Gross Morphology of Diseased Tissues in Nezara viridula (Hemiptera: Pentatomidae) and Molecular Characterization of an Associated Microsporidian.

Nezara viridula (L.) (Hemiptera: Pentatomidae), commonly known in the U.S. as the southern green stink bug (SGSB), is a cosmopolitan, highly polyphagous feeder that causes severe damage to a wide range of agronomically important crops such as fruit, vegetable, grain, tobacco, and cotton, throughout much of the United States, and is a global pest of considerable ecological, agricultural, and economical interest. During dissection of female Nz. viridula, conspicuous black and brown spots or lesions were observed on various internal organs. To determine the cause of these spots or lesions, tissues of fat body, spermatheca, ovaries, and ovulated eggs were collected from healthy and infected individuals. The gross morphology of the spots was characterized, and the microorganisms associated with the infection were identified by amplicon sequencing of the V4 region of the small subunit rRNA gene. The presence of a microsporidian pathogen Nosema maddoxi, Becnel, Solter, Hajek, Huang, Sanscrainte, & Estep (Microsporidia: Nosematidae) which has been observed on other species of stink bug, was evidenced for the first time. The characterization of the gross morphology of this associated microsporidian may enable more rapid determination of microsporidia infection in stink bug colonies and field populations.

Trypanosomatids Associated in the Alimentary Canal of Bagrada hilaris (Hemiptera: Pentatomidae).

Bagrada hilaris (Burmeister) is an invasive pest of economically important crops in the United States. During physiological investigations of B. hilaris, a flagellated protozoan was discovered in the alimentary canal of many specimens. This manuscript characterizes the morphology and molecular identification of the trypanosomatid, which appears similar to trypanosomatids identified in other stink bug species. It has been identified as a species in the Blastocrithidia genus based on morphological characteristics and molecular analyses.

Allantoin Crystal Formation in Bagrada hilaris (Burmeister) (Heteroptera: Pentatomidae) Females.

Bagrada hilaris is a polyphagous herbivore reported as an invasive pest in the United States. During the course of dissecting Burmeister hilaris unique crystals were observed in both the midgut and oviducts. Crystals were identified using X-ray diffraction techniques. Both acicular (i.e., needle-like, slender, and/or tapered) and cubic (i.e., cube shaped) crystals were observed in six of 75 individuals examined (8.0%). The crystals were mainly observed in females (6.7%), followed by males (1.3%) with no crystals observed in the minimal number of nymphs examined (0%). Crystals of both types were detected in the midgut and lateral oviducts of the females and midgut in males. The acicular crystals often appeared as distinct bundles when present in the midgut and oviducts. Crystals varied in size with the acicular crystals ranging from 0.12 mm to 0.5 mm in length although the cubic crystals ranged in length from 0.25 mm to over 1.0 mm with widths of ∼0.25 mm. The cubic crystals were identified as allantoin although the acicular crystals were most likely dl-allantoin in combination with halite. While allantoin in a soluble form is often found in insect tissues and excreta; being present as a crystal, especially in such a large form, is curious and raises some interesting questions. More research is warranted to further understand mechanisms associated with such crystal formation in B. hilaris and can lead to a better understanding of the excretory process in this species and the role allantoin plays in the elimination of excess nitrogen.

Co-Occurrence of Wing Deformity and Impaired Mobility of Alates with Deformed Wing Virus in Solenopsis invicta Buren (Hymenoptera: Formicidae).

