Mechanisms of resistance to three mite growth inhibitors of Tetranychus urticae in hops.

Mite growth inhibitors (MGIs), such as etoxazole and hexythiazox, are valuable IPM tools for Tetranychus urticae control in hops due to their unique mode of action and selectivity. Hence, it is necessary to standardize bioassay methods to evaluate the efficacy of MGIs, monitor resistance, and identify mechanisms underlying MGI resistance in the field. Here, we developed a three-tiered approach for evaluating ovicidal toxicity of MGIs to T. urticae, which simulated different MGI exposure scenarios in the field. The most effective bioassay method was direct exposure of T. urticae eggs to MGIs. With this method, four field-collected T. urticae populations showed low-to-moderate resistance to MGIs. Cross-resistance among MGIs and from MGIs to bifenazate and bifenthrin was detected. Besides target site insensitivity, enhanced cytochrome P450 and esterase activities also contribute to the MGI resistance in hop yard-collected T. urticae populations. Low-to-moderate MGI resistance in T. urticae populations may be mediated by multiple mechanisms. Positive selection pressure on the I1017F mutation is moderate in field-collected T. urticae populations. Further studies are required to identify metabolic detoxification genes that confer resistance to MGIs for precise resistance monitoring.

Invasion and egress by the obligate intracellular parasite Toxoplasma gondii: potential targets for the development of new antiparasitic drugs.

Intracellular protozoan parasites are a great threat to animal and human health. To successfully disseminate through an organism these parasites must be able to enter and exit host cells efficiently and rapidly. The inhibition of invasion or egress of obligate intracellular parasites is regarded as a goal for drug development since these processes are essential for their survival and likely to require proteins unique to the parasites. Thus, a more comprehensive knowledge of invasion and egress proteins will aid in the development of drugs and vaccines against these intracellular pathogens. In recent years, the study of a particular parasite, Toxoplasma gondii, has yielded valuable information on how invasion and egress are achieved by some protozoan parasites. Besides being a good model system for the study of parasite biology, Toxoplasma is an important human pathogen capable of causing devastating disease in both immunocompromised individuals and developing fetuses. The lack of effective, inexpensive and tolerable drugs against Toxoplasma makes the development of new therapies an imperative. The following review describes how the identification and in depth study, using proteomics, forward genetics and pharmacology of the Toxoplasma proteins involved in entering and exiting human cells provide an important starting point in identifying targets for drug discovery.

Case Study Part 2: Probabilistic modelling of long-term effects of pesticides on individual breeding success in birds and mammals.

Long term exposure of skylarks to a fictitious insecticide and of wood mice to a fictitious fungicide were modelled probabilistically in a Monte Carlo simulation. Within the same simulation the consequences of exposure to pesticides on reproductive success were modelled using the toxicity-exposure-linking rules developed by R.S. Bennet et al. (2005) and the interspecies extrapolation factors suggested by R. Luttik et al. (2005). We built models to reflect a range of scenarios and as a result were able to show how exposure to pesticide might alter the number of individuals engaged in any given phase of the breeding cycle at any given time and predict the numbers of new adults at the season's end.

Case Study Part 1: How to calculate appropriate deterministic long-term toxicity to exposure ratios (TERs) for birds and mammals.

In the European Union, first-tier assessment of the long-term risk to birds and mammals from pesticides is based on calculation of a deterministic long-term toxicity/exposure ratio (TER(lt)). The ratio is developed from generic herbivores and insectivores and applied to all species. This paper describes two case studies that implement proposed improvements to the way long-term risk is assessed. These refined methods require calculation of a TER for each of five identified phases of reproduction (phase-specific TERs) and use of adjusted No Observed Effect Levels (NOELs) to incorporate variation in species sensitivity to pesticides. They also involve progressive refinement of the exposure estimate so that it applies to particular species, rather than generic indicators, and relates spraying date to onset of reproduction. The effect of using these new methods on the assessment of risk is described. Each refinement did not necessarily alter the calculated TER value in a way that was either predictable or consistent across both case studies. However, use of adjusted NOELs always reduced TERs, and relating spraying date to onset of reproduction increased most phase-specific TERs. The case studies suggested that the current first-tier TER(lt )assessment may underestimate risk in some circumstances and that phase-specific assessments can help identify appropriate risk-reduction measures. The way in which deterministic phase-specific assessments can currently be implemented to enhance first-tier assessment is outlined.

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens.

