Development of RNAi methods to control the harlequin bug, Murgantia histrionica.

The harlequin bug (HB), Murgantia histrionica, is a major pest of cabbage family plants throughout its range in the United States. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism that is showing promise as a biopesticide due to the ability to target species-specific genes necessary for growth and/or survival with synthetic double-stranded RNA (dsRNA). In the present study, dsRNA stability assays revealed that nucleases present in the saliva of harlequin bugs did not rapidly degrade dsRNA. We tracked the movement and localization of radioactively labeled dsRNA in both mustard plant seedlings and harlequin bug nymphs that fed on treated host plants. Movement of 32 P-labeled-dsRNA from soil to plant and plant to insect was detected. The efficacy of RNAi in inducing mortality in harlequin bug adults and nymphs injected or fed with dsRNA targeting inhibitor of apoptosis (IAP), ATPase N2B (ATPase), serine/threonine-protein phosphatase PP1-ß catalytic subunit (PP1), signal recognition particle 54 kDa protein (SRP), and G protein-coupled receptor 161-like (GPCR) genes was evaluated. Injection of dsRNA targeting candidate genes into adults caused between 40% and 75% mortality and induced significant knockdown of target gene expression. Feeding dsRNA targeting the IAP gene to nymphs by plant-mediated and droplet feeding methods induced knockdown of the target gene and caused 40-55% mortality. These findings suggest that RNAi may be a viable approach for managing this pest.

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference.

Salivary enzymes of many piercing-sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3-4) and adults (PG3-5), using siRNA injection-based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA-treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3-4 and PG3-5 siRNA-treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury.

A Deep Insight Into the Sialotranscriptome of the Chagas Disease Vector, Panstrongylus megistus (Hemiptera: Heteroptera).

Saliva of blood-sucking arthropods contains a complex cocktail of pharmacologically active compounds that assists feeding by counteracting their hosts' hemostatic and inflammatory reactions. Panstrongylus megistus (Burmeister) is an important vector of Chagas disease in South America, but despite its importance there is only one salivary protein sequence publicly deposited in GenBank. In the present work, we used Illumina technology to disclose and publicly deposit 3,703 coding sequences obtained from the assembly of >70 million reads. These sequences should assist proteomic experiments aimed at identifying pharmacologically active proteins and immunological markers of vector exposure. A supplemental file of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/P_megistus/Pmeg-web.xlsx.

[Ultrastructural observation on nymphal Armillifer sp. by scanning electron microscopy and phylogenetic analysis based on 18S rRNA].

OBJECTIVE: To observe the ultrastructure of nymphal Armillifer sp. isolated from Macaca fascicularis by using scanning electron microscope (SEM), and analyze the phylogenetic relationships based on 18S rRNA gene sequences. METHODS: The parasite samples stored in 70% alcohol were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the 18S rRNA gene were performed following the extraction of total genome DNA. Sequence analysis was performed based on multiple alignment using ClustalX1.83, while phylogenetic analysis was made by Neighbor-Joining method using MEGA4.0. RESULTS: The nymphs were in cylindrical shape, the body slightly claviform tapering to posterior end. Abdominal annuli were gradually widened from anterior to posterior parts, the 12th-13th abdominal annuli of which were similar in width. The annuli ranged closer in the front half body, whereas in the latter part there were certain gaps between them. The circular-shaped mouth located in the middle of head ventrally. Folds were seen in inner margin of the mouth with a pair of curved hooks on both sides above it which practically disposed in a straight line. Two pairs of large sensory papillae were observed symmetrically over the last thoracic annulus of cephalothoraxs lying below the outer hook, and the first abdominal annulus was near the median ventral line. The number of abdominal annuli was 29, not including 2 incomplete terminal annuli. Rounded sensory papillae were fully distributed on the body surface, except the dorsal side of head and the ventral part of the terminal annulus. Agglomerate-like anus opening was observed at the end of ventral abdominal annuli and distinctly sub-terminal. These morphological features demonstrated that the nymphs were highly similar with that of Armillifer moniliformis Diesing, 1835. A fragment of 18SrRNA gene (1 836 bp) sequences was obtained by PCR combined with sequencing, and was registered to the GeneBank database with an accession number HM048870. The phylogenetic tree indicated that A. moniliformis, A.agkistrodon and A.armillatus were at the same clade with a bootstrap value at 95%, and A. moniliformis and A. agkistrodon were solo at a clade with a bootstrap value of 75%. CONCLUSION: The nymphs isolated from Macaca fascicularis are identified as A. moniliformis temporarily.

Diet-Induced Plasticity of Linear Static Allometry Is Not So Simple for Grasshoppers: Genotype-Environment Interaction in Ontogeny Is Masked by Convergent Growth.

