Environmental DNA assays for Laricobius beetles (Coleoptera: Derodontidae), biocontrol agents of the hemlock woolly adelgid in North America.

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), is an invasive pest causing significant ecological and economic damage to certain hemlock tree (Tsuga (Endlicher) Carrière, Pinales:Pinaceae) species. In response to this invasive threat, biological control strategies have been implemented, introducing natural predators such as Laricobius nigrinus Fender (Coleoptera: Derodontidae) and, more recently, Laricobius osakensis Montgomery and Shiyake (Coleoptera: Derodontidae), as specialist predators against A. tsugae. However, the genetic and morphological similarities between L. osakensis and both L. nigrinus and the native beetle, Laricobius rubidus LeConte (Coleoptera: Derodontidae), pose challenges in their identification. Effective monitoring of released predators is integral to evaluating the success of biological control measures. Environmental DNA (eDNA) holds potential for various detection applications, including species monitoring. In this study, we developed specific primers and probes targeting the mitochondrial cytochrome oxidase 1 gene sequences, achieving high specificity despite their 95% sequence similarity. With an optimal annealing temperature of 60 °C, our tools effectively differentiated L. osakensis from the other 2 beetles and demonstrated eDNA detection sensitivity down to 2 copies/µl. This research underscores the potential of precise molecular tools for advancing biological control and biodiversity assessment against invasive threats like A. tsugae.

Observation of key phenological stages of hemlock woolly adelgid (Hemiptera: Adelgidae): using citizen science as a tool to inform research and management.

Increasing efficiency of data gathering at the landscape scale on the growing number of pests and pathogens threatening forests worldwide has potential to improve management outcomes. Citizen science is expanding, with growing support and utility in environmental and conservation fields. We present a case study showing how citizen science observations can be used to inform research and management of a devastating forest pest. Hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), was introduced to eastern North America, leading to decline and mortality of eastern [Tsuga canadensis (L.) Carrière] (Pinales: Pinaceae) and Carolina hemlock (Tsuga caroliniana Engelmann) trees. Management activities, most notably biological control, rely on observations of A. tsugae phenology to inform the timing of releases and monitoring surveys of their highly synchronized specialist predators. In this article, we outline a citizen science program and report phenological observations on A. tsugae. Additionally, we report data comparing A. tsugae estivation break in Virginia (VA) and New York (NY) State, revealing that estivation break is synchronized between NY and VA. This observation is supported by 6 years of citizen scientist observations, showing similar patterns throughout NY, with egg laying shown to be much more variable than estivation break.

Phenology of Leucotaraxis argenticollis, a specialist predator of the invasive hemlock woolly adelgid, in the eastern United States.

In hemlock stands within eastern US forests, classical biological control has been one of the main strategies used to manage the hemlock woolly adelgid, Adelges tsugae Annand. Specialist predator species may offer a management solution to help regulate A. tsugae populations. In the Pacific Northwest, a suite of specialist predators has been a focus of research and includes 2 species of silver fly, Leucotaraxis argenticollis (Zetterstedt) and Leucotaraxis piniperda (Malloch) (Diptera: Chamaemyiidae). Leucotaraxis spp. phenology has been documented in the Pacific Northwest, but the phenology of either western Leucotaraxis species is unknown in the eastern United States. This study sought to document the phenology of Le. argenticollis in NY in 2021 and in VA in 2021 and 2022. Nylon mesh cages were applied over eastern hemlock branches infested with A. tsugae to contain Le. argenticollis adults. Biweekly and monthly branch samples were taken in 2021 and 2022, documenting all life stages of A. tsugae and of Le. argenticollis that were observed. In 2021 and 2022, Le. argenticollis adults and eggs were present during the oviposition stage of the 2 generations of A. tsugae. In addition, Le. argenticollis larvae were present when A. tsugae ovisacs had eggs and while A. tsugae nymphs of both generations were present. These observations indicate that Le. argenticollis phenology is well synchronized with A. tsugae in the eastern United States.

