Low-temperature exposure has immediate and lasting effects on the stress tolerance, chemotaxis and proteome of entomopathogenic nematodes

Temperature is one of the most important factors affecting soil organisms, including the infective stages of parasites and entomopathogenic nematodes, which are important biological control agents. We investigated the response of 2 species of entomopathogenic nematodes to different storage regimes: cold (9°C), culture temperature (20°C) and temperature swapped from 9 to 20°C. For Steinernema carpocapsae, cold storage had profound effects on chemotaxis, stress tolerance and protein expression that were retained in temperature-swapped individuals. These effects included reversal of chemotactic response for 3 (prenol, methyl salicylate and hexanol) of the 4 chemicals tested, and enhanced tolerance to freezing (−10°C) and desiccation (75% RH). Label-free quantitative proteomics showed that cold storage induced widespread changes in S. carpocapsae, including an increase in heat-shock proteins and late embryogenesis abundant proteins. For Heterorhabditis megidis, cold storage had a less dramatic effect on chemotaxis (as previously shown for proteomic expression) and changes were not maintained on return to 20°C. Thus, cold temperature exposure has significant effects on entomopathogenic nematodes, but the nature of the change depends on the species. Steinernema carpocapsae, in particular, displays significant plasticity, and its behaviour and stress tolerance may be manipulated by brief exposure to low temperatures, with implications for its use as a biological control agent

Interobserver Agreement in the Clinical Assessment of Children With Blunt Abdominal Trauma

Objectives The objective was to determine the interobserver agreement of historical and physical examination findings assessed during the emergency department (ED) evaluation of children with blunt abdominal trauma. Methods This was a planned substudy of a multicenter, prospective cohort study of children younger than 18 years of age evaluated for blunt abdominal trauma. Patients were excluded if injury occurred more than 24 hours prior to evaluation or if computed tomography (CT) imaging was obtained at another hospital prior to transfer to a study site. Two clinicians independently recorded their clinical assessments of a convenience sample of patients onto data collection forms within 60 minutes of each other and prior to CT imaging (if obtained) or knowledge of laboratory results. The authors categorized variables as either subjective symptoms (i.e., patient history) or objective findings (i.e., physical examination). For each variable recorded by the two observers, the agreement beyond that expected by chance was estimated, using the kappa (κ) statistic for categorical variables and weighted κ for ordinal variables. Variables with 95% lower confidence limits (LCLs) κ ≥ 0.4 (moderate agreement or better) were considered to have acceptable agreement. Results A total of 632 pairs of physician observations were obtained on 23 candidate variables. Acceptable agreement was achieved in 16 (70%) of the 23 variables tested. For six subjective symptoms, κ ranged from 0.48 (complaint of shortness of breath) to 0.90 (mechanism of injury), and only the complaint of shortness of breath had a 95% LCL κ < 0.4. For the 17 objective findings, κ ranged from –0.01 (pelvis instability) to 0.82 (seat belt sign present). The 95% LCL for κ was <0.4 for flank tenderness, abnormal chest auscultation, suspicion of alcohol or drug intoxication, pelvis instability, absence of bowel sounds, and peritoneal irritation. Conclusions Observers can achieve at least acceptable agreement on the majority of historical and physical examination variables in children with blunt abdominal trauma evaluated in the ED. Those variables are candidates for consideration for development of a clinical prediction rule for intra‐abdominal injury in children with blunt trauma. Resumen Concordancia Interobservador en la Valoración Clínica de los Niños con Traumatismo Abdominal Cerrado Objetivos Determinar la concordancia interobservador de los hallazgos de la historia clínica y la exploración física obtenidos durante la valoración de los niños con traumatismo abdominal de alta energía en el servicio de urgencias (SU). Metodología Se diseñó un subestudio de un estudio de cohorte prospectivo y multicéntrico de niños de 18 años o menos evaluados por traumatismo abdominal cerrado. Se excluyeron los pacientes si el traumatismo había ocurrido más de 24 horas antes de la primera valoración, o si las imágenes de la tomografía computarizada (TC) se obtuvieron en otro hospital previamente a trasladarse al lugar del estudio. Dos clínicos recogieron de forma independiente su valoración clínica en un formulario de datos, de una muestra de conveniencia de pacientes, en los primeros 60 minutos, y previamente a las imágenes de la TC (si ésta se realizó) o al conocimiento de los resultados del laboratorio. Se clasificaron las variables como síntomas subjetivos (ej.: historia del paciente) o hallazgos objetivos (ej.: exploración física). Para cada variable recogida por los dos observadores, se estimó la concordancia más allá de la esperada por el azar usando el índice kappa (κ) para las variables categóricas y índice κ ponderado para las variables ordinales. Se consideró que existía una concordancia aceptable para las variables con una κ ≥ 0,4 (concordancia moderada o buena) en el límite inferior del intervalo de confianza del 95% (IC 95%). Resultados Se obtuvieron 632 pares de observaciones clínicas en 23 variables candidatas. Se alcanzó la concordancia aceptable en 16 (70%) de ellas. Para los seis síntomas subjetivos, el rango de κ fue de 0,48 (queja de dificultad respiratoria) a 0,90 (mecanismo de la lesión), y sólo la queja de dificultad respiratoria tuvo una κ < 0,4 en el límite inferior del IC 95%. Para los 17 hallazgos objetivos, el rango de κ fue desde ‐0,01 (inestabilidad pelvis) a 0,82 (presencia del signo del cinturón de seguridad). El dolor en el flanco, la auscultación torácica alterada, la sospecha de intoxicación por alcohol o tóxicos, la inestabilidad de pelvis, la ausencia de ruidos intestinales y la irritación peritoneal tuvieron una κ < 0,4 en el límite inferior del IC 95%. Conclusiones Los observadores pueden alcanzar al menos una concordancia aceptable en la mayoría de las variables de la historia clínica y la exploración física en los niños con traumatismo abdominal cerrado evaluado en el SU. Estas variables son candidatas para considerarse en el desarrollo de una regla de predicción clínica para la lesión intrabdominal en los niños con traumatismo de cerrado.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98339/1/acem12132.pd

