Allison K. Hansen

A new Huanglongbing Species, "Candidatus Liberibacter psyllaurous," found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc).

ABSTRACT A new huanglongbing (HLB) “Candidatus Liberibacter” species is genetically characterized, and the bacterium is designated “Candidatus Liberibacter psyllaurous.” This bacterium infects the psyllid Bactericera cockerelli and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.

Aphid genome expression reveals host–symbiont cooperation in the production of amino acids

The evolution of intimate symbiosis requires the coordination of gene expression and content between the distinct partner genomes; this coordination allows the fusion of capabilities of each organism into a single integrated metabolism. In aphids, the 10 essential amino acids are scarce in the phloem sap diet and are supplied by the obligate bacterial endosymbiont (Buchnera), which lives inside specialized cells called bacteriocytes. Although Buchnera’s genome encodes most genes for essential amino acid biosynthesis, several genes in essential amino acid pathways are missing, as are most genes for production of nonessential amino acids. Additionally, it is unresolved whether the supply of nitrogen for amino acid biosynthesis is supplemented by recycling of waste ammonia. We compared pea aphid gene expression between bacteriocytes and other body tissues using RNA sequencing and pathway analysis and exploiting the genome sequences available for both partners. We found that 26 genes underlying amino acid biosynthesis were up-regulated in bacteriocytes. Seven of these up-regulated genes fill the gaps of Buchnera’s essential amino acid pathways. In addition, genes underlying five nonessential amino acid pathways lost from Buchnera are up-regulated in bacteriocytes. Finally, our results reveal that two genes, glutamine synthetase and glutamate synthase, which potentially work together in the incorporation of ammonium nitrogen into glutamate (GOGAT) cycle to assimilate ammonia into glutamate, are up-regulated in bacteriocytes. Thus, host gene expression and symbiont capabilities are closely integrated within bacteriocytes, which function as specialized organs of amino acid production. Furthermore, the GOGAT cycle may be a key source of nitrogen fueling the integrated amino acid metabolism of the aphid–Buchnera partnership.

Distinctive gut microbiota of honey bees assessed using deep sampling from individual worker bees

Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to “Candidatus Gilliamella apicola”) one within Betaproteobacteria (“Candidatus Snodgrassella alvi”), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype.

The impact of microbial symbionts on host plant utilization by herbivorous insects

Herbivory, defined as feeding on live plant tissues, is characteristic of highly successful and diverse groups of insects and represents an evolutionarily derived mode of feeding. Plants present various nutritional and defensive barriers against herbivory; nevertheless, insects have evolved a diverse array of mechanisms that enable them to feed and develop on live plant tissues. For decades, it has been suggested that insect‐associated microbes may facilitate host plant use, and new molecular methodologies offer the possibility to elucidate such roles. Based on genomic data, specialized feeding on phloem and xylem sap is highly dependent on nutrient provisioning by intracellular symbionts, as exemplified by Buchnera in aphids, although it is unclear whether such symbionts play a substantive role in host plant specificity of their hosts. Microorganisms present in the gut or outside the insect body could provide more functions including digestion of plant polymers and detoxification of plant‐produced toxins. However, the extent of such contributions to insect herbivory remains unclear. We propose that the potential functions of microbial symbionts in facilitating or restricting the use of host plants are constrained by their location (intracellular, gut or environmental), and by the fidelity of their associations with insect host lineages. Studies in the next decade, using molecular methods from environmental microbiology and genomics, will provide a more comprehensive picture of the role of microbial symbionts in insect herbivory.

Frequency of secondary symbiont infection in an invasive psyllid relates to parasitism pressure on a geographic scale in California.