Grasshoppers, Melanoplus sanguinipes (Orthoptera: Acrididae), develop larger head width (HW) and shorter leg length, relative to body size, when fed low nutrient, lignin-rich grasses compared to sibs fed a diet of high nutrient grasses. To elucidate how underlying genetic variation and plasticity of growth generate plasticity of this linear static allometry within coarse-grained environments, I measured head and leg size of three nymphal instars and adult grasshoppers raised on either a low or high nutrient diet within a half-sib quantitative genetic experiment. Doubly-multivariate repeated measures multiple analysis of variance (MANOVA) of head, mandible, and hind leg size and their rate of growth (mm/period) and growth period (days) through ontogeny were used to analyze how the ontogeny of diet-induced plasticity for these variables and additive genetic variation for plasticity (genotype × environment interaction [G×E]) contribute to plasticity in functional linear static allometry. Genetic variation for diet-induced plasticity (G×E) of head and leg size varied through ontogeny, as did genetic variation for plasticity of growth in third and fourth instar nymphs. Despite extensive genetic variation in plasticity of HW and leg length in fourth instar nymphs, the static allometry between head and leg was stable within each diet because the patterns of G×E were similar for HW, leg length and their coordinated growth. Nutrient sensitive plasticity in growth shifted the intercept but not the slope of static allometry, a result consistent with one outcome of a graphical model of the relationships between G× E and plasticity of within environment static allometry. In addition, G×E of fourth instar head and leg size was reduced in adults by negatively size-dependent, convergent growth in the last period of ontogeny. Consequently, the bivariate reaction norms of head and leg size for adults exhibited no G×E and, again, plasticity in the intercept but not in the slope of static allometry. The ontogeny of seemingly simple diet-induced linear static allometry between functional body parts in grasshoppers arises from a complex combination of differing patterns of nutrient-sensitive growth, duration of growth, convergent growth, and G×E, all relevant to understanding the development and evolution of functional allometry in hemimetabolous insects.

Liposome encapsulation and EDTA formulation of dsRNA targeting essential genes increase oral RNAi-caused mortality in the Neotropical stink bug Euschistus heros.

BACKGROUND: The Neotropical stink bug Euschistus heros is a major pest in soybean fields. Development of highly species-specific pesticides based on RNA interference (RNAi) could provide a new sustainable and environmentally friendly control strategy. RESULTS: Here, the potential of RNAi as a pest control tool against E. heros was assessed. First, target gene selection using a microinjection approach was performed. Seven of the 15 candidate genes tested exhibited > 95% mortality after hemolymph injection of 27.5 ng dsRNA. Subsequently, dsRNA was administered orally using different formulations: naked dsRNA, liposome-encapsulated-dsRNA and dsRNA formulated with EDTA. Liposome-encapsulated dsRNA targeting vATPase A and muscle actin led to significant mortality after 14 days (45% and 42%, respectively), whereas EDTA-formulated dsRNA did so for only one of the target genes. Ex vivo analysis of the dsRNA stability in collected saliva indicated a strong dsRNA-degrading capacity by E. heros saliva, which could explain the need for dsRNA formulations. CONCLUSION: The results demonstrate that continuous ingestion of dsRNA with EDTA or liposome-encapsulated dsRNA can prevent dsRNA from being degraded enzymatically and suggest great potential for using these formulations in dsRNA delivery to use RNAi as a functional genomics tool or for pest management of stink bugs. © 2018 Society of Chemical Industry.

Redescription of the adult stages of Ixodes (Afrixodes) rasus Neumann 1899, with notes on its phylogenetic position within the genus Ixodes.

The tick subgenus Afrixodes Morel 1966 (Ixodidae) is distributed in sub-Saharan Africa. It consists of about 60 species which are described based on a few specimens and/or only some developmental stages, mostly adults. Because of this, the diagnosis of the African species of Ixodes is in many aspects complicated. Ixodes rasus Neumann 1899 is a common species, widely distributed in tropical and subtropical moist broadleaved forests in central Africa, whose morphology remains poorly described. We redescribe the adults of the species and provide molecular data for 16S rDNA based on three questing females and one male collected in the Central African Republic. Both sexes have an anal groove rounded and closed. The female has syncoxae on coxae I to III, a dental formula of 2/2, and long, curved auriculae. The male has moderate syncoxae on coxae I-III, coxae I-II carry one single internal spur each: coxae III and IV unarmed. The cornua are absent in the male, and the auriculae are small lobes. The male pregenital plate is longer than broad, with an anterior margin widely rounded; the median plate is large and clearly divergent posteriorly, with adanal plates fused posteriorly to the anus, and a dental formula of 4/4 (one row of 5/5). The molecular features of 16S rDNA place I. rasus near I. aulacodi (the only other Afrixodes species for which 16S rDNA is available) and near I. turdus and I. frontalis; two species of ticks of the Palearctic region, which commonly infest birds. The paucity of data regarding the subgenus Afrixodes makes it necessary to provide reliable and comparable re-descriptions of several species, as well as description of the immature stages for many of the currently known taxa.

Identification of Lyme borreliae proteins promoting vertebrate host blood-specific spirochete survival in Ixodes scapularis nymphs using artificial feeding chambers.

