Yu Matsuura

Laccase2 is required for cuticular pigmentation in stinkbugs.

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa.

Novel clade of alphaproteobacterial endosymbionts associated with stinkbugs and other arthropods.

ABSTRACT Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade.

Evolution of symbiotic organs and endosymbionts in lygaeid stinkbugs

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone’ in the middle of each germarium and in the ‘symbiont ball’ at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri’, the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola’ is proposed for the endosymbiont clade.

Diversity of Bacterial Endosymbionts Associated with Macrosteles Leafhoppers Vectoring Phytopathogenic Phytoplasmas

ABSTRACT Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, “Candidatus Sulcia muelleri” and “Candidatus Nasuia deltocephalinicola,” and five facultative endosymbionts, Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and a novel bacterium belonging to the Rickettsiaceae, from the leafhoppers. “Ca. Sulcia muelleri” and “Ca. Nasuia deltocephalinicola” exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, “Candidatus Phytoplasma asteris,” associated with aster yellows and related plant diseases, and “Candidatus Phytoplasma oryzae,” associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.

Intrasperm vertical symbiont transmission

Significance Diverse organisms are commonly associated with bacterial endosymbionts, which often affect hosts’ biology and phenotypes in a variety of ways. The majority of these symbionts are generally present in the host cell cytoplasm and maternally transmitted through host generations. Here, however, this conventional knowledge is countered by our discovery of intrasperm vertical transmission of nuclear-targeting bacterial symbiont (Rickettsia) in an insect (leafhopper Nephotettix cincticeps), which potentially erodes the nuclear-cytoplasmic conflict that governs the majority of endosymbiotic associations. The molecular and cellular mechanisms underlying the sperm head infection without disturbing sperm functioning are of not only basic but also applied interest, which may provide insights into the development of sperm-mediated genetic transformation and/or material delivery technologies. Symbiotic bacteria are commonly associated with cells and tissues of diverse animals and other organisms, which affect hosts’ biology in a variety of ways. Most of these symbionts are present in the cytoplasm of host cells and maternally transmitted through host generations. The paucity of paternal symbiont transmission is likely relevant to the extremely streamlined sperm structure: the head consisting of condensed nucleus and the tail made of microtubule bundles, without the symbiont-harboring cytoplasm that is discarded in the process of spermatogenesis. Here, we report a previously unknown mechanism of paternal symbiont transmission via an intrasperm passage. In the leafhopper Nephotettix cincticeps, a facultative Rickettsia symbiont was found not only in the cytoplasm but also in the nucleus of host cells. In male insects, strikingly, most sperm heads contained multiple intranuclear Rickettsia cells. The Rickettsia infection scarcely affected the host fitness including normal sperm functioning. Mating experiments revealed both maternal and paternal transmission of the Rickettsia symbiont through host generations. When cultured with mosquito and silkworm cell lines, the Rickettsia symbiont was preferentially localized within the insect cell nuclei, indicating that the Rickettsia symbiont itself must have a mechanism for targeting nucleus. The mechanisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic and applied interest.

Bacterial Symbionts of a Devastating Coffee Plant Pest, the Stinkbug Antestiopsis thunbergii (Hemiptera: Pentatomidae)

ABSTRACT Stinkbugs of the genus Antestiopsis, so-called antestia bugs or variegated coffee bugs, are notorious pests of coffee plants in Africa. We investigated the symbiotic bacteria associated with Antestiopsis thunbergii, a major coffee plant pest in Rwanda. PCR, cloning, sequencing, and phylogenetic analysis of bacterial genes identified four distinct bacterial lineages associated with A. thunbergii: a gammaproteobacterial gut symbiont and symbionts representing the genera Sodalis, Spiroplasma, and Rickettsia. In situ hybridization showed that the gut symbiont densely occupied the lumen of midgut crypts, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont sparsely and sporadically infected various cells and tissues. Diagnostic PCR survey of 154 A. thunbergii individuals collected at 8 localities in Rwanda revealed high infection frequencies (100% for the gut symbiont, 51.3% for the Sodalis symbiont, 52.6% for the Spiroplasma symbiont, and 24.0% for the Rickettsia symbiont). These results suggest that the gut symbiont is the primary symbiotic associate of obligate nature for A. thunbergii, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont are the secondary symbiotic associates of facultative nature. We observed high coinfection frequencies, i.e., 7.8% of individuals with quadruple infection with all the symbionts, 32.5% with triple infections with the gut symbiont and two of the secondary symbionts, and 39.6% with double infections with the gut symbiont and any of the three secondary symbionts, which were statistically not different from the expected coinfection frequencies and probably reflected random associations. The knowledge of symbiotic microbiota in A. thunbergii will provide useful background information for controlling this devastating coffee plant pest.

