Wild lime psyllid Leuronota fagarae Burckhardt (Hemiptera: Psylloidea) picorna-like virus full genome annotation and classification.

Picorna-like viruses of the order Picornavirales are a poorly defined group of positive-sense, single-stranded RNA viruses that include numerous pathogens known to infect plants, animals, and insects. A new picorna-like viral species was isolated from the wild lime psyllid (WLP), Leuronota fagarae, in the state of Florida, USA, and labelled: Leuronota fagarae picorna-like virus isolate FL (LfPLV-FL). The virus was found to have homology to a picorna-like virus identified in the Asian Citrus Psyllid (ACP), Diaphorina citri, collected in the state of Florida. Computational analysis of RNA extracts from WLP adult heads identified a 10,006-nucleotide sequence encoding a 2,942 amino acid polyprotein with similar functional domain structure to polyproteins of both Dicistroviridae and Iflaviridae. Sequence comparisons of nucleic acid and amino acid translations of the conserved RNA-dependent RNA polymerase, along with the entire N-terminal nonstructural coding region, provided insight into an evolutionary relationship of LfPLV-FL to insect-infecting iflaviruses. Viruses belonging to the family Iflaviridae encode a polyprotein of around 3000 amino acids in length that is processed post-translationally to produce components necessary for replication. The classification of a novel picorna-like virus in L. fagarae, with evolutionary characteristics similar to picorna-like viruses infecting Bactericera cockerelli and D. citri, provides an opportunity to examine virus host specificity, as well as identify critical components of the virus' genome required for successful transmission, infection, and replication. This bioinformatic classification allows for further insight into a novel virus species, and aids in the research of a closely related virus of the invasive psyllid, D. citri, a major pest of Floridian citriculture. The potential use of viral pathogens as expression vectors to manage the spread D. citri is an area that requires additional research; however, it may bring forth an effective control strategy to reduce the transmission of Candidatus Liberibacter asiaticus (CLas), the causative agent of Huanglongbing (HLB).

Lessons learned about the biology and genomics of Diaphorina citri infection with "Candidatus Liberibacter asiaticus" by integrating new and archived organ-specific transcriptome data.

BACKGROUND: Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of CLas and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to CLas infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from CLas-positive and CLas-negative D. citri using the chromosomal length D. citri genome assembly. RESULTS: Each organ had a unique transcriptome profile and response to CLas infection. Though most psyllids were infected with the bacterium, CLas-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNA-sequencing data interpretation. CONCLUSIONS: Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue-specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression.

Statistical analysis reveals the onset of synchrony in sparse swarms of Photinus knulli fireflies.

Flash synchrony within firefly swarms is an elegant but elusive manifestation of collective animal behaviour. It has been observed, and sometimes demonstrated, in a few populations across the world, but exactly which species are capable of large-scale synchronization remains unclear, especially for low-density swarms. The underlying question which we address here is: how does one qualify a collective flashing display as synchronous, given that the only information available is the time and location of flashes? We propose different statistical approaches and apply them to high-resolution stereoscopic video recordings of the collective flashing of Photinus knulli fireflies, hence establishing the occurrence of synchrony in this species. These results substantiate detailed visual observations published in the early 1980s and made at the same experimental site: Peña Blanca Canyon, Coronado National Forest, AZ, USA. We also remark that P. knulli's collective flashing patterns mirror those observed in Photinus carolinus fireflies in the Eastern USA, consisting of synchronous flashes in periodic bursts with rapid accretion and quick decay.

Evaluating firefly extinction risk: Initial red list assessments for North America.

Fireflies are a family of charismatic beetles known for their bioluminescent signals. Recent anecdotal reports suggest that firefly populations in North America may be in decline. However, prior to this work, no studies have undertaken a systematic compilation of geographic distribution, habitat specificity, and threats facing North American fireflies. To better understand their extinction risks, we conducted baseline assessments according to the categories and criteria of the International Union for Conservation of Nature (IUCN) Red List for 132 species from the United States and Canada (approximately 79% of described species in the region). We found at least 18 species (14%) are threatened with extinction (e.g. categorized as Critically Endangered, Endangered, or Vulnerable) due to various pressures, including habitat loss, light pollution, and climate change (sea level rise and drought). In addition, more than half of the species (53%) could not be evaluated against the assessment criteria due to insufficient data, highlighting the need for further study. Future research and conservation efforts should prioritize monitoring and protecting populations of at-risk species, preserving and restoring habitat, gathering data on population trends, and filling critical information gaps for data deficient species suspected to be at risk.

