A diversity of endosymbionts across Australian aphids and their persistence in aphid cultures.

There is increasing interest in the use of endosymbionts in pest control, which will benefit from the identification of endosymbionts from potential donor species for transfer to pest species. Here, we screened for endosymbionts in 123 Australian aphid samples across 32 species using 16S DNA metabarcoding. We then developed a qPCR method to validate the metabarcoding data set and to monitor endosymbiont persistence in aphid cultures. Pea aphids (Acyrthosiphon pisum) were frequently coinfected with Rickettsiella and Serratia, and glasshouse potato aphids (Aulacorthum solani) were coinfected with Regiella and Spiroplasma; other secondary endosymbionts detected in samples occurred by themselves. Hamiltonella, Rickettsia and Wolbachia were restricted to a single aphid species, whereas Regiella was found in multiple species. Rickettsiella, Hamiltonella and Serratia were stably maintained in laboratory cultures, although others were lost rapidly. The overall incidence of secondary endosymbionts in Australian samples tended to be lower than recorded from aphids overseas. These results indicate that aphid endosymbionts probably exhibit different levels of infectivity and vertical transmission efficiency across hosts, which may contribute to natural infection patterns. The rapid loss of some endosymbionts in cultures raises questions about factors that maintain them under field conditions, while endosymbionts that persisted in laboratory culture provide candidates for interspecific transfers.

A rapidly spreading deleterious aphid endosymbiont that uses horizontal as well as vertical transmission.

Endosymbiotic bacteria that live inside the cells of insects are typically only transmitted maternally and can spread by increasing host fitness and/or modifying reproduction in sexual hosts. Transinfections of Wolbachia endosymbionts are now being used to introduce useful phenotypes into sexual host populations, but there has been limited progress on applications using other endosymbionts and in asexual populations. Here, we develop a unique pathway to application in aphids by transferring the endosymbiont Rickettsiella viridis to the major crop pest Myzus persicae. Rickettsiella infection greatly reduced aphid fecundity, decreased heat tolerance, and modified aphid body color, from light to dark green. Despite inducing host fitness costs, Rickettsiella spread rapidly through caged aphid populations via plant-mediated horizontal transmission. The phenotypic effects of Rickettsiella were sensitive to temperature, with spread only occurring at 19 °C and not 25 °C. Body color modification was also lost at high temperatures despite Rickettsiella maintaining a high density. Rickettsiella shows the potential to spread through natural M. persicae populations by horizontal transmission and subsequent vertical transmission. Establishment of Rickettsiella in natural populations could reduce crop damage by modifying population age structure, reducing population growth and providing context-dependent effects on host fitness. Our results highlight the importance of plant-mediated horizontal transmission and interactions with temperature as drivers of endosymbiont spread in asexual insect populations.

Barley Yellow Dwarf Virus Influences Its Vector's Endosymbionts but Not Its Thermotolerance.

The barley yellow dwarf virus (BYDV) of cereals is thought to substantially increase the high-temperature tolerance of its aphid vector, Rhopalosiphum padi, which may enhance its transmission efficiency. This is based on experiments with North American strains of BYDV and R. padi. Here, we independently test these by measuring the temperature tolerance, via Critical Thermal Maximum (CTmax) and knockdown time, of Australian R. padi infected with a local BYDV isolate. We further consider the interaction between BYDV transmission, the primary endosymbiont of R. padi (Buchnera aphidicola), and a transinfected secondary endosymbiont (Rickettsiella viridis) which reduces the thermotolerance of other aphid species. We failed to find an increase in tolerance to high temperatures in BYDV-infected aphids or an impact of Rickettsiella on thermotolerance. However, BYDV interacted with R. padi endosymbionts in unexpected ways, suppressing the density of Buchnera and Rickettsiella. BYDV density was also fourfold higher in Rickettsiella-infected aphids. Our findings indicate that BYDV does not necessarily increase the temperature tolerance of the aphid transmission vector to increase its transmission potential, at least for the genotype combinations tested here. The interactions between BYDV and Rickettsiella suggest new ways in which aphid endosymbionts may influence how BYDV spreads, which needs further testing in a field context.

Fungicides have transgenerational effects on Rhopalosiphum padi but not their endosymbionts.