Deformed wing virus (DWV), a major honey bee pathogen, is a generalist insect virus detected in diverse insect phyla, including numerous ant genera. Its clinical symptoms have only been reported in honey bees, bumble bees, and wasps. DWV is a quasispecies virus with three main variants, which, in association with the ectoparasitic mite, Varroa destructor, causes wing deformity, shortened abdomens, neurological impairments, and colony mortality in honey bees. The red imported fire ant, Solenopsis invicta Buren, is one of the most-invasive and detrimental pests in the world. In this study, we report the co-occurrence of DWV-like symptoms in S. invicta and DWV for the first time and provide molecular evidence of viral replication in S. invicta. Some alates in 17 of 23 (74%) lab colonies and 9 of 14 (64%) field colonies displayed deformed wings (DWs), ranging from a single crumpled wing tip to twisted, shriveled wings. Numerous symptomatic alates also exhibited altered locomotion ranging from an altered gait to the inability to walk. Deformed wings may prevent S. invicta alates from reproducing since mating only occurs during a nuptial flight. The results from conventional RT-PCR and Sanger sequencing confirmed the presence of DWV-A, and viral replication of DWV was confirmed using a modified strand-specific RT-PCR. Our results suggest that S. invicta can potentially be an alternative and reservoir host for DWV. However, further research is needed to determine whether DWV is the infectious agent that causes the DW syndrome in S. invicta.

Complete chromosome-level genome assembly data from the tawny crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae).

The tawny crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae) has a native range that extends from northern Argentina to southern Brazil. In the U.S.A. this species has often been misidentified as Nylanderia (Paratrechina) pubens or N. cf. pubens and has likely been present in Florida and Texas for several decades [1]. In the early 2000's explosive population growth in Texas and neighboring states drew renewed taxonomic focus. Genetic analyses [2,3] aided in identifying the pest species as N. fulva. This species poses an invasive threat to native flora and fauna and human structures. In its invasive range it has been reported to displace another invasive species, the red imported fire ant. The specimens used for genome sequencing were obtained from the coastal region of Mississippi. DNA was extracted from pupae. The genome data set was deposited to the National Center for Biotechnology Information as submission ID: SUB10775679, Project ID: PRJNA796544, Accession IDs: SAMN24895442 and JAKFQQ000000000. The organism taxid is 613905, locus tag prefixes are L1K79. The assembly, USDA_Nfulva_1.0, was generated in collaboration with Dovetail Genomics (now Cantata Bio) to yield a chromosome-level assembly of 375 Mb with a 15.67 Mb N50 and 78X coverage and revealing 16 putative chromosomes. This high-quality chromosome-level genome assembly was released prior to publication as a public service to the research community.

Expression and RNA interference of salivary polygalacturonase genes in the tarnished plant bug, Lygus lineolaris.

Three genes encoding polygalacturonase (PG) have been identified in Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). Earlier studies showed that the three PG gene transcripts are exclusively expressed in the feeding stages of L. lineolaris. In this report, it is shown that all three transcripts are specifically expressed in salivary glands indicating that PGs are salivary enzymes. Transcriptional profiles of the three PGs were evaluated with respect to diet, comparing live cotton plant material to artificial diet. PG2 transcript levels were consistently lower in cotton-fed insects than those reared on artificial diet. RNA interference was used to knock down expression of PG1 mRNA in adult salivary glands providing the first demonstration of the use of this method in the non-model insect, L. lineolaris.

Near-Complete Genome Sequences of New Strains of Nylanderia Fulva Virus 1 from Solenopsis invicta.

Nylanderia fulva virus 1 (NfV-1) is a single-stranded positive-sense RNA virus that infects the tawny crazy ant. Three near-complete genomes of NfV-1SI (92% to 94% nucleotide identity to reference strain NfV-1) found infecting the red imported fire ant were determined. The genomes have 10,904 to 10,918 nucleotides and include most of the coding region for the polyprotein.

Distribution patterns of imported fire ants (Hymenoptera: Formicidae) on a sheep and goat farm in Oklahoma.

Imported fire ant colonies were quantified in 1,000-m(2) circular subplots spaced approximately 125 m apart on a sheep and goat farm in Oklahoma. Social form (percent polygyny), mound density, cumulative above-ground mound volume, and average mound volume were subjected to multiple regression analyses to examine trends related to landscape metrics and habitat characteristics. Monogyne populations were spatially autocorrelated, and polygyne mounds tended to be smaller and more numerous. A model incorporating the effects of percent polygyny, canopy cover, and 1-d cumulative incident solar radiation explained 34% of the variation in mound density. Percent polygyny was not significantly related to cumulative mound volume, which provides a better estimate of overall ant biomass. A model incorporating the effects of 1-d cumulative incident solar radiation on the summer solstice, elevation, canopy cover, distance from cisterns, distance from water, and distance from trees explained 42% of the variation in cumulative mound volume. Monogyne mounds in areas that were flat and close to water in low-lying areas were largest. Results indicate that remotely sensed data in combination with publicly available U.S. Geological Survey data may be useful in predicting areas of high and low fire ant abundance at a field scale.

