华南地区柑橘木虱与柑橘粉虱内共生菌检测及其Wolbachia共生菌的系统发育关系分析

【目的】探明不同地理种群的柑橘木虱Diaphorina citri Kuwayama和柑橘粉虱Dialeurodes citri Ashmead体内昆虫内共生菌的种类及其感染率,并以Wolbachia共生菌为代表,对其系统发育关系进行分析,为今后自共生菌角度研发柑橘木虱和柑橘粉虱的新型防控技术奠定基础。【方法】以16S r DNA、23S r DNA以及wsp为目标基因,利用PCR技术检测采自于广州、湛江、南宁、桂林、厦门的柑橘木虱以及采自广州的柑橘粉虱体内共生菌的种类及其感染率;利用多位点序列分型(MLST)技术和MEGA 5.0软件对不同昆虫样本中的Wolbachia进行系统发育关系分析。【结果】本研究采集的柑橘木虱和柑橘粉虱均含有原生共生菌Portiera和次生共生菌Wolbachia、Cardinium、Rickettsia,但该3种次生共生菌在不同木虱与粉虱种群的感染率有所不同;Arsenophonus只在广州和湛江种群的柑橘木虱中检出。基于wsp基因及MLST基因序列的Wolbachia系统发育分析表明,华南地区柑橘木虱和柑橘粉虱体内的Wolbachia均属于Wolbachia的B大组Con亚组。【结论】不同地理种群的柑橘木虱与柑橘粉虱体内感染的共生菌种类及其感染率不同;Wolabchia共生菌与柑橘木虱寄主不存在协同进化关系,在同一采集点存在Wolbachia通过柑橘寄主在柑橘木虱之间、柑橘木虱与柑橘粉虱之间水平传播的可能性。     

Population Dynamics and Growth Rates of Endosymbionts During Diaphorina citri (Hemiptera,Liviidae) Ontogeny

The infection density of symbionts is among the major parameters to understand their biological effects in host–endosymbionts interactions. Diaphorina citri harbors two bacteriome-associated bacterial endosymbionts (Candidatus Carsonella ruddii and Candidatus Profftella armatura), besides the intracellular reproductive parasite Wolbachia. In this study, the density dynamics of the three endosymbionts associated with the psyllid D. citri was investigated by real-time quantitative PCR (qPCR) at different developmental stages. Bacterial density was estimated by assessing the copy number of the 16S rRNA gene for Carsonella and Profftella, and of the ftsZ gene for Wolbachia. Analysis revealed a continuous growth of the symbionts during host development. Symbiont growth and rate curves were estimated by the Gompertz equation, which indicated a negative correlation between the degree of symbiont–host specialization and the time to achieve the maximum growth rate (t*). Carsonella densities were significantly lower than those of Profftella at all host developmental stages analyzed, even though they both displayed a similar trend. The growth rates of Wolbachia were similar to those of Carsonella, but Wolbachia was not as abundant. Adult males displayed higher symbiont densities than females. However, females showed a much more pronounced increase in symbiont density as they aged if compared to males, regardless of the incorporation of symbionts into female oocytes and egg laying. The increased density of endosymbionts in aged adults differs from the usual decrease observed during host aging in other insect–symbiont systems.     

Molecular Characterization of Wolbachia Strains Associated with the Invasive Asian Citrus Psyllid Diaphorina citri in Brazil

Wolbachia is a symbiont intensively studied due to its ability to interfere with their host’s reproduction, and it has been recently proposed as an alternative tool to control insect pests or vectors of diseases. The Asian citrus psyllid Diaphorina citri is an important pest of citrus since it vectors the bacterium that causes the "Huanglongbing" disease in citrus. The frequency and diversity of Wolbachia associated with D. citri is unknown, limiting the utilization of Wolbachia as an alternative strategy for insect management. Thus, we aimed to determine the natural rate of infection, to characterize the Wolbachia strains associated with this psyllid by "multilocus sequencing typing” (MLST) and wsp analysis, and to verify the association of the symbiont to particular genotypes of the host. Analysis indicated Wolbachia infects 100 % of all specimens tested from all 15 sampled populations. MLST revealed the occurrence of five new sequence types (STs) of Wolbachia, while analysis based on the wsp sequences indicated only four different types of Wolbachia. ST-173 was predominant, while the remaining STs were population specific. Analysis of the host–symbiont relationship did not reveal any particular association of Wolbachia and haplotypes or a decrease in nucleotide diversity of D. citri in populations in which more than one ST was recorded. The consequences of the diversity of STs reported are still unknown, but the fact that Wolbachia infection is fixed and that there is one ST with a broad distribution highlights the use of this symbiont as an alternative strategy to control D. citri.     

Comparative microbiome analysis of Diaphorina citri and its associated parasitoids Tamarixia radiata and Diaphorencyrtus aligarhensis reveals Wolbachia as a dominant endosymbiont

Microbiome analysis in a host–parasitoid interaction network was conducted to compare the taxonomic composition of bacterial communities of Diaphornia citri, Tamarixia radiata, and Diaphorencyrtus aligarhensis. The comparative analysis revealed differences in the composition and diversity of the symbiont populations across the host and its associated parasitoids. Proteobacteria was the most dominant phylum, representing 67.80% of the total bacterial community, while Candidatus Profftella armature and Wolbachia were the dominant genera across the host and parasitoids. There were clear differences observed in alpha and beta diversity of microbiota through the host and its associated parasitoids. The function prediction of bacterial communities and Pearson correlation analysis showed that specific bacterial communities displayed positive correlations with the carbohydrate metabolism pathway. Furthermore, when symbiotic bacteria were eliminated using a broad-spectrum antibiotic, tetracycline hydrochloride, the parasitoids' median survival time and longevity were significantly reduced. We confirmed the physiological effects of symbiotic bacteria on the fitness of parasitoids and demonstrated the effect of antibiotics in decreasing the food intake and measurement of amino acids in the hemolymph. This study sheds light on basic information about the mutualism between parasitoids and bacteria, which may be a potential source for biocontrol strategies for citrus psyllid, especially D. citri.     