ABSTRACT Two endosymbionts, an obligate primary symbiont and a facultative secondary symbiont, are harbored within the invasive red gum (eucalyptus) lerp psyllid, Glycaspis brimblecombei, in California. An extensive survey of diversity and frequency of G. brimblecombeis secondary symbiont in multiple populations throughout the state of California was conducted using PCR detection, restriction enzymes, cloning, and sequencing. A total of 380 G. brimblecombei individuals in 19 populations were screened for secondary symbionts. Based on molecular screening results, only one type of secondary symbiont was present in G. brimblecombei populations in California. Overall, 40% of the 380 psyllids screened were infected with the secondary symbiont. Interestingly, secondary symbiont infection frequencies in G. brimblecombei populations varied dramatically from 0 to 75% and were significantly related to parasitism pressure by Psyllaphaegus bliteus, a solitary endoparasitoid of the psyllid.

Independent Studies Using Deep Sequencing Resolve the Same Set of Core Bacterial Species Dominating Gut Communities of Honey Bees

Starting in 2003, numerous studies using culture-independent methodologies to characterize the gut microbiota of honey bees have retrieved a consistent and distinctive set of eight bacterial species, based on near identity of the 16S rRNA gene sequences. A recent study [Mattila HR, Rios D, Walker-Sperling VE, Roeselers G, Newton ILG (2012) Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse. PLoS ONE 7(3): e32962], using pyrosequencing of the V1–V2 hypervariable region of the 16S rRNA gene, reported finding entirely novel bacterial species in honey bee guts, and used taxonomic assignments from these reads to predict metabolic activities based on known metabolisms of cultivable species. To better understand this discrepancy, we analyzed the Mattila et al. pyrotag dataset. In contrast to the conclusions of Mattila et al., we found that the large majority of pyrotag sequences belonged to clusters for which representative sequences were identical to sequences from previously identified core species of the bee microbiota. On average, they represent 95% of the bacteria in each worker bee in the Mattila et al. dataset, a slightly lower value than that found in other studies. Some colonies contain small proportions of other bacteria, mostly species of Enterobacteriaceae. Reanalysis of the Mattila et al. dataset also did not support a relationship between abundances of Bifidobacterium and of putative pathogens or a significant difference in gut communities between colonies from queens that were singly or multiply mated. Additionally, consistent with previous studies, the dataset supports the occurrence of considerable strain variation within core species, even within single colonies. The roles of these bacteria within bees, or the implications of the strain variation, are not yet clear.

Manipulation of plant defense responses by the tomato psyllid (Bactericerca cockerelli) and its associated endosymbiont Candidatus Liberibacter psyllaurous.

Some plant pathogens form obligate relationships with their insect vector and are vertically transmitted via eggs analogous to insect endosymbionts. Whether insect endosymbionts manipulate plant defenses to benefit their insect host remains unclear. The tomato psyllid, Bactericerca cockerelli (Sulc), vectors the endosymbiont “Candidatus Liberibacter psyllaurous” (Lps) during feeding on tomato (Solanum lycopersicum L.). Lps titer in psyllids varied relative to the psyllid developmental stage with younger psyllids harboring smaller Lps populations compared to older psyllids. In the present study, feeding by different life stages of B. cockerelli infected with Lps, resulted in distinct tomato transcript profiles. Feeding by young psyllid nymphs, with lower Lps levels, induced tomato genes regulated by jasmonic acid (JA) and salicylic acid (SA) (Allene oxide synthase, Proteinase inhibitor 2, Phenylalanine ammonia-lyase 5, Pathogenesis-related protein 1) compared to feeding by older nymphs and adults, where higher Lps titers were found. In addition, inoculation of Lps without insect hosts suppressed accumulation of these defense transcripts. Collectively, these data suggest that the endosymbiont-like pathogen Lps manipulates plant signaling and defensive responses to benefit themselves and the success of their obligate insect vector on their host plant.