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A (Pi-cecA), lebocin (Pi-leb) and lysozyme (Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Haemocytes from Pseudoplusia includens express multiple alpha and beta integrin subunits.

Cellular immune responses such as encapsulation involve the adhesion of one or more classes of haemocytes. How insect haemocytes recognize encapsulation targets as foreign or the identity of the molecules regulating haemocyte adhesion is unknown. One of the most important classes of adhesion receptors in mammalian immune cells is the integrins, which form functional heterodimers through different combinations of alpha and beta subunits. Prior studies with the moth Pseudoplusia includens indicated that encapsulation depends on two classes of haemocytes called granulocytes and plasmatocytes. Here we report the cloning and identification of three alpha integrin subunits (alphaPi1-3) and one beta subunit (betaPi1) from P. includens. Northern blot analysis indicated that all four subunits are expressed in granulocytes and that three of the four subunits are expressed in plasmatocytes. Quantification of transcription patterns using real-time PCR revealed that expression of alphaPi2 and betaPi1 increased in granulocytes and plasmatocytes when binding to a foreign surface or forming a capsule. alphaPi2 transcription in plasmatocytes was further increased by granulocyte conditioned medium, plasmatocyte spreading peptide, and the integrin recognition peptide RGD. Collectively, these results suggest that one or more integrins play an important role in regulating haemocyte adhesion during encapsulation.

Insect hemocytes and their role in immunity.

The innate immune system of insects is divided into humoral and cellular defense responses. Humoral defenses include antimicrobial peptides, the cascades that regulate coagulation and melanization of hemolymph, and the production of reactive intermediates of oxygen and nitrogen. Cellular defenses refer to hemocyte-mediated responses like phagocytosis and encapsulation. In this review, we discuss the cellular immune responses of insects with emphasis on studies in Lepidoptera and Diptera. Insect hemocytes originate from mesodermally derived stem cells that differentiate into specific lineages identified by morphology, function, and molecular markers. In Lepidoptera, most cellular defense responses involve granular cells and plasmatocytes, whereas in Drosophila they involve primarily plasmatocytes and lamellocytes. Insect hemocytes recognize a variety of foreign targets as well as alterations to self. Both humoral and cell surface receptors are involved in these recognition events. Once a target is recognized as foreign, hemocyte-mediated defense responses are regulated by signaling factors and effector molecules that control cell adhesion and cytotoxicity. Several lines of evidence indicate that humoral and cellular defense responses are well-coordinated with one another. Cross-talk between the immune and nervous system may also play a role in regulating inflammation-like responses in insects during infection.

Surface characteristics of foreign targets that elicit an encapsulation response by the moth Pseudoplusia includens.

Hemocytes from the moth Pseudoplusia includens encapsulate a variety of biotic and abiotic targets. Prior studies indicated that granular cells are usually the first hemocyte type to attach to foreign targets. Thereafter, large numbers of plasmatocytes attach to the target and form a capsule. To identify surface features that induce an encapsulation response, chromatography beads that differed in matrix composition, charge, and functional groups were tested using in vitro and in vivo bioassays. We first conducted in vitro assays using hemocytes with no plasma components present. These experiments indicated that bead types having sulfonic, diethylaminoethyl, and quaternary amine functional groups were encapsulated significantly more often than beads with other functional groups. Charge also significantly affected encapsulation with positively charged beads being encapsulated more often than negatively charged or neutral beads. In vitro assays using purified populations of hemocytes confirmed that these targets were recognized as foreign by granular cells, and that plasmatocytes only formed capsules after granular cells attached to the target. Bead types that were encapsulated under these in vitro conditions were always rapidly encapsulated when injected into P. includens larvae. However, some bead types, like CM-Sephadex, not encapsulated in vitro were encapsulated in vivo if left in the insect hemocoel for a longer period of time (ca. 24 h). Purified plasmatocytes encapsulated these beads in vitro if they were preincubated in plasma. Basic characterization studies suggest these humoral recognition molecules are proteins or small peptides. Comparative studies with other species of noctuid moths also indicated that encapsulation of some bead types differed significantly among species. Collectively, these results reveal that P. includens recognizes some targets as foreign by pattern recognition receptors on granular cells, whereas others are recognized by pattern recognition molecules in plasma. The binding affinities of these recognition molecules also appear to differ among closely related species of Lepidoptera.

Vesicoureteral reflux after ureteroneocystostomy: indications for postoperative voiding cystography.