BACKGROUND: While several agricultural fungicides are known to directly affect invertebrate pests, including aphids, the mechanisms involved are often unknown. One hypothesis is that fungicides with antibacterial activity suppress bacterial endosymbionts present in aphids which are important for aphid survival. Endosymbiont-related effects are expected to be transgenerational, given that these bacteria are maternally inherited. Here, we test for these associations using three fungicides (chlorothalonil, pyraclostrobin and trifloxystrobin) against the bird cherry-oat aphid, Rhopalosiphum padi, using a microinjected strain that carried both the primary endosymbiont Buchnera and the secondary endosymbiont Rickettsiella. RESULTS: We show that the fungicide chlorothalonil did not cause an immediate effect on aphid survival, whereas both strobilurin fungicides (pyraclostrobin and trifloxystrobin) decreased survival after 48 h exposure. However, chlorothalonil substantially reduced the lifespan and fecundity of the F1 generation. Trifloxystrobin also reduced the lifespan and fecundity of F1 offspring, however, pyraclostrobin did not affect these traits. None of the fungicides consistently altered the density of Buchnera or Rickettsiella in whole aphids. CONCLUSIONS: Our results suggest fungicides have sublethal impacts on R. padi that are not fully realized until the generation after exposure, and these sublethal impacts are not associated with the density of endosymbionts harbored by R. padi. However, we cannot rule out other effects of fungicides on endosymbionts that might influence fitness, like changes in their tissue distribution. We discuss these results within the context of fungicidal effects on aphid suppression across generations and point to potential field applications. © 2022 Society of Chemical Industry.

High Incidence of Related Wolbachia across Unrelated Leaf-Mining Diptera.

The maternally inherited endosymbiont, Wolbachia pipientis, plays an important role in the ecology and evolution of many of its hosts by affecting host reproduction and fitness. Here, we investigated 13 dipteran leaf-mining species to characterize Wolbachia infections and the potential for this endosymbiont in biocontrol. Wolbachia infections were present in 12 species, including 10 species where the Wolbachia infection was at or near fixation. A comparison of Wolbachia relatedness based on the wsp/MLST gene set showed that unrelated leaf-mining species often shared similar Wolbachia, suggesting common horizontal transfer. We established a colony of Liriomyza brassicae and found adult Wolbachia density was stable; although Wolbachia density differed between the sexes, with females having a 20-fold higher density than males. Wolbachia density increased during L. brassicae development, with higher densities in pupae than larvae. We removed Wolbachia using tetracycline and performed reciprocal crosses between Wolbachia-infected and uninfected individuals. Cured females crossed with infected males failed to produce offspring, indicating that Wolbachia induced complete cytoplasmic incompatibility in L. brassicae. The results highlight the potential of Wolbachia to suppress Liriomyza pests based on approaches such as the incompatible insect technique, where infected males are released into populations lacking Wolbachia or with a different incompatible infection.

Thoracic Compression Fracture as a Result of Taser® Discharge.

A conducted electrical device (CED), usually Taser®, is commonly used by law enforcement officers to aid in the incapacitation of subjects. While CEDs are considered "safe" for use on subjects, adverse events may rarely occur. We report a case of a 23-year-old male presenting with severe back pain following deployment of a CED with resulting acute compression fractures of the thoracic sixth, seventh, and eighth vertebral bodies. To the best of our knowledge, this represents the third case of traumatic injury from CED discharge to be reported in the literature since 1995.

Evidence for the benefits of food chain interventions on E. coli O157:H7/NM prevalence in retail ground beef and human disease incidence: A success story.

OBJECTIVES: Human infection with Escherichia coli O157:H7/NM has historically been associated with consumption of undercooked ground beef. The purpose of this paper is to investigate the correlation of the decline in E. coli O157:H7/NM infections in Canada with the introduction of control efforts in ground beef by industry. METHODS: The human incidence of E. coli O157:H7/NM, prevalence in ground beef and interventions from 1996 to 2014 were analyzed. Pathogen prevalence data were obtained from federal government and industry surveillance and inspection/compliance programs. A survey of the largest ground beef producers in Canada was conducted to identify when interventions were implemented. RESULTS: The incidence of E. coli O157:H7/NM infections in Canada declined from ≈4 cases/100 000 to ≈1 case/100000 from 2000 to 2010. Verotoxigenic Escherichia coli (VTEC) prevalence in ground beef sold at retail declined from about 30% around the year 2000 to <2% since 2012. Other measures of the prevalence of E. coli, VTEC, and E. coli O157:H7/NM in beef and ground beef also declined. The number and types of interventions implemented in the major beef processing establishments in Canada increased from 1996 to 2016. CONCLUSION: The observed decline in human illnesses and pathogen levels in relation to retail meats was associated with the introduction of control efforts by industry, federal and provincial/territorial governments, and the general population. Industry-led changes in beef processing along with the introduction of food safety policies, regulations, and public education have led to improved food safety in Canada.