The Molecular Signatures of Adaptive Plasticity in Parasitic Nematodes

Entomopathogenic nematodes (EPN; Steinernema spp. and Heterorhabditis spp.) are parasites which kill and reproduce within insects. The infective juvenile (IJ) is a developmentally arrested, nonfeeding, stress tolerant stage which forages in the soil for new insects to infect. IJs carry symbiotic bacteria which aid killing the insect and converts the host’s tissues to a nutritive liquid which the EPN can consume. EPN are promising biocontrol agents for the control of insect pests and are also model organisms for the study of parasitism. The objective of this project was to characterise the plasticity of IJ behaviour, and to investigate some of the associated molecular processes. There are two broad components: firstly, the identification of molecules (ascarosides) externally secreted by IJs and their effects on dispersal, and secondly changes in behaviour and stress tolerance of IJs over time during storage at different temperatures, and associated changes in the proteome. When the host cadaver becomes overcrowded, there is a build-up of signals which encourage newly formed IJs to disperse, thus reducing competition between the many IJs arising from the same cadaver. Among these chemicals are members of a class of pheromones, ascarosides, which have profound effects on IJ behaviour and development. Whether or not these IJs continue producing ascarosides into the surrounding medium, the composition of the mixture, the abundance of these pheromones and whether they affect the behaviour of conspecifics and heterospecifics was investigated. The IJs of four EPN species, Steinernema carpocapsae, Steinernema longicaudum, Steinernema feltiae, and Heterorhabditis megidis, were collected and stored in water at 5000 IJs/ml. The worm conditioned water was analysed with LC-MS/MS after storage in 20°C for specific timepoints. Various ascarosides were detected, generally increasing in abundance the longer the IJs were stored in the water, indicating that these IJs secrete ascarosides consistently after emergence from the host. The composition of ascarosides detected was species specific however the most abundant ascaroside detected in all species was Ascr#9, similar to many other insect-associated species. The worm-conditioned water from both conspecifics and heterospecifics was shown to induce dispersal behaviour in each of the species in agar plate assays. The species-specificity and complexity of ascaroside secretion, despite the similar effects on dispersal, implies that it has other ecological or biological functions. The second part of the thesis investigates the effects of time and temperature on stress tolerance, behaviour (chemotaxis) and the proteome of S. carpocapsae and H. megidis IJs. Previously it has been shown that conditioning IJs in low temperatures (<10°C) enhances not only their freezing tolerance, but their tolerance to other abiotic stressors. This cross tolerance is presumed to be due to the accumulation of non-specific protectants within the IJs. The IJs of H. megidis and S. carpocapsae were stored in 20°C and 9°C for up to 12 weeks, and at specific timepoints, assays were performed, and their proteins were extracted and analysed via LC-MS/MS. More proteins were detected within S. carpocapsae IJs (2422) than in H. megidis IJs (1582). The S. carpocapsae proteome was more strongly affected by low temperature storage (9°C), whereas the H. megidis proteome changed over time in a similar manner, less influenced by the ambient temperature. Compared to freshly emerged IJs, the highest abundance proteins detected within S. carpocapsae IJs at 20°C were proteins related to the cytoskeleton, cell signalling, and infection proteins such as proteases, protease inhibitors and a chitinase. The proteins that were detected at the highest abundance after conditioning at 9°C were late embryogenesis abundant proteins, heat shock proteins, and proteins related to stress tolerance. The proteins which were decreased in abundance to the greatest extent after conditioning at 20°C and 9°C were those related to the cytoskeleton and stress related proteins. The highest abundance proteins detected in H. megidis after conditioning in both 20°C and in 9°C were those related to the