PURPOSE: The aim of this study was to determine the risk factors for vesicoureteral reflux following ureteral reimplantation to identify a population that can be safely excluded from postoperative voiding cystography. MATERIALS AND METHODS: We retrospectively reviewed the medical records of 273 patients who underwent ureteroneocystostomy for vesicoureteral reflux between 1990 and 1998 and recorded the postoperative renal ultrasonography and voiding cystography results. RESULTS: There were 273 patients (534 ureters) who underwent ureteral reimplantation. We recorded the grade of preoperative hydronephrosis and vesicoureteral reflux and noted several preoperative and intraoperative variables, such as dysfunctional voiding, breakthrough infections, renal scarring, bladder trabeculations, type of reimplant, and postoperative urinary tract infections. With a mean follow-up of 20.6 months, persistent postoperative vesicoureteral reflux was noted in 11 patients (4%). Persistent postoperative reflux was noted in 11 patients (4%) or 12 renal units (2.2%). Reflux resolution rates for 534 renal units and 273 patients after routine follow-up voiding cystourethrogram (VCUG) was 97.8% (renal units) and 96% (patients), respectively. Contralateral vesicoureteral reflux developed in 4 (5.1%) of the 78 patients who underwent unilateral reimplantation. Two patients (0.7%) had postoperative ureteral obstruction. The risk factors for persistent postoperative reflux were identified as preoperative and postoperative hydronephrosis, renal scarring, and tapered reimplantations. The type of reimplant did not correlate with outcome. CONCLUSIONS: Vesicoureteral reflux after ureteral reimplantation is uncommon (4%). Because of the high success rate of ureteral reimplants and the benign course of those patients with persistent low-grade postoperative reflux, it is safe and efficient to eliminate postoperative VCUG in most patients who had a simple ureteral reimplantation for reflux. However, in some higher-risk patients, such as those with preoperative hydronephrosis, renal scarring, and ureteral tapering, postoperative voiding cystography may be indicated to assure resolution of vesicoureteral reflux.

Hematological and pulmonary responses to high altitude in Quechuas: a multivariate approach.

This study investigates the relationships among hematological variables, pulmonary function, and age in a sample of high-altitude natives. The following anthropometric and physiological variables were examined in 77 adult Quechua males from the Peruvian Central Andes (Huancavelica, 3,680 m): height, weight, sitting height, chest diameters, chest and abdominal circumferences, forced vital capacity (FVC), forced expiratory volume at 1 sec (FEV1), peak expiratory flow (PEF), hemoglobin concentration (Hb), red blood cells (RBC), hematocrit (Htc), diastolic and systolic blood pressure, body temperature, pulmonary rate, and pulse rate. The means of these variables for the Huancavelica sample fall within the range of variability previously observed in Andean populations. Principal components analysis and canonical correlation analysis suggest that in this native Andean population: 1) aging decreases lung function but does not affect hematological features, and 2) there is a negative age-independent correlation between lung function (FVC, FEV1, PEF) and hematological traits (Hb, RBC, Htc).

Net primary production of a forest ecosystem with experimental CO2 enrichment

The concentration of atmospheric carbon dioxide was increased by 200 microliters per liter in a forest plantation, where competition between organisms, resource limitations, and environmental stresses may modulate biotic responses. After 2 years the growth rate of the dominant pine trees increased by about 26 percent relative to trees under ambient conditions. Carbon dioxide enrichment also increased litterfall and fine-root increment. These changes increased the total net primary production by 25 percent. Such an increase in forest net primary production globally would fix about 50 percent of the anthropogenic carbon dioxide projected to be released into the atmosphere in the year 2050. The response of this young, rapidly growing forest to carbon dioxide may represent the upper limit for forest carbon sequestration.

Polydnaviruses: potent mediators of host insect immune dysfunction.

Endoparasitic insects are used as biological control agents to kill many species of insect pest. One key to the success of parasitoids that develop in the hemocoel of their host is their ability to knock out the host's immune system, inducing a decline in the responsiveness of a variety of cellular and humoral components so that parasitoid eggs are not encapsulated. In many species parasitized by braconid and ichneumonid wasps, host immunosuppression appears to be mediated by polydnaviruses (PDVs) injected by the female parasitoid into the host hemocoel. The viruses exhibit a complex and intimate genetic relationship with the wasp, since viral sequences are integrated within the wasp's chromosomal DNA. Here Mark Lavine and Nancy Beckage summarize the current evidence for mechanisms of virally induced host immunosuppression in parasitized insects, as well as the roles of other factors including wasp ovarian proteins and venom components, in suppressing hemocyte-mediated and humoral immune responses. Interestingly, in some species, the PDV-induced host immunosuppression appears transitory, with older parasitoid larvae probably exploiting other mechanisms to protect themselves from the host's immune system during the final stages of parasitism. During the final stages of parasitism, the parasitoids likely exploit other mechanisms of immunoevasion via antigen masking, antigen mimicry, or production of active inhibitors of the hemocyte-mediated encapsulation response as well as inhibiting melanization.