Infection prevalence of Sodalis symbionts among stinkbugs.

IntroductionDiverse insects and other organisms are associated with microbial symbionts, which often significantly contribute to growth and survival of their hosts and/or drastically affect phenotypes of their hosts in a variety of ways. Sodalis glossinidius was first identified as a facultative bacterial symbiont of tsetse flies, and recent studies revealed that Sodalis-allied bacteria encompass diverse ecological niches ranging from free-living bacteria through facultative symbionts to obligate symbionts associated with a diverse array of insects. Despite potential ecological and evolutionary relevance of the Sodalis symbionts, their infection prevalence in natural insect populations has been poorly investigated.ResultsHere we surveyed diverse stinkbugs and allied terrestrial heteropteran bugs, which represented 17 families, 77 genera, 108 species, 310 populations and 960 individuals, for infection with the Sodalis symbionts. Diagnostic PCR detected relatively low infection frequencies of the Sodalis symbionts: 13.6% (14/103) of the species, 7.5% (22/295) of the populations, and 4.3% (35/822) of the individuals of the stinkbugs except for those belonging to the family Urostylididae. Among the urostylidid stinkbugs, strikingly, the Sodalis symbionts exhibited very high infection frequencies: 100% (5/5) of the species, 100% (15/15) of the populations, and 94.2% (130/138) of the individuals we examined. Molecular phylogenetic analysis based on bacterial 16S rRNA gene sequences revealed that all the symbionts were placed in the clade of Sodalis-allied bacteria while the symbiont phylogeny did not reflect the systematics of their stinkbug hosts. Notably, the Sodalis symbionts of the urostylidid stinkbugs were not clustered with the Sodalis symbionts of the other stinkbug groups on the phylogeny, suggesting their distinct evolutionary trajectories.ConclusionsThe relatively low infection frequency and the overall host-symbiont phylogenetic incongruence suggest that the Sodalis symbionts are, in general, facultative symbiotic associates in the majority of the stinkbug groups. On the other hand, it is conceivable, although speculative, that the Sodalis symbionts may play some substantial biological roles for their host stinkbugs of the Urostylididae.

Huge Symbiotic Organs in Giant Scale Insects of the Genus Drosicha (Coccoidea: Monophlebidae) Harbor Flavobacterial and Enterobacterial Endosymbionts

Giant scale insects (Drosicha: Coccoidea: Monophlebidae) were investigated for their symbiotic organs and bacterial endosymbionts. Two types of bacterial 16S rRNA gene sequences, flavobacterial and enterobacterial, were consistently detected in D. corpulenta and D. pinicola. The former sequences formed a compact clade in the Bacteroidetes, allied to the symbionts of cushion and armored scales. The latter sequences formed a robust clade in the &ggr;-Proteobacteria, allied to enteric bacteria like Enterobacter aerogenes and Escherichia coli. Another type of 16S sequence derived from Wolbachia was also detected in D. pinicola. In-situ hybridization demonstrated that the flavobacterial and enterobacterial symbionts were localized in a pair of huge bacteriomes in the abdomen, the former in uninucleated peripheral bacteriocytes and the latter in syncytial central bacteriocytes. Electron microscopy confirmed the endocellular locations of the pleomorphic flavobacterial symbiont and the rod-shaped enterobacterial symbiont, and also revealed the location and fine structure of the Wolbachia symbiont in D. pinicola. Infection frequencies of the flavobacterial and enterobacterial symbionts were consistently 100% in populations of D. corpulenta and D. pinicola, while the Wolbachia symbiont exhibited 0% and 100% infection frequencies in D. corpulenta and D. pinicola, respectively. Neither the flavobacterial symbiont nor the enterobacterial symbiont exhibited AT-biased nucleotide composition or accelerated molecular evolution. The huge bacteriomes of Drosicha giant scales would provide a useful system for investigating biochemical, physiological, and genomic aspects of the host-symbiont and symbiont-symbiont interactions.

