divergence of two sympatric sister elm leaf beetles, Pyrrhalta maculicollis and P. aenescens, which feed on different age classes of the elm Ulmus pumila L. (seedling versus adult trees). Using a field survey coupled with preference and performance trials, we show that these beetle species are highly divergent in both feeding and oviposition preference and specialize on either seedling or adult stages of their host plant. An experiment using artificial leaf discs painted with leaf surface wax extracts showed that host plant chemistry is a critical element that shapes preference. Specialization appears to be driven by adaptive divergence as there was also evidence of divergent selection; beetles had significantly higher survival and fecundity when reared on their natal host plant age class. Together, the results identify the first probable example of divergence induced by host plant age, thus extending how phytophagous insects might diversify in the absence of host shifts.

Male mate recognition via cuticular hydrocarbons facilitates sexual isolation between sympatric leaf beetle sister species.

Chemical signals in insects have been documented to play an important role in mate recognition, and divergence in chemical signals can often cause sexual isolation between closely related species or populations within species. We investigated the role of cuticular hydrocarbons (CHCs), short distance chemical signals, in male mate recognition between the two sympatric elm leaf beetles, Pyrrhalta maculicollis and Pyrrhaltaaenescens. Mating experiments demonstrated that strong sexual isolation between the two species was driven by CHCs divergence. Males preferred to mate with conspecific females with intact conspecific CHCs or conspecific CHCs reapplied after removal. Males also preferred heterospecific females that were treated with conspecific CHCs. Chemical analysis showed that the CHC profiles differ significantly between species. In P. maculicollis dimethyl-branched alkanes between C29 and C35 account for the majority of the saturated alkanes while the CHC profile of P. aenescens mostly consisted of monomethyl-branched alkanes between C22 and C29. Additionally, some compounds, such as 12,18-diMeC32, 12,18-diMeC34, are unique to P. maculicollis.

Assortative mating between two sympatric closely-related specialists: inferred from molecular phylogenetic analysis and behavioral data.

Host plant shifting of phytophagous insects can lead to the formation of host associated differentiation and ultimately speciation. In some cases, host plant specificity alone acts as a nearly complete pre-mating isolating barrier among insect populations. We here test whether effective pre-mating isolation and host-independent behavioral isolation have evolved under the condition of extreme host specilization using two sympatric flea beetles with incomplete post-mating isolation under laboratory conditions. Phylogenetic analysis and coalescent simulation results showed that there is a limited interspecific gene flow, indicating effctive isolation between these species. Three types of mating tests in the absence of host plant cues showed that strong host-independent behavioral isolation has evolved between them. We conclude that almost perfect assortative mating between these two extreme host specialists results from a combination of reduced encounter rates due to differential host preference and strong sexual isolation.

Notes on black elytron species of Pyrrhalta Joannis and the description of a new species from China (Coleoptera, Chrysomelidae, Galerucinae).

Thirteen species of Pyrrhalta Joannis, 1865 with black elytron are reviewed. A key to species, photographs of aedeagus and habitus are provided. Pyrrhalta qianana sp. n. is described from Guizhou, China. Pyrrhalta martensi Medvedev & Sprecher-Uebersax, 1999 is newly recorded from China (Tibet).

Recent speciation in three closely related sympatric specialists: inferences using multi-locus sequence, post-mating isolation and endosymbiont data.

Shifting between unrelated host plants is relatively rare for phytophagous insects, and distinct host specificity may play crucial roles in reproductive isolation. However, the isolation status and the relationship between parental divergence and post-mating isolation among closely related sympatric specialists are still poorly understood. Here, multi-locus sequence were used to estimate the relationship among three host plant-specific closely related flea beetles, Altica cirsicola, A. fragariae and A. viridicyanea (abbreviated as AC, AF and AV respectively). The tree topologies were inconsistent using different gene or different combinations of gene fragments. The relationship of AF+(AC+AV) was supported, however, by both gene tree and species tree based on concatenated data. Post-mating reproductive data on the results of crossing these three species are best interpreted in the light of a well established phylogeny. Nuclear-induced but not Wolbachia-induced unidirectional cytoplasmic incompatibility, which was detected in AC-AF and AF-AV but not in AC-AV, may also suggest more close genetic affinity between AC and AV. Prevalence of Wolbachia in these three beetles, and the endosymbiont in most individuals of AV and AC sharing a same wsp haplotype may give another evidence of AF+(AC+AV). Our study also suggested that these three flea beetles diverged in a relative short time (0.94 My), which may be the result of shifting between unrelated host plants and distinct host specificity. Incomplete post-mating isolation while almost complete lineage sorting indicated that effective pre-mating isolation among these three species should have evolved.

Common volatiles are major attractants for neonate larvae of the specialist flea beetle Altica koreana (Coleoptera: Chrysomelidae).

Olfactory stimuli play an important role in the host searching of larval phytophagous insects. Previous studies indicate that larvae that have to find feeding sites after hatching are generally attracted to host volatiles. However, there are few studies on the olfactory responses of neonate larvae to host volatiles in cases when those larvae hatched on the host plant. In the present study, we determined the olfactory responses of neonate larvae of the specialist flea beetle, Altica koreana Ogloblin, to host and six non-host plants, using a static-air "arena." Larvae responded significantly to the host plant Potentilla chinensis Ser. and five of six non-host plants, compared to the control. Larvae did not prefer the host plant over the non-host plants (except Artemisia sp.) when offered a choice. Additionally, odours of a non-host plant, which were unattractive to neonate larvae, may have masked the attractive odour of the host plant. These results indicate that common volatiles can play a major role in attracting larvae of this specialist to plants, but attraction to such odours may not be the major mechanism of host choice.

Host plant use in sympatric closely related flea beetles.

Studies on strategies of host plant use in sympatric-related species are significant to the theory of sympatric speciation. Altica fragariae Nakane and Altica koreana Ogloblin are sympatric closely related flea beetles found in Beijing, northern China. All their recorded host plants are in the subfamily Rosoideae of the Rosaceae, so we regard them as a model system to study interactions between herbivorous insects and plant-insect co-evolution. We conducted a set of experiments on the host preference and performance of these flea beetles to study whether these closely related species have the ability to use sympatric novel host plants and whether monophagous and oligophagous flea beetles use the same strategy in host plant use. Oviposition preference experiments showed that A. koreana, a monophagous flea beetle, displayed high host fidelity. However, A. fragariae, which is oligophagous, often made "oviposition mistakes," ovipositing on nonhost plants such as Potentilla chinensis, the host plant of A. koreana, although normal host plants were preferred over novel ones. Larval performance studies suggested that A. fragariae was able to develop successfully on P. chinensis. Feeding experiences of larvae had no effect on feeding preference, oviposition preference, and fecundity of adults. However, females were impaired in their reproductive ability when fed on nonhost plants. Therefore, A. fragariae finished their development of larval stages on P. chinensis and came back to their primary host plant, Duchesnea indica, for feeding and reproduction after eclosion.