Transcriptome Sequencing of Immature Ants Reveals the Complete Genome Sequence of a New Isolate of Solenopsis invicta Virus 2 from the Mississippi Delta.

Solenopsis invicta virus 2 (SINV-2) is an RNA virus that infects red imported fire ants. I report the genome sequence of SINV-2MSD, an isolate infecting ants collected from Mississippi. The obtained genome is 11,303 nucleotides, including six open reading frames encoding four structural proteins, a helicase, and an RNA-dependent RNA polymerase.

Molecular cloning and expression of three polygalacturonase cDNAs from the tarnished plant bug, Lygus lineolaris.

Three unique cDNAs encoding putative polygalacturonase enzymes were isolated from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The three nucleotide sequences were dissimilar to one another, but the deduced amino acid sequences were similar to each other and to other polygalacturonases from insects, fungi, plants, and bacteria. Four conserved segments characteristic of polygalacturonases were present, but with some notable semiconservative substitutions. Two of four expected disulfide bridge-forming cysteine pairs were present. All three inferred protein translations included predicted signal sequences of 17 to 20 amino acids. Amplification of genomic DNA identified an intron in one of the genes, Llpg1, in the 5' untranslated region. Semiquantitative RT-PCR revealed expression in all stages of the insect except the eggs. Expression in adults, male and female, was highly variable, indicating a family of highly inducible and diverse enzymes adapted to the generalist polyphagous nature of this important pest.

Differential Gene Expression in Red Imported Fire Ant (Solenopsis invicta) (Hymenoptera: Formicidae) Larval and Pupal Stages.

Solenopsis invicta Buren is an invasive ant species that has been introduced to multiple continents. One such area, the southern United States, has a history of multiple control projects using chemical pesticides over varying ranges, often resulting in non-target effects across trophic levels. With the advent of next generation sequencing and RNAi technology, novel investigations and new control methods are possible. A robust genome-guided transcriptome assembly was used to investigate gene expression differences between S. invicta larvae and pupae. These life stages differ in many physiological processes; of special importance is the vital role of S. invicta larvae as the colonies' "communal gut". Differentially expressed transcripts were identified related to many important physiological processes, including digestion, development, cell regulation and hormone signaling. This dataset provides essential developmental knowledge that reveals the dramatic changes in gene expression associated with social insect life stage roles, and can be leveraged using RNAi to develop effective control methods.

Comparison of RNAi Sequences in Insect-Resistant Plants to Expressed Sequences of a Beneficial Lady Beetle: A Closer Look at Off-Target Considerations.

Sequences obtained from transcriptomes of the lady beetle Coleomegilla maculata were compared to those designed for incorporation into crops. Searches of the transcriptomes identified sequences as the most likely to be closely similar to the sequences described in RNAi plant incorporated products. Some proposed prime RNAi pest management targets were also used to identify predicted orthologs from C. maculata. The lady beetle sequences were aligned with sequences from corn rootworms and Colorado potato beetles and, as appropriate in the case of targets, regions of similarity were compared with the genetic model organism for beetles, Tribolium castaneum. Some high levels of nucleotide identity were identified, particularly with an actin-derived sequence from Colorado potato beetle. This actin-derived sequence shared identical sequences with the lady beetle and a parasitic wasp.

Quality of transgenic laboratory strains of Cochliomyia hominivorax (Diptera: Calliphoridae).