Estivation and Postestivation Development of Hemlock Woolly Adelgid (Adelges tsugae) (Hemiptera: Adelgidae) at Different Temperatures.

Hemlock woolly adelgid (Adelges tsugae) is the most important pest of hemlocks in the eastern United States, where it completes three generations a year. We investigated the impact of temperature (8, 12, 16, and 20°C) on the estivation and postaestivation stages of the A. tsugae sistens generation. Temperature significantly impacted development and survival of this generation. The highest mortality occurred at the coolest temperature (8°C). Adelges tsugae developed rapidly as the temperature increased and optimum temperatures for development ranged between 17 and 22°C for the different instars. The estimated lower temperature threshold was 0°C for second instar nymphs and 3 -5°C for the other instars and the preoviposition period. Estivating first-instar sistentes resumed development (as evidenced by segments becoming visible) after 40-100 d at the constant temperatures (fastest at 16°C) then required only 105 degree-days (DD) for 50% of the individuals to molt. Subsequent instars developed rapidly (another 470 DD total to reach adult), and oviposition began at ~623 DD from the time the first instars resumed development. This study provides valuable data required to develop an annual phenology model for A. tsugae which will assist in timing monitoring and control treatments.

Hymenopteran Parasitoids of Leucotaraxis argenticollis (Diptera: Chamaemyiidae) and Leucotaraxis piniperda: Implications for Biological Control of Hemlock Woolly Adelgid (Hemiptera: Adelgidae).

The hemlock woolly adelgid (Adelges tsugae (Annand)) is a serious invasive pest of hemlock trees in eastern North America. Multiple biological control agents have been the focus of research aimed at pest management and conserving hemlock communities. Three promising A. tsugae specialist predators are the beetle Laricobius nigrinus (Fender) (Coleoptera: Derodontidae) and flies in the genus Leucotaraxis (Diptera: Chamaemyiidae), Leucotaraxis argenticollis (Zetterstedt), and Leucotaraxis piniperda (Malloch). However, these flies are vulnerable to parasitism by wasps in the genera Pachyneuron (Walker) (Hymenoptera: Pteromalidae) and Melanips (Walker) (Hymenoptera: Figitidae). This study explores parasitoid wasp interactions with these Leucotaraxis species in their native western North American range and potential impacts on the biological control program in the East. Leucotaraxis, La. nigrinus, and parasitoid emergences were observed from adelgid-infested foliage collected from Washington State and British Columbia in 2018, 2019, and 2020. Undescribed species of Pachyneuron and Melanips emerged from puparia as solitary parasitoids. Parasitoid emergence was positively correlated with Leucotaraxis emergence. Percent parasitism increased between February and July, with the months of June and July experiencing higher parasitoid emergence than Leucotaraxis. Differences in emergence patterns suggest that Pachyneuron may be more closely associated with Le. argenticollis as a host, and that Melanips may be associated with Le. piniperda. High parasitism in Leucotaraxis had no effect on La. nigrinus larval abundance, whereas the combined emergence of parasitoids and Leucotaraxis was positively correlated with La. nigrinus. This suggests that there is limited competition among these predators.

The Lari-Leuco Container: A Novel Collection Arena for Separating Insects Ascending or Descending From a Plant Foliage Sample.

Efficient separation of insects from plant material for quantification and collection is an important component of entomological research. This paper reports on a novel, easily replicable container designed to efficiently collect two different biological control agents dispersing from hemlock (Tsuga spp.) foliage infested with the invasive hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). The container utilizes a simplified Berlese-style funnel design to collect Laricobius spp. (Coleoptera: Derodontidae) larvae dropping from the foliage into a removable bottom jar, a central jar to house the foliage sample, and a removable top jar to collect adult silver flies (Leucopis spp., Diptera: Chamaemyiidae) emerging from puparia on the twigs. The efficacy of two designs (with and without a funnel leading to the top collection jar) was evaluated using western hemlock [Tsuga heterophylla (Raf.) Sarg.] foliage naturally colonized with HWA and the two predator genera. All Laricobius larvae were effectively collected in the bottom jar, and the addition of an inverted funnel leading to the top collection jar increased the proportion of Leucopis flies reaching the target jar from 60% to 94%. This 'Lari-Leuco' container is presented as a research and motoring tool to benefit the integrated pest management program for HWA in eastern North America and for potential use in simultaneously separating ascending and descending life stages in other insect-plant or predator-prey systems.