A stationary tweezer platform for high throughput dissections of minute arthropods and extirpation of their minute organs.

A homemade platform satisfied the need for fast, efficient dissection of minute arthropods and extirpation of their key organs, such as salivary glands and midguts, involved in agricultural disease transmission pathways. With its implementation, ~200 organs could be extirpated per 8 h workday while the subjects are submerged in protein or transcript protectant. A vacuum wand is used to capture insects and position them in the field of view. Two stationary tweezers are positioned on an adjustable scaffold that spans the microscope stage transversely such that their tips, and the insects they immobilize, can be submerged in select dissection media. High tensile strength fishlines are attached to the stationary tweezers for opening and closing with the 5th fingers while hand-held dissection tweezers load insects from the wand to their tines, then extirpate the target organs. Organs are lifted out with glass splints or plastic toothpicks into a final tube of select preservation media for freezing at session end.•Constructed from common retail materials•Adjustable design fits many microscopes•Can also be used in a wide variety of applications, including materials science.

Reinterpretation of 'sperm pump' or 'sperm syringe' function with notes on other male internal reproductive organs in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae).

Reproduction is a critical feature in the search for means to manage the Asian citrus psyllid, vector of a devastating bacterial pathogen of citrus. The importance of accuracy in functional, anatomical descriptions and interpretations for use by other disciplines, particularly molecular genetics, cannot be overstressed. The term 'sperm pump' was coined by classical authors on observational appearance of the endoskeleton of the male reproductive apparatus. They described a thimble-shaped cuticle with smooth, cylindrical columns, interpreted as muscles, that ran longitudinally around a central cylinder. They detected transverse lines on the cylinder giving the false impression of a coiled spring. These features fostered the teleological interpretation that the device is a contractile pump. Now obsolete, the term is replaced by 'drum/spout complex'. It is a hypodermis with a sclerotized cuticle that houses the phallus which transports seminal fluid through its lumen to the female for insemination. Between the spout and the external genitalia is a spout extension, conferring flexibility to the apparatus about the abdominal apex. Approximately 21 longitudinal columns extend circumferentially around the cylinder's hemolymph-side, from the thimble's basal plate to its apical plate. These columns are correctly muscle cells, and reinterpreted to exude a lipaceous, lubricating substance for mating.

New Anatomical Evidence from the Male Asian Citrus Psyllid (Hemiptera: Liviidae) Invokes Controversy Over the Accepted Function of Some Male Reproductive Organs in Psylloidea.

Males of many Psylloidea are known to possess a characteristic structure at the functional hub of their reproductive apparatus, between afferent and efferent passage of seminal fluid. The structure is a squat, cylindrical endoskeleton consisting of two sections. Classical authors named them as 'sperm pump' and 'ejaculatory duct', based on superficial resemblance to a spring-loaded, thimble-shaped cylinder, encircled by smooth, vertical columns interpreted to be muscles which, when contracted, compress the cylinder and affect seminal fluid discharge. The discovery of numerous spherules of unknown composition and function in and around the columns of the Asian citrus psyllid male genitalia invoked rigorous scrutiny of the classical literature for evidence to support its claims, and determined that the grounds for vetting the structure as a sperm pump were fully teleological. This paper raises several objections to modern acceptance of this classical interpretation, presenting them as problematic, thought-provoking, and sometimes controversial anatomical features. The two sections are herein called 'drum' and 'spout'. As an endoskeleton, the sections are an invagination of the exoskeleton and therefore cannot receive seminal fluid into their hollow. A phallus is identified inside an aedeagal tube, indicating that it is the ejaculatory duct-the tube, drum, and spout are considered its housing. A sheath envelopes the drum and is directly continuous with the spout hypodermis, another problematical feature raising the question of whether it is detached from adherence to the drum cuticles. Also, there are four afferent tubes but only two openings in the drum to receive their seminal fluids.

Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph.