Genetically modified, mass reared insects present novel possibilities for the future of insect control. One concern about manipulation of insects is a possible loss of strain quality due to the introduction of a foreign gene of any sort into the insect genome. Eight transgenic strains of screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), were compared with the wild-type parental laboratory strain in laboratory culture. Measurements of average fertility, fecundity, larval productivity, and longevity were analyzed. Two transgenic strains had significantly lower larval productivity than controls, one of which was explained by a homozygous lethal insertion of the transgene. Another strain produced significantly fewer eggs than controls. Overall strain characteristics, including measurements from egg, larva, pupa, and adult stages, were compared. Transgenic colonies did not consistently show significantly lower individual or aggregate strain quality characteristics than the control parental colony; hence, the presence of the transgene used to produce the strains tested did not incur a discrete cost to the colonies of laboratory-reared C. hominivorax.

Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae).

A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A GFP marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus. Transformation was confirmed by Mendelian genetics. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the flight muscles.

Postlarval fitness of transgenic strains of Cochliomyia hominivorax (Diptera: Calliphoridae).

Eight transgenic strains of Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) were compared with the wild-type parental laboratory strain (P95) in colony. Measurements of average weight of pupae, percentage of adults emerging from pupae, ratio of males to total emerged adults, and mating competitiveness were analyzed. The parental strain colony was subcultured and exposed to handling procedures equivalent to transgenic strains for valid comparison of overall colony fitness. None of the transgenic colonies exhibited significantly lower fitness characteristics than the control parental colony. One transgenic colony had a higher ratio of adults emerging from pupae, and five colonies had higher average pupal weight; because fitness cost would only be indicated by lower values, the statistical variations were not significant. Males of one transgenic strain were shown to mate with equal frequency compared with males of the parental strain. Hence, the presence of the transgene used to produce the strains tested did not incur a fitness cost to the colonies of laboratory-reared C. hominivorax.

Saliva of Lygus lineolaris digests double stranded ribonucleic acids.

The prospects for development of highly specific pesticides based on double stranded ribonucleic acid have been a recent focus of scientific research. Creative applications have been proposed and demonstrated. However, not all insects are sensitive to double stranded RNA (dsRNA) gene knockdown effects; applications in the order Lepidoptera, for example, have met with varied success. Gene knockdown has been demonstrated in several species in the order Hemiptera. In our laboratory, knockdown experiments relied on microinjection of dsRNA into the hemocoel of the tarnished plant bug, Lygus lineolaris. Subsequent experiments delivering dsRNA to insects by feeding were repeatedly unsuccessful in demonstrating knockdown, and a hypothesis was formulated that the dsRNA was digested and degraded by the insect prior to contact with the insect cells. Exposure of dsRNA to insect saliva, insect salivary glands, and insect hemolymph was compared with commercial RNAase III. The saliva of L. lineolaris was found to rapidly digest double stranded RNA. RNAase inhibitor did not affect the activity but heat treatment slowed enzymatic activity.

Mutations in AKAP5 disrupt dendritic signaling complexes and lead to electrophysiological and behavioral phenotypes in mice.

AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIalpha or RIIbeta as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and beta-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.

Critical role of cAMP-dependent protein kinase anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons.

The cAMP-dependent protein kinase (PKA) regulates a wide array of cellular functions. In brain and heart PKA increases the activity of the L-type Ca2+ channel Cav1.2 in response to beta-adrenergic stimulation. Cav1.2 forms a complex with the beta2-adrenergic receptor, the trimeric GS protein, adenylyl cyclase, and PKA wherein highly localized signaling occurs [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Burette, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. PKA primarily phosphorylates Cav1.2 on serine 1928 of the central, pore-forming alpha11.2 subunit. Here we demonstrate that the A-kinase anchor protein 150 (AKAP150) is critical for PKA-mediated regulation of Cav1.2 in the brain. AKAP150 and MAP2B specifically co-immunoprecipitate with Cav1.2 from rat brain. Recombinant AKAP75, the bovine homologue to rat AKAP150, binds directly to three different sites of alpha11.2. MAP2B from rat brain also interacts with these same sites in pull-down assays. Gene disruption of AKAP150 in mice dramatically reduces co-immunoprecipitation of PKA with Cav1.2 and prevents phosphorylation of serine 1928 upon beta-adrenergic stimulation in vivo. These results demonstrate the physiological relevance of PKA anchoring by AKAPs in general and AKAP150 specifically in the regulation of Cav1.2 in vivo.