Apparent seasonal rhythms in hypothalamic neuropeptides in rats without photoperiod changes.

An apparent seasonal or circannual rhythm in the hypothalamic content of CRF, TRH, neurotensin, and neuromedin N has been observed in 12 separate monthly coherts (n = 10@ or 130 total) adult, male Sprague-Dawley rats obtained at the same time each month from a single commercial supplier and held under constant (12:12) photoperiod conditions since birth. Both annual and 4-month (terannual) harmonics can be statistically discerned in these apparent rhythms, which exhibit cycles containing concentration changes up to 3-fold the lowest levels across the year (CRF increases 390%, TRH increases 173%, neurotensin increases 136%, and neuromedin N increases 150%). Hypothalamic somatostatin did not exhibit these statistically significant robust rhythms nor did any peptide in regions outside the hypothalamus. These data indicate that a mechanism allowing enhanced or diminished physiological availability of these regulatory neuropeptides at different times of the year may exist and may display distinct cycles even in the absence of normal photoperiod cues. Possible regulatory responses of pituitary receptor populations for these hypothalamic peptides must be considered. As certain of these neuropeptides also appear to be altered in the cerebrospinal fluid of patients with major depression or schizophrenia, similar hypothalamic cyclic changes may underly psychiatric symptoms with seasonal periodicity.

Poor prognosis associated with thrombocytosis in patients with cervical cancer.

The medical records of 113 patients treated with radiation therapy for invasive cervical cancer between 1975 and 1985 were retrospectively reviewed. Patients with thrombocytosis (platelet count greater than 400,000/microliters) were compared with those with normal platelet counts. Thrombocytosis was present at the initiation of therapy in 20 (17.7%) of the 113 patients. The 5-year cumulative survival rate for the 93 patients with normal platelet counts was 65%, whereas it was 25% for the 20 with thrombocytosis (P less than 0.0001, log-rank test). Using Cox's proportional hazards model, thrombocytosis continued to correlate strongly with poor survival even when adjusted for histologic type, patient age, and disease stage (P less than 0.001). Thrombocytosis is an independent indicator of poor prognosis in patients with cervical cancer.

The relative accuracies of two automated noninvasive arterial pressure measurement devices.

We compared the accuracies of two types of noninvasive blood pressure devices. Thirty-two patients requiring an intraarterial catheter for anesthetic management underwent simultaneous monitoring with Dinamap 1846SX and Ohmeda Finapres 3700 devices. For the first 10 minutes of recording, new Dinamap determinations were performed every 60 seconds; subsequent recordings were made at 3-minute intervals. Data were obtained at the time of new Dinamap readings, and twice between new readings to approximate the real-time performance of the two monitors. We defined superior accuracy as a statistically significant difference in mean absolute error greater than 5 mm Hg. With these criteria, pooled data from all patients revealed no difference in performance, even in real time. Pooled data can be misleading since there was a significant amount of variation in accuracy for both monitors. Therefore, we used nonparametric analysis to determine how many individual patients were monitored better by either device. When we compared only data from new Dinamap readings, the Finapres monitor showed superior performance for systolic readings in 13 patients, versus 6 patients for the Dinamap (P less than 0.05, chi-square test). Similar analysis for diastolic and mean pressure performance did not reach statistical significance. However, in real time, the Finapres unit monitored more patients more accurately for systolic (14 Finapres versus 3 Dinamap), diastolic (11 Finapres versus 3 Dinamap), and mean (10 Finapres versus 3 Dinamap) pressure determinations. The magnitude of these differences were, however, less dramatic than expected. This was probably due to stabilization of arterial pressure during the anesthetic, which minimized the error due to intermittent sampling. We conclude that continuous Finapres readings and new Dinamap determinations are equally accurate for diastolic and mean arterial pressures. The accuracy of Finapres appears to be slightly superior for systolic pressure. The intermittent sampling of oscillometric devices compromises their performance relative to the Finapres in many, but not all, cases.

The effect of short-term refrigeration of milk and addition of breast milk fortifier on the delivery of lipids during tube feeding.