Genomic basis of endosymbiont-conferred protection against an insect parasitoid

Bacterial endosymbionts exert a variety of beneficial effects on insect hosts. In pea aphids (Acyrthosiphon pisum), several inherited endosymbiont species protect their hosts against parasitoid wasps, which are major natural enemies. However, strains of these symbiont species vary in their ability to confer protection against parasitoids, with some conferring almost complete protection and others conferring almost none. In this study, two strains of the endosymbiont Regiella insecticola (R. insecticola 5.15 and R. insecticola LSR1) were found to differ in ability to protect pea aphids attacked by the parasitoid Aphidius ervi. Parasitism trials reveal that R. insecticola 5.15, but not R. insecticola LSR1, significantly reduced parasitoid success and increased aphid survivorship. To address the potential genetic basis of protection conferred by R. insecticola 5.15 we sequenced the genome of this symbiont strain, and then compared its gene repertoire with that of the already sequenced nonprotective strain R. insecticola LSR1. We identified striking differences in gene sets related to eukaryote pathogenicity. The protective strain R. insecticola 5.15 encoded five categories of pathogenicity factors that were missing or inactivated in R. insecticola LSR1. These included genes encoding the O-antigen biosynthetic pathway, an intact Type 1 Secretion System and its secreted RTX toxins, an intact SPI-1 Type 3 Secretion System and its effectors, hemin transport, and the two-component system PhoPQ. These five pathogenicity factors and translocation systems are hypothesized to collectively play key roles in the endosymbionts virulence against parasitoids, resulting in aphid protection. Mechanisms through which these factors may target parasitoids are discussed.

Widespread expression of conserved small RNAs in small symbiont genomes.

Genome architecture of a microbe markedly changes when it transitions from a free-living lifestyle to an obligate symbiotic association within eukaryotic cells. These symbiont genomes experience numerous rearrangements and massive gene loss, which is expected to radically alter gene regulatory networks compared with those of free-living relatives. As such, it remains unclear whether and how these small symbiont genomes regulate gene expression. Here, using a label-free mass-spec quantification approach we found that differential protein regulation occurs in Buchnera, a model symbiont with a reduced genome, when it transitions between two distinct life stages. However, differential mRNA expression could not be detected between Buchnera life stages, despite the presence of a small number of putative transcriptional regulators. Instead a comparative analysis of small RNA expression profiles among five divergent Buchnera lineages, spanning a variety of Buchnera life stages, reveals 140 novel intergenic and antisense small RNAs and 517 untranslated regions that were significantly expressed, some of which have been conserved for ∼65 million years. In addition, the majority of these small RNAs exhibit both sequence covariation and thermodynamic stability, indicators of a potential structural RNA role. Together, these data suggest that gene regulation at the post-transcriptional level may be important in Buchnera. This is the first study to empirically identify Buchnera small RNAs, and we propose that these novel small RNAs may facilitate post-transcriptional regulation through translational inhibition/activation, and/or transcript stability. Ultimately, post-transcriptional regulation may shape metabolic complementation between Buchnera and its aphid host, thus impacting the animal’s ecology and evolution.

Midgut fungal and bacterial microbiota of Aedes triseriatus and Aedes japonicus shift in response to La Crosse virus infection.

Understanding how midgut microbial communities of field‐collected mosquitoes interact with pathogens is critical for controlling vector infection and disease. We used 16S rRNA and internal transcribed spacer sequencing to characterize the midgut bacterial and fungal communities of adult females of Aedes triseriatus and Aedes japonicus collected as pupae in tree holes, plastic bins and waste tires and their response to La Crosse virus (LACV) infection. For both mosquito species and across all habitat and virus treatments, a total of 62 bacterial operational taxonomic units (OTUs) from six phyla and 21 fungal OTUs from two phyla were identified. The majority of bacterial (92%) and fungal (71%) OTUs were shared between the mosquito species; however, several OTUs were unique to each species. Bacterial and fungal communities of individuals that took either infectious or noninfectious bloodmeals were less diverse and more homogeneous compared to those of newly emerged adults. Interestingly, LACV‐infected A. triseriatus and A. japonicus had higher bacterial richness and lower fungal richness compared to individuals that took a noninfectious bloodmeal, suggesting that viral infection was associated with an increase in bacterial OTUs and a decrease in fungal OTUs. For both mosquito species, several OTUs were identified that had both high fidelity and specificity to mosquito midguts that were infected with LACV. Overall, these findings demonstrate that bacterial and fungal communities that reside in mosquito midguts respond to host diet and viral infection and could play a role in modulating vector susceptibility to LACV.