cytoskeleton, cell signalling, and carbon metabolism. The proteins decreased to the greatest extent after conditioning the IJs in 20°C and 9°C were those related to metabolism, heat shock proteins and ribosomal proteins. Storage of IJs at low temperatures prolongs their survival. S. carpocapsae IJs exhibited increased molecular chaperones over time, and to a greater extent in 9°C. The H. megidis IJs exhibited decreased abundance of proteins associated with translation over time. These two responses may represent a species-specific response to proteostatic collapse as the IJs age. Proteostatic collapse is one of the consequences of aging in cells and likely a contributing factor to nematode longevity, as misfolded and damaged proteins and toxic aggregations of proteins begin to accumulate within the cells. Similarly, as nematodes age, reactive oxygen species (ROS) accumulate. Both species demonstrated an increase in abundance of proteins which enhance ROS tolerance, and to a greater extent in 9°C. Storage of EPN IJs in low temperatures also affects various behaviours such as dispersal, infectivity, and response to host volatiles. If these behaviours are adversely affected, then cold storage of nematodes will be a trade-off between longevity of the nematodes in storage and the efficacy of these parasites when applied to the field as biocontrol agents. After conditioning at 20°C, IJs of both species were attracted to hexanol, methyl salicylate and acetone, and were repelled by prenol (an odour associated with infected hosts). The chemotaxis responses of H. megidis to each odorant was enhanced after storage in 9°C, becoming more strongly attracted or repelled to each odour than after storage at 20°C. S. carpocapsae IJs tended to act in the opposite manner after exposure to low temperatures, becoming repelled by attractants and strongly attracted to the repellent, prenol. Storage of H. megidis IJs at temperatures below the culture temperature of 20°C resulted in IJs with a gradual change in chemotaxis from 9°C to 12°C to 15°C, whereas S. carpocapsae showed a binary response, as all temperatures below culture temperature tested resulted in IJs with similar responses. To investigate whether IJs retain these altered chemotaxis responses following a return to culture temperature, and how long a period of cold it takes to induce them, IJs from both species were stored at 9°C for brief periods, 3 hours, 1 day and 1 week, and were transferred to 20°C for the duration of the experiment (3 weeks). At the 3-week timepoint, all of the conditioned IJs were exposed to a strong attractant or a strong repellent, and their responses were compared to that of IJs which were stored at 9°C or 20°C for the full 3 weeks. H. megidis did not retain cold-altered chemotaxis responses after transfer to 20°C, while S. carpocapsae IJs stored at 9°C and transferred to 20°C after just 3 hours retained their altered chemotaxis responses for 3 weeks. Storage at 9°C for a week enhanced the freezing (-10°C for 6 hours) and desiccation (75% relative humidity) tolerance of S. carpocapsae IJs, and this enhanced stress tolerance was retained by those IJs which were transferred to 20°C when tested after 2 weeks at the temperature. I next explored whether the cold-induced changes in the S. carpocapsae proteome were also maintained following return to 20°C, as was found for behaviour and stress tolerance. IJs which were stored at 9°C for the full 3 weeks, and those stored for just for 1 week and then transferred to 20°C for 2 weeks had similar proteomes and were both different to that of IJs stored at 20°C for the full 3 weeks. The first two groups displayed hundredfold increases in the abundance of many molecular chaperones when compared to IJs that were stored at 20°C for the full duration. Conversely, the highest abundance protein in the IJs stored at 20°C for 3 weeks relative to the IJs stored at 9°C was a chitinase, implicated in many roles in nematodes including fungal defence and infection. The plasticity of EPN IJs enables them to adapt to diverse and changing environments. Proteomic profiling is a useful guide for further elucidating the molecular mechanisms behind these phenotypes, which will facilitate their use as biocontrol agents, and as models for parasitism