Ultrabithorax is essential for bacteriocyte development

Significance Among the most fundamental questions in developmental biology is how novel cell types have emerged in the metazoan evolution. Among the most challenging questions in evolutionary biology is how sophisticated symbiotic associations have evolved through less intimate interorganismal interactions. These fundamental biological issues are crystalized in the evolution and development of insect’s bacteriocytes specialized for harboring symbiotic bacteria. Here, we report that a conserved transcription factor Ultrabithorax is essential for bacteriocyte development in an insect, thereby uncovering a molecular mechanism underlying the emergence of the novel host cells for symbiosis. Our finding highlights the importance of developmental cooption of preexisting transcription factors and sheds new light on a long-lasting enigma in evolutionary developmental biology. Symbiosis often entails the emergence of novel adaptive traits in organisms. Microbial symbionts are indispensable for diverse insects via provisioning of essential nutrients, wherein novel host cells and organs for harboring the microbes, called bacteriocytes and bacteriomes, have evolved repeatedly. Molecular and developmental mechanisms underpinning the emergence of novel symbiotic cells and organs comprise an unsolved question in evolutionary developmental biology. Here, we report that a conserved homeotic gene, Ultrabithorax, plays a pivotal role in the bacteriocyte differentiation in a hemipteran insect Nysius plebeius. During embryonic development, six pairs of aggregated presumptive bacteriocytes appear on both sides of six abdominal segments, incorporate the symbiotic bacteria at the stage of germband retraction, and fuse into a pair of lateral bacteriomes at the stage of germband flip, where bacteriocyte-associated Ultrabithorax expression coincides with the symbiont infection process. Suppression of Ultrabithorax expression by maternal RNA interference results in disappearance of the bacteriocytes and the symbiont localization therein, suggesting that Ultrabithorax is involved in differentiation of the host cells for symbiosis. Suppression of other homeotic genes abdominal-A and Antennapedia disturbs integrity and positioning of the bacteriomes, affecting the configuration of the host organs for symbiosis. Our findings unveil the molecular and developmental mechanisms underlying the bacteriocyte differentiation, which may have evolved either via cooption of the transcription factors for inducing the novel symbiotic cells, or via revival of the developmental pathway for the bacteriocytes that had existed in the ancestral hemipterans.

Burkholderia of Plant-Beneficial Group are Symbiotically Associated with Bordered Plant Bugs (Heteroptera: Pyrrhocoroidea: Largidae)

A number of phytophagous stinkbugs (order Heteroptera: infraorder Pentatomomorpha) harbor symbiotic bacteria in a specific midgut region composed of numerous crypts. Among the five superfamilies of the infraorder Pentatomomorpha, most members of the Coreoidea and Lygaeoidea are associated with a specific group of the genus Burkholderia, called the “stinkbug-associated beneficial and environmental (SBE)” group, which is not vertically transmitted, but acquired from the environment every host generation. A recent study reported that, in addition to these two stinkbug groups, the family Largidae of the superfamily Pyrrhocoroidea also possesses a Burkholderia symbiont. Despite this recent finding, the phylogenetic position and biological nature of Burkholderia associated with Largidae remains unclear. Based on the combined results of fluorescence in situ hybridization, cloning analysis, Illumina deep sequencing, and egg inspections by diagnostic PCR, we herein demonstrate that the largid species are consistently associated with the “plant-associated beneficial and environmental (PBE)” group of Burkholderia, which are phylogenetically distinct from the SBE group, and that they maintain symbiosis through the environmental acquisition of the bacteria. Since the superfamilies Coreoidea, Lygaeoidea, and Pyrrhocoroidea are monophyletic in the infraorder Pentatomomorpha, it is plausible that the symbiotic association with Burkholderia evolved at the common ancestor of the three superfamilies. However, the results of this study strongly suggest that a dynamic transition from the PBE to SBE group, or vice versa, occurred in the course of stinkbug evolution.