Lyme borreliosis, the most common vector-borne illness in Europe and the United States, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex and transmitted by Ixodes ticks. In humans, the spirochetes disseminate from the tick bite site to multiple tissues, leading to serious clinical manifestations. The ability of spirochetes to survive in ticks during blood feeding is thought to be essential for Lyme borreliae to be transmitted to different vertebrate hosts. This ability is partly attributed to several B. burgdorferi proteins, including BBA52 and Lp6.6, which promote spirochete survival in nymphal ticks feeding on mice. One of the strategies to identify such proteins without using live animals is to feed B. burgdorferi-infected ticks on blood via artificial feeding chambers. In previous studies, ticks were only fed on bovine blood in the feeding chambers. In this study, we used this chamber model and showed that I. scapularis ticks will not only acquire bovine blood but human and quail blood as well. The latter two are the incidental host and an avian host of Lyme borreliae, respectively. We also investigated the roles that BBA52 and Lp6.6 play in promoting spirochete survival in nymphal ticks fed on human or quail blood. After feeding on human blood, spirochete burdens in ticks infected with an lp6.6-deficient B. burgdorferi were significantly reduced, while bba52-deficient spirochete burdens in ticks remained unchanged, similar to the wild-type strain. No strain showed a change in spirochete burdens in ticks fed on quail blood. These results indicate that Lp6.6 plays a role for B. burgdorferi in nymphs fed on human but not quail blood. Such information also demonstrates that the artificial feeding chamber is a powerful tool to identify B. burgdorferi proteins that promote vertebrate host blood-specific spirochete survival in I. scapularis ticks.

Genomics of interaction between the brown planthopper and rice.

Rice (Oryza sativa L.) and the brown planthopper (Nilaparvata lugens (Stål)) form a model system for dissection of the mechanism of interaction between insect pest and crop. In this review, we focus on the genomics of BPH-rice interaction. On the side of rice, a number of BPH-resistance genes have been identified genetically. Thirteen of these genes have been cloned which shed a light on the molecular basis of the interaction. On the aspect of BPH, a lot of salivary proteins have been identified using transcriptome and proteome techniques. The genetic loci of virulence were mapped in BPH genome based on the linkage map. The understanding of interaction between BPH and rice will provide novel insights into efficient control of this pest.

Gene expression changes in the tyrosine metabolic pathway regulate caste-specific cuticular pigmentation of termites.

In social insects, all castes have characteristic phenotypes suitable for their own tasks and to engage in social behavior. The acquisition of caste-specific phenotypes was a key event in the course of social insect evolution. However, understanding of the genetic basis and the developmental mechanisms that produce these phenotypes is still very limited. In particular, termites normally possess more than two castes with specific phenotypes (i.e. workers, soldiers, and reproductives), but proximate developmental mechanisms are far from being fully understood. In this study, we focused on the pigmentation of the cuticle as a model trait for caste-specific phenotypes, during the molts of each caste; workers, soldiers, presoldiers (intermediate stage of soldiers), and alates (primary reproductives) in Zootermopsis nevadensis. Expression patterns of cuticular tanning genes (members of the tyrosine metabolic pathway) were different among each molt, and high expression levels of several "key genes" were observed during each caste differentiation. For the differentiation of castes with well-tanned cuticles (i.e. soldiers and alates), all focal genes except DDC in the former were highly expressed. On the other hand, high expression levels of yellow and aaNAT were observed during worker and presoldier molts, respectively, but most other genes in the pathway were expressed at low levels. RNA interference (RNAi) of these key genes affected caste-specific cuticular pigmentation, leading to soldiers with yellowish-white heads and pigmented mandibular tips, presoldiers with partly pigmented head cuticles, and alates with the yellow head capsules. These results suggest that the pigmentation of caste-specific cuticles is achieved by the regulation of gene expression in the tyrosine metabolic pathway.

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants.

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study.

BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis.

Description of a new argasid tick (Acari: Ixodida) from bat caves in Brazilian Amazon.

Nothoaspis amazoniensis n. sp. (Acari: Ixodida: Argasidae) is described from adult and immature ticks (nymph II, nymph I, larva) collected from bat caves in the Brazilian Amazon. Also, 16S rDNA sequences are provided. The diagnostic characters for adults are the presence of false shield or nothoaspis, an anteriorly projecting hood covering the capitulum, a medial extension of palpal article I (flaps), genital plate extending from coxa I to IV, absence of 2 setae on the internal margin of the flaps, a minute hypostome without denticles, presence of a central pore in the base of hypostome, and a reticulate surface pattern on the posterior half of the nothoaspis in males. The nymph II stage is characterized by a hood that is small in relation to the capitulum, short coxal setae, palpal flaps lacking setae on the internal margin, long hypostome, pointed with dentition 4/4 apically, and the anterior half of the body is covered by a cell-like configuration. Nymph I stage is characterized by a hood, small in relation to the capitulum, dorsum of the body covered by a cell-like configuration, venter integument covered by a cell-like configuration, and hypostome dentition 4/4 with apices that are "V"-shaped. Diagnostic characters of the larvae are the number and size of dorsal setae, and the shape of scutum and hypostome. The new species appears to have a life cycle with a larva that feeds on bats, a non-feeding nymphal stage (nymph I), a feeding nymphal stage (nymph II), and adults that probably represent non-feeding stages.