The Molecular Signatures of Adaptive Plasticity in Parasitic Nematodes

Entomopathogenic nematodes (EPN; Steinernema spp. and Heterorhabditis spp.) are parasites which kill and reproduce within insects. The infective juvenile (IJ) is a developmentally arrested, nonfeeding, stress tolerant stage which forages in the soil for new insects to infect. IJs carry symbiotic bacteria which aid killing the insect and converts the host’s tissues to a nutritive liquid which the EPN can consume. EPN are promising biocontrol agents for the control of insect pests and are also model organisms for the study of parasitism. The objective of this project was to characterise the plasticity of IJ behaviour, and to investigate some of the associated molecular processes. There are two broad components: firstly, the identification of molecules (ascarosides) externally secreted by IJs and their effects on dispersal, and secondly changes in behaviour and stress tolerance of IJs over time during storage at different temperatures, and associated changes in the proteome. When the host cadaver becomes overcrowded, there is a build-up of signals which encourage newly formed IJs to disperse, thus reducing competition between the many IJs arising from the same cadaver. Among these chemicals are members of a class of pheromones, ascarosides, which have profound effects on IJ behaviour and development. Whether or not these IJs continue producing ascarosides into the surrounding medium, the composition of the mixture, the abundance of these pheromones and whether they affect the behaviour of conspecifics and heterospecifics was investigated. The IJs of four EPN species, Steinernema carpocapsae, Steinernema longicaudum, Steinernema feltiae, and Heterorhabditis megidis, were collected and stored in water at 5000 IJs/ml. The worm conditioned water was analysed with LC-MS/MS after storage in 20°C for specific timepoints. Various ascarosides were detected, generally increasing in abundance the longer the IJs were stored in the water, indicating that these IJs secrete ascarosides consistently after emergence from the host. The composition of ascarosides detected was species specific however the most abundant ascaroside detected in all species was Ascr#9, similar to many other insect-associated species. The worm-conditioned water from both conspecifics and heterospecifics was shown to induce dispersal behaviour in each of the species in agar plate assays. The species-specificity and complexity of ascaroside secretion, despite the similar effects on dispersal, implies that it has other ecological or biological functions. The second part of the thesis investigates the effects of time and temperature on stress tolerance, behaviour (chemotaxis) and the proteome of S. carpocapsae and H. megidis IJs. Previously it has been shown that conditioning IJs in low temperatures (<10°C) enhances not only their freezing tolerance, but their tolerance to other abiotic stressors. This cross tolerance is presumed to be due to the accumulation of non-specific protectants within the IJs. The IJs of H. megidis and S. carpocapsae were stored in 20°C and 9°C for up to 12 weeks, and at specific timepoints, assays were performed, and their proteins were extracted and analysed via LC-MS/MS. More proteins were detected within S. carpocapsae IJs (2422) than in H. megidis IJs (1582). The S. carpocapsae proteome was more strongly affected by low temperature storage (9°C), whereas the H. megidis proteome changed over time in a similar manner, less influenced by the ambient temperature. Compared to freshly emerged IJs, the highest abundance proteins detected within S. carpocapsae IJs at 20°C were proteins related to the cytoskeleton, cell signalling, and infection proteins such as proteases, protease inhibitors and a chitinase. The proteins that were detected at the highest abundance after conditioning at 9°C were late embryogenesis abundant proteins, heat shock proteins, and proteins related to stress tolerance. The proteins which were decreased in abundance to the greatest extent after conditioning at 20°C and 9°C were those related to the cytoskeleton and stress related proteins. The highest abundance proteins detected in H. megidis after conditioning in both 20°C and in 9°C were those related to the cytoskeleton, cell signalling, and carbon metabolism. The proteins decreased to the greatest extent after conditioning the IJs in 20°C and 9°C were those related to metabolism, heat shock proteins and ribosomal proteins. Storage of IJs at low temperatures prolongs their survival. S. carpocapsae IJs exhibited increased molecular chaperones over time, and to a greater extent in 9°C. The H. megidis IJs exhibited decreased abundance of proteins associated with translation over time. These two responses may represent a species-specific response to proteostatic collapse as the IJs age. Proteostatic collapse is one of the consequences of aging in cells and likely a contributing factor to nematode longevity, as misfolded and damaged proteins and toxic aggregations of proteins begin to accumulate within the cells. Similarly, as nematodes age, reactive oxygen species (ROS) accumulate. Both species demonstrated an increase in abundance of proteins which enhance ROS tolerance, and to a greater extent in 9°C. Storage of EPN IJs in low temperatures also affects various behaviours such as dispersal, infectivity, and response to host volatiles. If these behaviours are adversely affected, then cold storage of nematodes will be a trade-off between longevity of the nematodes in storage and the efficacy of these parasites when applied to the field as biocontrol agents. After conditioning at 20°C, IJs of both species were attracted to hexanol, methyl salicylate and acetone, and were repelled by prenol (an odour associated with infected hosts). The chemotaxis responses of H. megidis to each odorant was enhanced after storage in 9°C, becoming more strongly attracted or repelled to each odour than after storage at 20°C. S. carpocapsae IJs tended to act in the opposite manner after exposure to low temperatures, becoming repelled by attractants and strongly attracted to the repellent, prenol. Storage of H. megidis IJs at temperatures below the culture temperature of 20°C resulted in IJs with a gradual change in chemotaxis from 9°C to 12°C to 15°C, whereas S. carpocapsae showed a binary response, as all temperatures below culture temperature tested resulted in IJs with similar responses. To investigate whether IJs retain these altered chemotaxis responses following a return to culture temperature, and how long a period of cold it takes to induce them, IJs from both species were stored at 9°C for brief periods, 3 hours, 1 day and 1 week, and were transferred to 20°C for the duration of the experiment (3 weeks). At the 3-week timepoint, all of the conditioned IJs were exposed to a strong attractant or a strong repellent, and their responses were compared to that of IJs which were stored at 9°C or 20°C for the full 3 weeks. H. megidis did not retain cold-altered chemotaxis responses after transfer to 20°C, while S. carpocapsae IJs stored at 9°C and transferred to 20°C after just 3 hours retained their altered chemotaxis responses for 3 weeks. Storage at 9°C for a week enhanced the freezing (-10°C for 6 hours) and desiccation (75% relative humidity) tolerance of S. carpocapsae IJs, and this enhanced stress tolerance was retained by those IJs which were transferred to 20°C when tested after 2 weeks at the temperature. I next explored whether the cold-induced changes in the S. carpocapsae proteome were also maintained following return to 20°C, as was found for behaviour and stress tolerance. IJs which were stored at 9°C for the full 3 weeks, and those stored for just for 1 week and then transferred to 20°C for 2 weeks had similar proteomes and were both different to that of IJs stored at 20°C for the full 3 weeks. The first two groups displayed hundredfold increases in the abundance of many molecular chaperones when compared to IJs that were stored at 20°C for the full duration. Conversely, the highest abundance protein in the IJs stored at 20°C for 3 weeks relative to the IJs stored at 9°C was a chitinase, implicated in many roles in nematodes including fungal defence and infection. The plasticity of EPN IJs enables them to adapt to diverse and changing environments. Proteomic profiling is a useful guide for further elucidating the molecular mechanisms behind these phenotypes, which will facilitate their use as biocontrol agents, and as models for parasitism