Genetic variation and potential coinfection of Wolbachia among widespread Asian citrus psyllid (Diaphorina citri Kuwayama) populations

Wolbachia can profoundly influence the survival, reproduction, and defenses of insect hosts. These interactions could potentially be harnessed for managing pests or insecttransmitted diseases. Diaphorina citri Kuwayama is a phloem-feeding pest capable of transmitting the putative causal agent of citrus greening, Candidatus Liberibacter asiaticus (CLas). Like many insects, D. citri is also infected with Wolbachia (wDi). Recent studies indicate that the relative abundance of wDi could be associated with the abundance of CLas, and that wDi may contribute to regulating expression of phage lytic cycle genes in CLas, suggesting the need for better understanding of wDi biology in general. This study investigated the genetic diversity of wDi among D. citri in populations spanning eleven countries and two U.S. territories. Six Wolbachia genes, wsp, coxA.fbpA.ftsZ, gatB, and hep A, were sequenced and compared across samples. Two prevalent wDi strains were identified across the samples, and screening of clone libraries revealed possible coinfection of wDi strains in specific populations. D. citri mitochondrial cytochrome oxidase subunit I gene (mtCOI) were more divergent between D. citri populations that were infected with different wDi strains or had different infection statuses (single infection vs. coinfection). While we could not eliminate the possibility that maternal transmission may contribute to such patterns, it is also possible that wDi may induce cytoplasmic incompatibility in their host. These fin dings should contribute to the understanding of wDi population ecology, which may facilitate manipulation of this endosymbiont for management of citrus greening disease worldwide.     

Assembly of a Marine Viral Metagenome after Physical Fractionation

Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.     

Temporal response and attraction of Diaphorina citri to visual stimuli

As the vector of the global disease of citrus greening or huanglongbing, Asian citrus pysllids, Diaphorina citri Kuwayama (Hemiptera: Liviidae), are the greatest threat to the worldwide citrus industry. Critical to management of D. citri and huanglongbing is optimization of surveillance methodologies. Although phytophagous insects may find host plants by multimodal cues, some appear to primarily use visual cues. In this study, we examined the behavior of Asian citrus psyllids toward light from light‐emitting diodes (LEDs) in the insect visible spectrum. The periodicity of attraction of psyllids to visual cues was evaluated in the field (yellow sticky traps) and laboratory (multi‐colored LEDs) with a strong peak of activity during the afternoon in both the field and the laboratory (both 14:00 to 18:00 hours). In laboratory evaluations of psyllids to differently colored LEDs, strongest attraction was to LEDs emitting ultraviolet (390 nm), green (525 nm), and yellow (590 nm) light. Male and female psyllids did not differ significantly in their responses to visual cues. These findings provide the basis for formulating better traps that reflect UV and yellow light and potentially incorporate UV LEDs for monitoring psyllids and a better understanding of Asian citrus psyllid visual behavior.     

Asymmetrical Interactions between Wolbachia and Spiroplasma Endosymbionts Coexisting in the Same Insect Host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

Importance of multiple mating to female reproductive output in Diaphorina citri

Abstract The importance of multiple mating to female reproductive output in Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is evaluated by grouping individual females with one or three males for 24 h (short duration) or 2 weeks (long duration) and examining oviposition over 18–19 days. For the short‐duration treatments, females lay more eggs per day when grouped with multiple males, whereas females in the long‐duration treatments lay more eggs when paired with one male. When held for 24 h with one or three males, females show a decline in fecundity beginning 10 and 15 days after mating, respectively. Total fecundity is relatively high for females paired with one male for 2 weeks, but fecundity is low and mortality high when females are held with three males for 2 weeks. In treatments in which females are held with males for 2 weeks, oviposition increases dramatically in the days after removal of males. For females paired with a male for 24 h and re‐paired for 24 h, 9 days later, fecundity remains high throughout the 18–19‐day observation period. Egg fertility does not differ among treatments, but varies over time in a manner that is similar among treatments. The present study demonstrates that, in D. citri, females require multiple matings over time to achieve high reproductive output, but oviposition is constrained by the presence of males.     

Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes,Sebastiscus) via Genome Survey Sequencing

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.     

In vitro egg hatching of Diaphorina citri,the vector of Huanglongbing

Citrus greening (Huanglongbing, HLB) is a destructive disease associated with the uncultivable, phloem-limited, gram-negative bacteria Candidatus Liberibacter spp., which affects citrus crops and other Rutaceae species. HLB is rapidly transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We developed an artificial medium on which D. citri eggs can hatch and first-instar nymphs can feed. The medium could be used to study insect physiology and screen molecules that may interfere with egg hatching and nymph development.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

Susceptibility of nymphs and adults of Diaphorina citri to the entomophathogenic fungus Hirsutella citriformis

The susceptibility of nymphs and adults of Diaphorina citri to infection by Hirsutella citriformis was evaluated. We also studied the ability of adult D. citri that had been contaminated with fungal conidia to transmit infection to nymphs. Diaphorina citri nymphs were more susceptible than adults. Adults were not able to transmit conidia to nymphs and cause infection.