Biological Control of Hemlock Woolly Adelgid: Implications of Adult Emergence Patterns of Two Leucopis spp. (Diptera: Chamaemyiidae) and Laricobius nigrinus (Coleoptera: Derodontidae) Larval Drop.

The hemlock woolly adelgid (Hemiptera: Adelgidae Adelges tsugae Annand) poses a serious threat to hemlocks in eastern North America, and ongoing research is focused on the identification and development of biological controls to protect and manage hemlock resources. Three predators native to the Pacific Northwest of North America that have been the focus of much research are Leucopis argenticollis (Zetterstedt), Leucopis piniperda (Malloch) (Diptera: Chamaemyiidae), and Laricobius nigrinus (Fender) (Coleoptera: Derodontidae). This study addresses the knowledge gap of adult Leucopis spp. emergence patterns, with comparisons to the timing of larval La. nigrinus drop for pupation. Adult Leucopis spp. emergence was observed in the lab from field-collected, adelgid-infested foliage from Washington state in 2019 and 2020. Adult Leucopis spp. were collected daily as they emerged from foliage collections and identified to species using morphological features; a subset was validated using DNA barcoding. Accumulated heating degree days were calculated to compare a standardized emergence timing across collections made at different locations and temperature regimes. The abundance of the two Leucopis spp. and of the combined Leucopis spp. and La. nigrinus varied among sites and years, and no species was consistently more abundant than the other. Evaluations of seasonal emergence trends of the three species determine the predator complex behaves in a temporally stratified and predictable way. Emergence of adult Le. argenticollis was observed first, followed by La. nigrinus larval drop, with Le. piniperda emerging at the end of larval drop, and finally a second emergence of Le. argenticollis.

Sticky traps as an early detection tool for crawlers of Adelges tsugae (Hemiptera: Adelgidae).

We developed an approach using sticky trap arrays as an early detection tool for populations of first-instar nymphs of the hemlock woolly adelgid (Adelges tsugae Annand), a pest of hemlocks (Tsuga spp. [Pinaceae]) in North America. We considered the detection rate of at least one nymph from trapping arrays consisting of one to six sticky panels, where we varied both the surface area of each trap that we assessed and the length of the trapping duration. We also estimated the time needed to set up, service, and assess groups of traps and attempted to relate capture of nymphs on traps to incidence and abundance of A. tsugae in the canopy above the traps. Arrays consisting of two traps provided a detection rate of 75% when 87.5% of the surface area of each trap was assessed, a process that required 38 min per array. The probability of detecting nymphs on traps left in the field for 5-6 d was similar to that for traps left for 12 d. The number of nymphs trapped in an array predicted the probability of finding A. tsugae in the canopy but only when all six traps were fully assessed. To reliably detect incipient A. tsugae infestations, we recommend placing arrays of traps at 1 km intervals along the perimeter of a stand during peak activity of first-instar sistentes nymphs and servicing these arrays every 5-7 d.

End-anchored polymers in good solvents from the single chain limit to high anchoring densities.