The Molecular Signatures of Adaptive Plasticity in Parasitic Nematodes

Entomopathogenic nematodes (EPN; Steinernema spp. and Heterorhabditis spp.) are parasites which kill and reproduce within insects. The infective juvenile (IJ) is a developmentally arrested, nonfeeding, stress tolerant stage which forages in the soil for new insects to infect. IJs carry symbiotic bacteria which aid killing the insect and converts the host’s tissues to a nutritive liquid which the EPN can consume. EPN are promising biocontrol agents for the control of insect pests and are also model organisms for the study of parasitism. The objective of this project was to characterise the plasticity of IJ behaviour, and to investigate some of the associated molecular processes. There are two broad components: firstly, the identification of molecules (ascarosides) externally secreted by IJs and their effects on dispersal, and secondly changes in behaviour and stress tolerance of IJs over time during storage at different temperatures, and associated changes in the proteome. When the host cadaver becomes overcrowded, there is a build-up of signals which encourage newly formed IJs to disperse, thus reducing competition between the many IJs arising from the same cadaver. Among these chemicals are members of a class of pheromones, ascarosides, which have profound effects on IJ behaviour and development. Whether or not these IJs continue producing ascarosides into the surrounding medium, the composition of the mixture, the abundance of these pheromones and whether they affect the behaviour of conspecifics and heterospecifics was investigated. The IJs of four EPN species, Steinernema carpocapsae, Steinernema longicaudum, Steinernema feltiae, and Heterorhabditis megidis, were collected and stored in water at 5000 IJs/ml. The worm conditioned water was analysed with LC-MS/MS after storage in 20°C for specific timepoints. Various ascarosides were detected, generally increasing in abundance the longer the IJs were stored in the water, indicating that these IJs secrete ascarosides consistently after emergence from the host. The composition of ascarosides detected was species specific however the most abundant ascaroside detected in all species was Ascr#9, similar to many other insect-associated species. The worm-conditioned water from both conspecifics and heterospecifics was shown to induce dispersal behaviour in each of the species in agar plate assays. The species-specificity and complexity of ascaroside secretion, despite the similar effects on dispersal, implies that it has other ecological or biological functions. The second part of the thesis investigates the effects of time and temperature on stress tolerance, behaviour (chemotaxis) and the proteome of S. carpocapsae and H. megidis IJs. Previously it has been shown that conditioning IJs in low temperatures (<10°C) enhances not only their freezing tolerance, but their tolerance to other abiotic stressors. This cross tolerance is presumed to be due to the accumulation of non-specific protectants within the IJs. The IJs of H. megidis and S. carpocapsae were stored in 20°C and 9°C for up to 12 weeks, and at specific timepoints, assays were performed, and their proteins were extracted and analysed via LC-MS/MS. More proteins were detected within S. carpocapsae IJs (2422) than in H. megidis IJs (1582). The S. carpocapsae proteome was more strongly affected by low temperature storage (9°C), whereas the H. megidis proteome changed over time in a similar manner, less influenced by the ambient temperature. Compared to freshly emerged IJs, the highest abundance proteins detected within S. carpocapsae IJs at 20°C were proteins related to the cytoskeleton, cell signalling, and infection proteins such as proteases, protease inhibitors and a chitinase. The proteins that were detected at the highest abundance after conditioning at 9°C were late embryogenesis abundant proteins, heat shock proteins, and proteins related to stress tolerance. The proteins which were decreased in abundance to the greatest extent after conditioning at 20°C and 9°C were those related to the cytoskeleton and stress related proteins. The highest abundance proteins detected in H. megidis after conditioning in both 20°C and in 9°C were those related to the cytoskeleton, cell signalling, and carbon metabolism. The proteins decreased to the greatest extent after conditioning the IJs in 20°C and 9°C were those related to metabolism, heat shock proteins and ribosomal proteins. Storage of IJs at low temperatures prolongs their survival. S. carpocapsae IJs exhibited increased molecular chaperones over time, and to a greater extent in 9°C. The H. megidis IJs exhibited decreased abundance of proteins associated with translation over time. These two responses may represent a species-specific response to proteostatic collapse as the IJs age. Proteostatic collapse is one of the consequences of aging in cells and likely a contributing factor to nematode longevity, as misfolded and damaged proteins and toxic aggregations of proteins begin to accumulate within the cells. Similarly, as nematodes age, reactive oxygen species (ROS) accumulate. Both species demonstrated an increase in abundance of proteins which enhance ROS tolerance, and to a greater extent in 9°C. Storage of EPN IJs in low temperatures also affects various behaviours such as dispersal, infectivity, and response to host volatiles. If these behaviours are adversely affected, then cold storage of nematodes will be a trade-off between longevity of the nematodes in storage and the efficacy of these parasites when applied to the field as biocontrol agents. After conditioning at 20°C, IJs of both species were attracted to hexanol, methyl salicylate and acetone, and were repelled by prenol (an odour associated with infected hosts). The chemotaxis responses of H. megidis to each odorant was enhanced after storage in 9°C, becoming more strongly attracted or repelled to each odour than after storage at 20°C. S. carpocapsae IJs tended to act in the opposite manner after exposure to low temperatures, becoming repelled by attractants and strongly attracted to the repellent, prenol. Storage of H. megidis IJs at temperatures below the culture temperature of 20°C resulted in IJs with a gradual change in chemotaxis from 9°C to 12°C to 15°C, whereas S. carpocapsae showed a binary response, as all temperatures below culture temperature tested resulted in IJs with similar responses. To investigate whether IJs retain these altered chemotaxis responses following a return to culture temperature, and how long a period of cold it takes to induce them, IJs from both species were stored at 9°C for brief periods, 3 hours, 1 day and 1 week, and were transferred to 20°C for the duration of the experiment (3 weeks). At the 3-week timepoint, all of the conditioned IJs were exposed to a strong attractant or a strong repellent, and their responses were compared to that of IJs which were stored at 9°C or 20°C for the full 3 weeks. H. megidis did not retain cold-altered chemotaxis responses after transfer to 20°C, while S. carpocapsae IJs stored at 9°C and transferred to 20°C after just 3 hours retained their altered chemotaxis responses for 3 weeks. Storage at 9°C for a week enhanced the freezing (-10°C for 6 hours) and desiccation (75% relative humidity) tolerance of S. carpocapsae IJs, and this enhanced stress tolerance was retained by those IJs which were transferred to 20°C when tested after 2 weeks at the temperature. I next explored whether the cold-induced changes in the S. carpocapsae proteome were also maintained following return to 20°C, as was found for behaviour and stress tolerance. IJs which were stored at 9°C for the full 3 weeks, and those stored for just for 1 week and then transferred to 20°C for 2 weeks had similar proteomes and were both different to that of IJs stored at 20°C for the full 3 weeks. The first two groups displayed hundredfold increases in the abundance of many molecular chaperones when compared to IJs that were stored at 20°C for the full duration. Conversely, the highest abundance protein in the IJs stored at 20°C for 3 weeks relative to the IJs stored at 9°C was a chitinase, implicated in many roles in nematodes including fungal defence and infection. The plasticity of EPN IJs enables them to adapt to diverse and changing environments. Proteomic profiling is a useful guide for further elucidating the molecular mechanisms behind these phenotypes, which will facilitate their use as biocontrol agents, and as models for parasitism

