Stability of nuclear and mitochondrial reference genes in selected tissues of the Ambrosia beetle Xylosandrus germanus

SIMPLE SUMMARY: The ambrosia beetle Xylosandrus germanus (Blandford) is a destructive wood-boring insect of horticultural tree crops. A fungal mutualist is cultivated within host trees that provides the sole source of nutrition for the larvae and adults of this beetle. Female X. germanus adults use a pouch-like structure (i.e., mycangium) to maintain and transport spores of their fungal mutualist. To facilitate future studies examining gene expression of X. germanus’ mycangium, the identification of stable genes unaffected by experimental treatments is needed to provide a standard reference during gene expression studies. Selected tissue types were dissected from laboratory-reared and field-collected specimens of the ambrosia beetle X. germanus to evaluate the stability of five reference genes, namely, 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). The reference genes CO1 and AK were identified as primary and secondary reference genes. By contrast, EF1α was considered unsuitable for use as a reference gene during gene expression studies with X. germanus. These results will aid in normalizing the expression of target genes during studies with X. germanus. ABSTRACT: The fungus-farming ambrosia beetle Xylosandrus germanus (Blandford) uses a pouch-like structure (i.e., mycangium) to transport spores of its nutritional fungal mutualist. Our current study sought to identify reference genes necessary for future transcriptome analyses aimed at characterizing gene expression within the mycangium. Complementary DNA was synthesized using selected tissue types from laboratory-reared and field-collected X. germanus consisting of the whole body, head + thorax, deflated or inflated mycangium + scutellum, inflated mycangium, and thorax + abdomen. Quantitative reverse-transcription PCR reactions were performed using primers for 28S ribosomal RNA (28S rRNA), arginine kinase (AK), carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase (CAD), mitochondrial cytochrome oxidase 1 (CO1), and elongation factor-1α (EF1α). Reference gene stability was analyzed using GeNorm, NormFinder, BestKeeper, ΔCt, and a comprehensive final ranking by RefFinder. The gene CO1 was identified as the primary reference gene since it was generally ranked in first or second position among the tissue types containing the mycangium. Reference gene AK was identified as a secondary reference gene. In contrast, EF1α was generally ranked in the last or penultimate place. Identification of two stable reference genes will aid in normalizing the expression of target genes for subsequent gene expression studies of X. germanus’ mycangium

Ethanol Injection of Ornamental Trees Facilitates Testing Insecticide Efficacy Against Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae)

Exotic ambrosia beetles are damaging pests in ornamental tree nurseries in North America. The species Xylosandrus crassiusculus (Motshulsky) and Xylosandrus germanus (Blandford) are especially problematic. Management of these pests relies on preventive treatments of insecticides. However, field tests of recommended materials on nursery trees have been limited because of unreliable attacks by ambrosia beetles on experimental trees. Ethanol-injection of trees was used to induce colonization by ambrosia beetles to evaluate insecticides and botanical formulations for preventing attacks by ambrosia beetles. Experiments were conducted in Ohio, Tennessee, and Virginia. Experimental trees injected with ethanol had more attacks by ambrosia beetles than un-injected control trees in all but one experiment. Xylosandrus crassiusculus and X. germanus colonized trees injected with ethanol. In most experiments, attack rates declined 8 d after ethanol-injection. Ethanol-injection induced sufficient pressure from ambrosia beetles to evaluate the efficacy of insecticides for preventing attacks. Trunk sprays of permethrin suppressed cumulative total attacks by ambrosia beetles in most tests. Trunk sprays of the botanical formulations Armorex and Veggie Pharm suppressed cumulative total attacks in Ohio. Armorex, Armorex + Permethrin, and Veggie Pharm + Permethrin suppressed attacks in Tennessee. The bifenthrin product Onyx suppressed establishment of X. germanus in one Ohio experiment, and cumulative total ambrosia beetle attacks in Virginia. Substrate drenches and trunk sprays of neonicotinoids, or trunk sprays of anthranilic diamides or tolfenpyrad were not effective. Ethanol-injection is effective for inducing attacks and ensuring pressure by ambrosia beetles for testing insecticide efficacy on ornamental trees

Preharvest Quarantine Treatments of Chlorantraniliprole, Clothianidin, and Imidacloprid-Based Insecticides for Control of Japanese Beetle (Coleoptera: Scarabaeidae) and Other Scarab Larvae in the Root Zone of Field-Grown Nursery Trees

Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), is an important quarantine pest of nurseries. Nursery plant movement from P. japonica-infested regions is regulated by the U.S. Domestic Japanese Beetle Harmonization Plan (DJHP), which classifies states by risk categories. Treatments for category 2 states include preharvest soil surface treatment of nursery plants grown in field soil using Discus SC, Marathon (1G or 60 WP), or Flagship (0.22G or 25 WG). In this study, Discus, Marathon 60 WP, or Flagship 0.22G DJHP standards were compared with labeled rates of non–DJHP-approved insecticides, including neonicotinoids clothianidin (Arena 50WDG), generic imidacloprid (Quali-Pro Imidacloprid 2 F T&O Insecticide, Mallet 2 F T&O Insecticide, and Lada 2 F Insecticide), and imidacloprid + bifenthrin (Allectus SC), as well as the anthranilic diamide, chlorantraniliprole (Acelepryn Insecticide). Arena provided 100% P. japonica control in May, June, and July over four test years, but had one larva recovered during August in two of those 4 yr. Acelepryn did not provide DJHP-acceptable P. japonica control. During July, Allectus provided 100% P. japonica control in three of four test years, but had four larvae in one test year. Other treatments tested only during July, which provided 100% P. japonica control, included Discus (five tests); Marathon, Quali-Pro, and Mallet (two tests); and Lada and Flagship (one test). Generic imidacloprid 2 F formulations were equivalent in P. japonica control to DJHP-approved insecticides. Insecticides generally performed poorly on other scarabs or curculionid larvae. The study supports Arena, Allectus, and generic imidacloprid 2 F products as suitable candidates for the DJHP

The competent sentinel node: an association with an axillary presentation and an occult or a small primary invasive breast carcinoma

The concept of the sentinel node describes a primary or sentinel lymph node (SLN), which exists and through which tumour cells from a primary tumour in a particular location must first travel to spread to a particular regional lymph node group. In this series we present three patients presenting with a pathological axillary node associated with either an occult or very small primary breast cancer. In each case the primary tumour was found to have metastasised to the palpable node, however despite the significant enlargement of this node, no other axillary nodes were found to be affected on axillary node clearance. This has led us to postulate that the SLN in some cases contains unique characteristics that enable it to prevent further spread of the tumour up the lymphatic chain. Hence the term the competent sentinel node

Trap Tree and Interception Trap Techniques for Management of Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae) in Nursery Production Get access Arrow

The majority of wood-boring ambrosia beetles are strongly attracted to ethanol, a behavior which could be exploited for management within ornamental nurseries. A series of experiments was conducted to determine if ethanol-based interception techniques could reduce ambrosia beetle pest pressure. In two experiments, trap trees injected with a high dose of ethanol were positioned either adjacent or 10–15 m from trees injected with a low dose of ethanol (simulating a mildly stressed tree) to determine if the high-dose trap trees could draw beetle attacks away from immediately adjacent stressed nursery trees. The high-ethanol-dose trees sustained considerably higher attacks than the low-dose trees; however, distance between the low- and high-dose trees did not significantly alter attack rates on the low-dose trees. In a third experiment, 60-m length trap lines with varying densities of ethanol-baited traps were deployed along a forest edge to determine if immigrating beetles could be intercepted before reaching sentinel traps or artificially stressed sentinel trees located 10 m further in-field. Intercept trap densities of 2 or 4 traps per trap line were associated with fewer attacks on sentinel trees compared to no traps, but 7 or 13 traps had no impact. None of the tested intercept trap densities resulted in significantly fewer beetles reaching the sentinel traps. The evaluated ethanol-based interception techniques showed limited promise for reducing ambrosia beetle pressure on nursery trees. An interception effect might be enhanced by applying a repellent compound to nursery trees in a push–pull strategy

Proof of principle for a high sensitivity search for the electric dipole moment of the electron using the metastable a(1)[^3\Sigma^+] state of PbO

The metastable a(1)[^3\Sigma^+] state of PbO has been suggested as a suitable system in which to search for the electric dipole moment (EDM) of the electron. We report here the development of experimental techniques allowing high-sensitivity measurements of Zeeman and Stark effects in this system, similar to those required for an EDM search. We observe Zeeman quantum beats in fluorescence from a vapor cell, with shot-noise limited extraction of the quantum beat frequencies, high counting rates, and long coherence times. We argue that improvement in sensitvity to the electron EDM by at least two orders of magnitude appears possible using these techniques.Comment: 5 pages, 3 figure