An increasing number of applications utilize grafted polymer layers to alter the interfacial properties of solid substrates, motivating refinement in our theoretical understanding of such layers. To assess existing theoretical models of them, we have investigated end-anchored polymer layers over a wide range of grafting densities, σ, ranging from a single chain to high anchoring density limits, chain lengths ranging over two orders of magnitude, for very good and marginally good solvent conditions. We compare Monte Carlo and molecular dynamics simulations, numerical self-consistent field calculations, and experimental measurements of the average layer thickness, h, with renormalization group theory, the Alexander-de Gennes mushroom theory, and the classical brush theory. Our simulations clearly indicate that appreciable inter-chain interactions exist at all simulated areal anchoring densities so that there is no mushroom regime in which the layer thickness is independent of σ. Moreover, we find that there is no high coverage regime in which h follows the predicted scaling, h ∼ Nσ1/3, for classical polymer brushes either. Given that no completely adequate analytic theory seems to exist that spans wide ranges of N and σ, we applied scaling arguments for h as a function of a suitably defined reduced anchoring density, defined in terms of the solution radius of gyration of the polymer chains and N. We find that such a scaling approach enables a smooth, unified description of h in very good solvents over the full range of anchoring density and chain lengths, although this type of data reduction does not apply to marginal solvent quality conditions.

Self-consistent field theory of tethered polymers: one dimensional, three dimensional, strong stretching theories and the effects of excluded-volume-only interactions.

We examine end-tethered polymers in good solvents, using one- and three-dimensional self-consistent field theory, and strong stretching theories. We also discuss different tethering scenarios, namely, mobile tethers, fixed but random ones, and fixed but ordered ones, and the effects and important limitations of including only binary interactions (excluded volume terms). We find that there is a "mushroom" regime in which the layer thickness is independent of the tethering density, σ, for systems with ordered tethers, but we argue that there is no such plateau for mobile or disordered anchors, nor is there one in the 1D theory. In the other limit of brushes, all approaches predict that the layer thickness scales linearly with N. However, the σ(1/3) scaling is a result of keeping only excluded volume interactions: when the full potential is included, the dependence is faster and more complicated than σ(1/3). In fact, there does not appear to be any regime in which the layer thickness scales in the combination Nσ(1/3). We also compare the results for two different solvents with each other, and with earlier Θ solvent results.

Doubly self-consistent field theory of grafted polymers under simple shear in steady state.

We present a generalization of the numerical self-consistent mean-field theory of polymers to the case of grafted polymers under simple shear. The general theoretical framework is presented, and then applied to three different chain models: rods, Gaussian chains, and finitely extensible nonlinear elastic (FENE) chains. The approach is self-consistent at two levels. First, for any flow field, the polymer density profile and effective potential are calculated self-consistently in a manner similar to the usual self-consistent field theory of polymers, except that the calculation is inherently two-dimensional even for a laterally homogeneous system. Second, through the use of a modified Brinkman equation, the flow field and the polymer profile are made self-consistent with respect to each other. For all chain models, we find that reasonable levels of shear cause the chains to tilt, but it has very little effect on the overall thickness of the polymer layer, causing a small decrease for rods, and an increase of no more than a few percent for the Gaussian and FENE chains. Using the FENE model, we also probe the individual bond lengths, bond correlations, and bond angles along the chains, the effects of the shear on them, and the solvent and bonded stress profiles. We find that the approximations needed within the theory for the Brinkman equation affect the bonded stress, but none of the other quantities.

Controlling microtube permeability via grafted polymers and solvent quality.

We examine pressure-driven flow through a microtube with grafted polymers using a "doubly self-consistent field" steady-state theory. Our focus is on the structure of the polymer layer, the tube permeability, and the effects of solvent quality, for different regimes of open and closed tubes. We find that, within experimentally attainable pressure gradients, the flow has very little effect on the grafted layer. However, the polymers, and in particular variations in the solvent quality and cylinder radii, can have large effects on the flow. We find that the permeability can either increase or decrease with either the radius or solvent quality, and we identify the regimes for different behaviors in terms of general parameters that can be used to generalize to other systems. This allows us to identify regimes where the systems are most sensitive to these "tuning" parameters, and we find that they correspond to the boundaries between open and closed tubes identified earlier.

Grafted polymers inside cylindrical tubes: chain stretching vs layer thickness.