Infective juveniles of entomopathogenic nematodes (Steinernema and Heterorhabditis) secrete ascarosides and respond to interspecific dispersal signals.

Ascarosides are a modular series of signalling molecules that are widely conserved in nematodes where they function as pheromones with both behavioural and developmental effects. Here we show that the developmentally arrested infective juvenile (IJ) stage of entomopathogenic nematodes (EPN) secrete ascarosides into the surrounding medium. The exometabolome of Steinernema carpocapsae and Heterorhabditis megidis was examined at 0, 1, 7 and 21 days of storage. The concentration of several ascarosides (ascr#11, ascr#9, ascr#12, ascr#1 and ascr#14 for both species, plus ascr#10 for H. megidis) showed a progressive increase over this period, while the concentration of longer chain ascarosides increased up to day 7, with an apparent decline thereafter. Ascr #9 was the main ascaroside produced by both species. Similar ascarosides were found over a 7-day period for Steinernema longicaudum and S. feltiae. Ascaroside blends have previously been shown to promote nematode dispersal. S. carpocapsae and H. megidis IJs were stored for up to 12 weeks and assayed at intervals. IJs where exometabolome was allowed to accumulate showed higher dispersal rates than those where water was changed frequently, indicating that IJ exometabolome maintained high dispersal. Infectivity was not affected. IJ exometabolome accumulated over 7 days promoted dispersal of freshly harvested IJs, both of their own and other EPN species. Similarly, extracts of nematode-infected cadavers promoted dispersal of con- and heterospecific IJs. Thus, IJs are encouraged to disperse from a source cadaver or from other crowded conditions by public information cues, a finding that may have application in enhancing biocontrol. However, the complexity of the ascaroside blend produced by IJs suggests that it may have ecological functions other than dispersal

The effect of temperature conditioning (9°C and 20°C) on the proteome of entomopathogenic nematode infective juveniles

Entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis are parasites which kill and reproduce within insects. While both have life cycles centred around their developmentally arrested, nonfeeding and stress tolerant infective juvenile (IJ) stage, they are relatively distantly related. These IJs are promising biocontrol agents, and their shelf life and stress tolerance may be enhanced by storage at low temperatures. The purpose of this study was to investigate how the proteome of the IJs of two distantly related EPN species is affected by storage at 9°C (for up to 9 weeks) and 20°C (for up to 6 weeks), using label-free quantitative proteomics. Overall, more proteins were detected in S. carpocapsae (2422) than in H. megidis (1582). The S. carpocapsae proteome was strongly affected by temperature, while the H. megidis proteome was affected by both time and temperature. The proteins which increased in abundance to the greatest extent in S. carpocapsae IJs after conditioning at 9°C were chaperone proteins, and proteins related to stress. The proteins which increased in abundance the most after storage at 20°C were proteins related to the cytoskeleton, cell signalling, proteases and their inhibitors, which may have roles in infection. The proteins which decreased in abundance to the greatest extent in S. carpocapsae after both 9°C and 20°C storage were those associated with metabolism, stress and the cytoskeleton. After storage at both temperatures, the proteins increased to the greatest extent in H. megidis IJs were those associated with the cytoskeleton, cell signalling and carbon metabolism, and the proteins decreased in abundance to the greatest extent were heat shock and ribosomal proteins, and those associated with metabolism. As the longest-lived stage of the EPN life cycle, IJs may be affected by proteostatic stress, caused by the accumulation of misfolded proteins and toxic aggregates. The substantial increase of chaperone proteins in S. carpocapsae, and to a greater extent at 9°C, and the general decrease in ribosomal and chaperone proteins in H. megidis may represent species-specific proteostasis mechanisms. Similarly, organisms accumulate reactive oxygen species (ROS) over time and both species exhibited a gradual increase in proteins which enhance ROS tolerance, such as catalase. The species-specific responses of the proteome in response to storage temperature, and over time, may reflect the phylogenetic distance and/or different ecological strategies

The effect of temperature conditioning (9°C and 20°C) on the proteome of entomopathogenic nematode infective juveniles

Entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis are parasites which kill and reproduce within insects. While both have life cycles centred around their developmentally arrested, nonfeeding and stress tolerant infective juvenile (IJ) stage, they are relatively distantly related. These IJs are promising biocontrol agents, and their shelf life and stress tolerance may be enhanced by storage at low temperatures. The purpose of this study was to investigate how the proteome of the IJs of two distantly related EPN species is affected by storage at 9°C (for up to 9 weeks) and 20°C (for up to 6 weeks), using label-free quantitative proteomics. Overall, more proteins were detected in S. carpocapsae (2422) than in H. megidis (1582). The S. carpocapsae proteome was strongly affected by temperature, while the H. megidis proteome was affected by both time and temperature. The proteins which increased in abundance to the greatest extent in S. carpocapsae IJs after conditioning at 9°C were chaperone proteins, and proteins related to stress. The proteins which increased in abundance the most after storage at 20°C were proteins related to the cytoskeleton, cell signalling, proteases and their inhibitors, which may have roles in infection. The proteins which decreased in abundance to the greatest extent in S. carpocapsae after both 9°C and 20°C storage were those associated with metabolism, stress and the cytoskeleton. After storage at both temperatures, the proteins increased to the greatest extent in H. megidis IJs were those associated with the cytoskeleton, cell signalling and carbon metabolism, and the proteins decreased in abundance to the greatest extent were heat shock and ribosomal proteins, and those associated with metabolism. As the longest-lived stage of the EPN life cycle, IJs may be affected by proteostatic stress, caused by the accumulation of misfolded proteins and toxic aggregates. The substantial increase of chaperone proteins in S. carpocapsae, and to a greater extent at 9°C, and the general decrease in ribosomal and chaperone proteins in H. megidis may represent species-specific proteostasis mechanisms. Similarly, organisms accumulate reactive oxygen species (ROS) over time and both species exhibited a gradual increase in proteins which enhance ROS tolerance, such as catalase. The species-specific responses of the proteome in response to storage temperature, and over time, may reflect the phylogenetic distance and/or different ecological strategies

Association Between the Seat Belt Sign and Intra‐abdominal Injuries in Children With Blunt Torso Trauma in Motor Vehicle Collisions