Both storage of breast milk for 3 days at 4 degrees C and the addition of breast milk fortifier containing lipid improved the delivery of lipids during continuous pump infusion. The percentage of the original lipid delivered after 8 h of pumping was 9, 25, 58, and 66% from the freshly collected milk, stored milk, freshly collected milk mixed with fortifier, and stored milk mixed with fortifier, respectively. Phospholipid and cholesterol levels also were measured. Their loss during delivery through the tube was similar to the loss of total lipid. While storage improved the delivery of lipids from breast milk, the additional lipid delivered from milk mixed with fortifier was primarily due to lipids from the fortifier.

Changing patterns of free fatty acids in breast milk during storage.

Milk was collected from eight mothers. Total lipid and free fatty acids were determined on collection. The samples were divided into aliquots and stored at 25, 4, or -11 degrees C for 6, 12, 24, or 48 h. Additional samples were stored at -11 or -70 degrees C for 1, 2, 4, 6, or 8 weeks. The effect of temperature, length of storage, and total milk lipid on the release of individual fatty acids during storage was determined. There was no measurable lipolysis in milk stored at -70 degrees C. In the other samples both storage temperature and length of storage significantly affected lipolysis of milk lipids. The accumulation of free fatty acids in the milk increased with the length of storage and with increased storage temperature. Total lipid was not a significant factor in the release of fatty acids during the initial 48 h of storage. In samples stored for greater than 48 h at -11 degrees C, total lipid was positively correlated with the release of fatty acids. The pattern of free fatty acids in milk changed as storage progressed with an increase in the proportion of free 18:2, 20:4, and the other long-chain polyenoic acids. This may have implications for infant nutrition as these fatty acids are required for normal growth and development of the neonate.

Within-day variation of lipolytic activity in human milk.

Milk was collected from six mothers during their 8th wk of lactation to determine the diurnal variation of bile salt-stimulated lipase and serum-stimulated lipase. On the day of collection, one breast was completely emptied at 0600, 1000, 1400, 1800, and 2000 h. Activities of bile-stimulated lipase and serum-stimulated lipase in the milk were determined. Activity of bile-stimulated lipase was constant throughout the day, whereas serum-stimulated lipase activity varied significantly with time. Both activities differed significantly in milk from different women. Time of collection must be considered when measuring activity of serum-stimulated lipase but is not a major variable when measuring activity of bile-stimulated lipase in human milk. These results may be helpful when designing future studies involving these enzymes.

Acute injuries of the lateral ligaments of the ankle: comparison of stress radiography and arthrography.

Anterior and inversion stress were applied to the lateral ligaments of the ankle and the findings compared with the arthrograms. Fifty-five patients underwent stress radiography of both ankles and arthrography of the injured ankle within 72 hours of acute injury to the lateral ligaments. Ten patients had normal arthrograms and 45 had evidence of ligamentous injury. Anterior stress measurements could not be correlated with arthrography, and no differentiation between single and double ligament tears could be made. Inversion stress testing was very accurate in predicting ligamentous injury when the angle of inversion was 10 degrees or greater but achieved this degree of accuracy in only 38.2% of patients.

Nitrogen retention, muscle creatine and orotic acid excretion in traumatized rats fed arginine and glycine enriched diets.

Male, Sprague-Dawley rats were subjected to the trauma of laparotomy under sodium pentothal anesthesia. Apparent N retention (N intake - Urinary N) was studied when these rats were fed a 25% casein diet either unsupplemented or enriched with arginine plus glycine or with ornithine plus glycine. These amino acids occur in particularly high concentrations in skin and connective tissue and might, therefore, be required in greater amounts for tissue repair. In one experiment muscle creatine content and orotic acid excretion in the urine were determined. We found that laparotomy carried out under sodium pentothal anesthesia was a highly reproducible form of trauma which resulted in a significant decrease in apparent N retention. Supplementing a 25% casein based diet with arginine and glycine significantly improved apparent N retention both in untraumatized as well as in traumatized rats. Ornithine was less effective than arginine in improving apparent N retention. Urinary orotic acid excretion was significantly increased in rats fed the unsupplemented casein diet, regardless of the imposition of trauma. Muscle creatine content was significantly increased by the supplementation of the diet with arginine plus glycine. The beneficial effect of arginine-plus-glycine enrichment in traumatized rats does not appear to be due to an arginine deficit needed for the detoxification of ammonia from excess amino acids but may be related to creatine synthesis and turnover.