We present a study of the detailed structure of grafted polymer chains and the layers they form inside cylindrical tubes, using the finitely extensible nonlinear elastic chain model and numerical self-consistent field theory. For very large tube radius, the chain stretching and layer thicknesses are the same as for polymers grafted to a planar surface. For decreasing radius, our calculations indicate that the layer almost always gets thinner, although there can be situations where it is very slightly thicker. However, we find that this thinning is not necessarily due to changes to the polymers: in fact, the root-mean-squared layer thickness would decrease even if the polymers themselves are completely unchanged. Furthermore, we find that the polymer stretching can increase at the same time that the layer thickness decreases. These apparent paradoxes are resolved by analyzing and distinguishing between the volume fraction profiles and monomer number distributions in these systems, including how they change and why. We also find that, in a given system, parts of each polymer move towards the curved surface and parts away from it, and that these differences are key to understanding the behavior.

Negative regulation of TLR-signaling pathways by activating transcription factor-3.

Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.

Enlarged spleens without enlarged lymph nodes in tlr3-/- pkr-/- mice.

Initial phenotypic studies in a mouse containing mutations in both toll-like receptor 3 (TLR3) and RNA-de-pendent protein kinase R (PKR) revealed comparable spleen and bone marrow cell populations in tlr3(-/)-, pkr(-/-), and tlr3(-/-)pkr(-/-) mice to wild-type controls. Splenomegaly developing between 8 and 10 weeks of age was observed in tlr3(-/-) and tlr3(-/-)pkr(-/-) mice but not in wild-type or pkr(-/-) mice. Palpably enlarged cervical, axillary, and inguinal lymph nodes accompanied by enlarged spleens were observed in 12-18-week-old tlr3(-/-) mice at a higher frequency compared with other genotypes. The enlarged spleens and lymph nodes observed in tlr3(-/-) mice were accompanied by destruction of organ architecture and lymphocyte infiltration. However, the enlargement of these organs was not the result of clonal proliferation of one lymphocyte subset. It is likely this phenotype is a result of TLR3 deficiency in combination with an additional, uncharacterized genetic defect or the presence of an infectious agent. These data also suggest that PKR may have a role in preventing progression from splenomegaly to lymphadenopathy in these mice.

Help! I've been sued: a guide to surviving a physician's nightmare.

Because many physicians will be sued for malpractice during their career, they need to know how to survive a lawsuit. This article is intended to assist doctors in understanding the phases of and players involved in a malpractice lawsuit. It covers discovery, the use of expert testimony, what happens during the trial, and the way malpractice law works in Minnesota. Having a better understanding of what happens during each phase of litigation helps to reduce the anxiety and uncertainty associated with being named in a malpractice suit.

Double-stranded RNA-dependent protein kinase (PKR) is downregulated by phorbol ester.

The double-stranded RNA-dependent protein kinase (PKR) is one of the key mediators of interferon (IFN) action against certain viruses. PKR also plays an important role in signal transduction and immunomodulation. Understanding the regulation of PKR activity is important for the use of PKR as a tool to discover and develop novel therapeutics for viral infections, cancer and immune dysfunction. We found that phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), decreased the level of autophosphorylated PKR in a dose- and time-dependent manner in IFN-treated mouse fibroblast cells. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced the activity of PKR induced by IFN, but did not overcome the PMA-induced reduction of PKR autophosphorylation. Western blot analysis with a monoclonal antibody to mouse PKR revealed that the decrease of PKR autophosphorylation in cells by PMA was a result of PKR protein degradation. Selective PKC inhibitors blocked the degradation of PKR stimulated by PMA, indicating that PKC activity was required for the effect. Furthermore, we also found that proteasome inhibitors prevented PMA-induced down regulation of PKR, indicating that an active proteasome is required. Our results identify a novel mechanism for the post-translational regulation of PKR.

Synergistic activation of innate immunity by double-stranded RNA and CpG DNA promotes enhanced antitumor activity.

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor alpha, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-alpha and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-alpha/beta receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.