Objectives The objective was to determine the association between the abdominal seat belt sign and intra‐abdominal injuries ( IAI s) in children presenting to emergency departments with blunt torso trauma after motor vehicle collisions ( MVC s). Methods This was a planned subgroup analysis of prospective data from a multicenter cohort study of children with blunt torso trauma after MVC s. Patient history and physical examination findings were documented before abdominal computed tomography ( CT ) or laparotomy. Seat belt sign was defined as a continuous area of erythema, ecchymosis, or abrasion across the abdomen secondary to a seat belt restraint. The relative risk ( RR ) of IAI with 95% confidence intervals ( CI s) was calculated for children with seat belt signs compared to those without. The risk of IAI in those patients with seat belt sign who were without abdominal pain or tenderness, and with Glasgow Coma Scale ( GCS ) scores of 14 or 15, was also calculated. Results A total of 3,740 children with seat belt sign documentation after blunt torso trauma in MVC s were enrolled; 585 (16%) had seat belt signs. Among the 1,864 children undergoing definitive abdominal testing ( CT , laparotomy/laparoscopy, or autopsy), IAI s were more common in patients with seat belt signs than those without (19% vs. 12%; RR  = 1.6, 95% CI  = 1.3 to 2.1). This difference was primarily due to a greater risk of gastrointestinal injuries (hollow viscous or associated mesentery) in those with seat belt signs (11% vs. 1%; RR  = 9.4, 95% CI  = 5.4 to 16.4). IAI was diagnosed in 11 of 194 patients (5.7%; 95% CI  = 2.9% to 9.9%) with seat belt signs who did not have initial complaints of abdominal pain or tenderness and had GCS scores of 14 or 15. Conclusions Patients with seat belt signs after MVC s are at greater risk of IAI than those without seat belt signs, predominately due to gastrointestinal injuries. Although IAI s are less common in alert patients with seat belt signs who do not have initial complaints of abdominal pain or tenderness, the risk of IAI is sufficient that additional evaluation such as observation, laboratory studies, and potentially abdominal CT scanning is generally necessary. Resumen Objetivos Determinar la asociación entre el signo del cinturón de seguridad ( SCS ) y las lesiones intra‐abdominales ( LIA ) en los niños atendidos en los servicios de urgencias por traumatismo torácico cerrado tras colisiones de vehículo de motor ( CVM ). Metodología Éste fue un análisis de subgrupo planificado de los datos prospectivos de un estudio de cohorte multicéntrico de niños con traumatismo torácico cerrado tras CVM . Se documentó la historia clínica y la exploración física del paciente antes de la tomografía computarizada ( TC ) abdominal o la laparotomía. El SCS se definió como un área continua de eritema, equimosis o abrasión a través del abdomen secundaria a la contención del cinturón de seguridad. Se calculó el riesgo relativo ( RR ) de LIA con los intervalos de confianza ( IC ) al 95% para los niños con SCS en comparación con aquéllos que no lo tenían. También se calculó el riesgo de LIA en aquellos pacientes con SCS que no tuvieron molestia o dolor abdominal con puntuaciones de 14 o 15 de la Escala de Coma de Glasgow ( ECG ). Resultados Se incluyeron 3.740 niños tras un traumatismo torácico cerrado en CVM ; 585 (16%) tuvieron SCS . Entre los 1.864 niños en los que se llevó a cabo un test diagnóstico abdominal definitivo ( TC , laparotomía/ laparoscopia, o autopsia), las LIA fueron más frecuentes en los pacientes con SCS que en aquéllos sin SCS (19% vs. 12%, RR  = 1,6; IC 95% = 1,3 a 2,1). Esta diferencia fue principalmente debida a un mayor riesgo de lesiones gastrointestinales (víscera hueca o asociadas al mesenterio) en aquéllos con SCS (11% vs. 1%, RR  = 9,4; IC 95% = 5,4 a 16,4). La LIA se diagnosticó en 11 de 194 pacientes (5,7%, IC 95% = 2,9% a 9,9%) con SCS que no tuvieron quejas iniciales de molestia o dolor abdominal y tuvieron puntuaciones de 14 o 15 en la ECG . Conclusiones Los pacientes con SCS tras una CVM tienen mayor riesgo de LIA que aquéllos sin SCS , debido fundamentalmente a lesiones gastrointestinales. Aunque las LIA son menos comunes en los pacientes con SCS que están alerta y que no tienen quejas iniciales de molestia o dolor abdominal, el riesgo de LIA es suficiente para que evaluaciones como la observación, las pruebas de laboratorio y potencialmente la TC abdominal sean generalmente necesarios.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109632/1/acem12506.pd

Identification of Microbial Populations Assimilating Nitrogen from RDX in Munitions Contaminated Military Training Range Soils by High Sensitivity Stable Isotope Probing

The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live-fire military training range soils contributes to significant pollution of groundwater. In situ microbial degradation has been proposed as a viable method for onsite containment of RDX. However, there is only a single report of RDX degradation in training range soils and the soil microbial communities involved in RDX degradation were not identified. Here we demonstrate aerobic RDX degradation in soils taken from a target area of an Eglin Air Force Base bombing range, C52N Cat’s Eye, (Eglin, Florida U.S.A.). RDX-degradation activity was spatially heterogeneous (found in less than 30% of initial target area field samples) and dependent upon the addition of exogenous carbon sources to the soils. Therefore, biostimulation (with exogenous carbon sources) and bioaugmentation may be necessary to sustain timely and effective in situ microbial biodegradation of RDX. High sensitivity stable isotope probing analysis of extracted soils incubated with fully labeled ¹⁵N-RDX revealed several organisms with ¹⁵N-labeled DNA during RDX-degradation, including <i>xplA</i>-bearing organisms. <i>Rhodococcus</i> was the most prominent genus in the RDX-degrading soil slurries and was completely labeled with ¹⁵N-nitrogen from the RDX. <i>Rhodococcus</i> and <i>Williamsia</i> species isolated from these soils were capable of using RDX as a sole nitrogen source and possessed the genes <i>xplB</i> and <i>xplA</i> associated with RDX-degradation, indicating these genes may be suitable genetic biomarkers for assessing RDX degradation potential in soils. Other highly labeled species were primarily Proteobacteria, including: <i>Mesorhizobium</i> sp., <i>Variovorax</i> sp., and <i>Rhizobium</i> sp