The parasitic mite Varroa destructor is the greatest single driver of the global honey bee health decline. Better understanding of the association of this parasite and its host is critical to developing sustainable management practices. Our work shows that this parasite is not consuming hemolymph, as has been the accepted view, but damages host bees by consuming fat body, a tissue roughly analogous to the mammalian liver. Both hemolymph and fat body in honey bees were marked with fluorescent biostains. The fluorescence profile in the guts of mites allowed to feed on these bees was very different from that of the hemolymph of the host bee but consistently matched the fluorescence profile unique to the fat body. Via transmission electron microscopy, we observed externally digested fat body tissue in the wounds of parasitized bees. Mites in their reproductive phase were then fed a diet composed of one or both tissues. Mites fed hemolymph showed fitness metrics no different from the starved control. Mites fed fat body survived longer and produced more eggs than those fed hemolymph, suggesting that fat body is integral to their diet when feeding on brood as well. Collectively, these findings strongly suggest that Varroa are exploiting the fat body as their primary source of sustenance: a tissue integral to proper immune function, pesticide detoxification, overwinter survival, and several other essential processes in healthy bees. These findings underscore a need to revisit our understanding of this parasite and its impacts, both direct and indirect, on honey bee health.

Asian citrus psyllid stylet morphology and applicability to the model for inter-instar stylet replacement in the potato psyllid.

In Hemiptera, presumptive stylets for each consecutive postembryonic instar are manufactured prior to ecdysis to replace the ecdysial stylets discarded with the exuviae. With the discovery that the bacterium "Candidatus" Liberibacter solanacearum accesses the tissues involved in the stylet replacement process of the potato psyllid, a hypothesis was formed that the bacterium could adhere to the stylets of freshly emerged instars and hence gain access to the host plant when feeding is resumed. Although unproven, it was imperative that a model for stylet replacement be built. Stylet morphology and the stylet replacement process of the Asian citrus psyllid (ACP), vector of "C." L. asiaticus, causal pathogen of citrus greening disease, are comparable to the potato psyllid model system. Morphology consists of a basal terminus with its tab-shaped auricle, a base, shaft, and an apical terminus. Each of the four auricles act as a platform for the replacement apparatus, which is compacted into a tight aggregate of cells, the 'end-cap'. As modeled, on apolysis of larval instar hypodermis, the aggregate 'deconstructs' and expands into a snail shell-shaped tube, the 'atrium', that houses the presumptive stylet as it is synthesized. Completed stylets then despool from the atrium and are fitted into their functional positions as the next instar emerges from its exuviae.

Stylet biogenesis in Bactericera cockerelli (Hemiptera: Triozidae).

The discovery of 'Ca. Liberibacter solanacearum', causal agent of certain solanaceous and apiaceous crop diseases, inside the functional (intrastadial) and pharate stylet anatomy of the potato psyllid prompted elucidation of the mechanism of stylet replacement as a novel exit portal in the transmission pathway. In Hemiptera, presumptive (formative) stylets, secreted during consecutive pharate instars, replace functional stylets lost with the exuviae. In potato psyllids, each functional stylet has a hollow core filled with a cytology that extends out of the core to form a hemispherical aggregate of cells, the 'end-cap', somewhat resembling a golf ball on a tee. A tightly folded mass of extremely thin cells, the 'matrix', occurs inside the end-cap. Micrograph interpretations indicate that during the pharate stage, the end-cap apolyses from the core and 'deconstructs' to release and expand the matrix into a long, coiled tube, the 'atrium'. Cells that were in contact with the inner walls of the functional stylet core maintain their position at the apex of the tube, and secrete a new stylet, apex first, the growing length of which descends into the tube until completed. They then despool from the coils into their functional position as the exuviae is shed.

Colonization and Intrusive Invasion of Potato Psyllid by 'Candidatus Liberibacter solanacearum'.

Previous studies have shown that the fastidious bacterial plant pathogen 'Candidatus Liberibacter solanacearum' (CLso) is transmitted circulatively and propagatively by the potato psyllid (PoP) Bactericera cockerelli. In this study, the temporal and spatial interrelationships between CLso PoP were investigated by scanning electron microscopy of the digestive system of PoP immature and adult instars and salivary glands of adults post CLso ingestion. CLso biofilms were not detectable on the outer midgut surface of the first and second instars; however, for third to fifth instars and teneral and mature adults, biofilms were observed in increasing numbers in each successive developmental stage. In adult PoP midguts, CLso cells were observed between the basal lamina and basal epithelial cell membranes; in basal laminar perforations, on the outer basal laminar surface, and in the ventricular lumen, epithelial cytosol, and filter chamber periventricular space. CLso were also abundantly visible in the salivary gland pericellular spaces and in the epidermal cell cytosol of the head. Collectively, these results point to an intrusive, systemic invasion of PoP by CLso that employs an endo/exocytosis-like mechanism, in the context of a propagative, circulative mode of transmission.

Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the insect vector of the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease, or Huanglongbing (HLB). The widespread invasiveness of the psyllid vector and HLB in citrus trees worldwide has underscored the need for non-traditional approaches to manage the disease. One tenable solution is through the deployment of RNA interference technology to silence protein-protein interactions essential for ACP-mediated CLas invasion and transmission. To identify psyllid interactor-bacterial effector combinations associated with psyllid-CLas interactions, cDNA libraries were constructed from CLas-infected and CLas-free ACP adults and nymphs, and analyzed for differential expression. Library assemblies comprised 24,039,255 reads and yielded 45,976 consensus contigs. They were annotated (UniProt), classified using Gene Ontology, and subjected to in silico expression analyses using the Transcriptome Computational Workbench (TCW) (http://www.sohomoptera.org/ACPPoP/). Functional-biological pathway interpretations were carried out using the Kyoto Encyclopedia of Genes and Genomes databases. Differentially expressed contigs in adults and/or nymphs represented genes and/or metabolic/pathogenesis pathways involved in adhesion, biofilm formation, development-related, immunity, nutrition, stress, and virulence. Notably, contigs involved in gene silencing and transposon-related responses were documented in a psyllid for the first time. This is the first comparative transcriptomic analysis of ACP adults and nymphs infected and uninfected with CLas. The results provide key initial insights into host-parasite interactions involving CLas effectors that contribute to invasion-virulence, and to host nutritional exploitation and immune-related responses that appear to be essential for successful ACP-mediated circulative, propagative CLas transmission.

Comparison of potato and asian citrus psyllid adult and nymph transcriptomes identified vector transcripts with potential involvement in circulative, propagative liberibacter transmission.

The potato psyllid (PoP) Bactericera cockerelli (Sulc) and Asian citrus psyllid (ACP) Diaphorina citri Kuwayama are the insect vectors of the fastidious plant pathogen, Candidatus Liberibacter solanacearum (CLso) and Ca. L. asiaticus (CLas), respectively. CLso causes Zebra chip disease of potato and vein-greening in solanaceous species, whereas, CLas causes citrus greening disease. The reliance on insecticides for vector management to reduce pathogen transmission has increased interest in alternative approaches, including RNA interference to abate expression of genes essential for psyllid-mediated Ca. Liberibacter transmission. To identify genes with significantly altered expression at different life stages and conditions of CLso/CLas infection, cDNA libraries were constructed for CLso-infected and -uninfected PoP adults and nymphal instars. Illumina sequencing produced 199,081,451 reads that were assembled into 82,224 unique transcripts. PoP and the analogous transcripts from ACP adult and nymphs reported elsewhere were annotated, organized into functional gene groups using the Gene Ontology classification system, and analyzed for differential in silico expression. Expression profiles revealed vector life stage differences and differential gene expression associated with Liberibacter infection of the psyllid host, including invasion, immune system modulation, nutrition, and development.

Implication of Bemisia tabaci heat shock protein 70 in Begomovirus-whitefly interactions.

The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally, membrane feeding of whiteflies with anti-HSP70 antibodies and TYLCV virions showed an increase in TYLCV transmission, suggesting an inhibitory role for HSP70 in virus transmission, a role that might be related to protection against begomoviruses while translocating in the whitefly.

Composite, haustellate mouthparts in netwinged beetle and firefly larvae (Coleoptera, Cantharoidea: Lycidae, Lampyridae).

During lycid embryogenesis, labral and mandibular anlagen develop separately. Each anterolateral corner of the labrum elongates into a stylet, while the whole of each mandible involutes into a flute that is open to the lateral aspects. The stylets and flutes interlock in such a way as to leave canals, fashioning left and right haustellate apparatuses. During lampyrid embryogenesis, only one pair of anlagen forms instead of two. The pair matures into sharp, arcuate appendages that appear to be typical mandibles, but nevertheless a canal forms internally. The canal is not formed by coadaptation of two appendages; instead, cells within the pair create the canal autonomously. Indications are that the canal is homologous between these two families. It is suggested that in lampyrids, labral and mandibular cell fields develop together in the same anlage but behave according to their own respective programs, forming the canal with morphogenetic movements that are comparable to those of lycids. © 1994 Wiley-Liss, Inc.