Variation in heat and pressure resistance of verotoxigenic and nontoxigenic Escherichia coli.

This study evaluated the heat and pressure resistance of 112 strains of Escherichia coli, including 102 strains of verotoxigenic E. coli (VTEC) representing 23 serotypes and four phylogenetic groups. In an initial screening, the heat and pressure resistance of 100 strains, including 94 VTEC strains, were tested in phosphate-buffered saline (PBS). Treatment at 60°C for 5 min reduced cell counts by 2.0 to 5.5 log CFU/ml; treatment at 600 MPa for 3 min at 25°C reduced the cell counts by 1.1 to 5.5 log CFU/ml. Heat or pressure resistance did not correlate to the phylogenetic group or the serotype. A smaller group of E. coli strains was evaluated for heat and pressure resistance in Luria-Bertani (LB) broth. Generally, the levels of heat resistance of E. coli strains in LB and PBS were similar; however, the levels of pressure resistance observed for treatments in LB broth or PBS were variable. The cell counts of pressure-resistant strains of VTEC were reduced by less than 1.5 log CFU/ml after treatment at 600 MPa for 3 min. E. coli strains were also treated with 600 MPa for 3 min in ground beef or inoculated into beef patties and grilled to 63 or 71°C. The cell counts of the VTEC E. coli O26:H11 strain 05-6544 were reduced by 2 log CFU/g by pressure treatment in ground beef. The cell counts of the heat-resistant E. coli strain AW1.7 were reduced by 1.4 and 3.4 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. The cell counts of E. coli 05-6544 were reduced by less than 3 and 6 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. To study whether the composition of the beef patties influenced heat resistance, E. coli strains AW1.7, AW1.7 Δ pHR1, MG1655, and LMM1030 were mixed into beef patties containing 15 or 35% fat and 0 or 2% NaCl, and the patties were grilled to an internal temperature of 63°C. The highest heat resistance of E. coli was observed in patties containing 15% fat and 2% NaCl.

Inhibition of polymerase chain reaction for the detection of Escherichia coli O157:H7 and Salmonella enterica on walnut kernels.

The aim of this study was to determine whether Escherichia coli O157:H7 can be reliably detected and isolated from walnut kernels using standard methods of analysis. The limit of detection approached 1 cell per analytical unit (25 g) for E. coli O157:H7 on walnut kernels enriched in modified tryptic soy broth with 20 µg/ml novobiocin and plating onto selective agar media. The presence of PCR inhibitors in walnut kernels was indicated by the failure to detect E. coli O157:H7 from culture positive enrichment broths analysed by PCR, with two separate polymerase and reagent compositions (Dupont BAX E. coli O157:H7 MP system, Promega GoTaq Green for stx) and three methods of template preparation (DuPont BAX, Qiagen DNeasy, Bio-Rad InstaGene). PCR inhibition was overcome by 1:100 dilution in TE buffer of the DNeasy or InstaGene template. PCR inhibition was not relieved by dilution of the BAX template. Similar results were observed for walnut kernels inoculated with Salmonella enterica and analysed for invA, indicating that PCR inhibition is not specific to the organism or primer/template. These results indicate that analysis of walnut kernels for pathogens should be with culture based methods or use protocols for DNA template preparation modified to remove or dilute inhibitors and the need for internal amplification controls in PCR methods.

Comparative examination of Escherichia coli O157:H7 survival on romaine lettuce and in soil at two independent experimental sites.

Little is known about the influence of abiotic factors such as climate and soil chemistry on the survival of Escherichia coli O157:H7 in field lettuce. We applied a nalidixic acid-resistant derivative of strain ATCC 700728 to field-grown romaine lettuce in two regions in Canada characterized by large variances in soil type and climate. Surviving populations in soil and on lettuce leaves were estimated on sorbitol MacConkey agar supplemented with nalidixic acid. Data were fitted with the Weibull decline function to permit comparison of decay rates in the two experimental sites. E. coli O157:H7 populations fell from 105 to <10² CFU/g on leaves, and <10³ CFU/g in soil within 7 days after inoculation. Analysis revealed there was no significant difference between decay rates at the two experimental sites in either environment. The results of this study suggest that the inherent ecological fitness of E. coli O157:H7 ATCC 700728 determines the extent of survival in the production environment.