The molecular basis of venom resistance in a rattlesnake‐squirrel predator‐prey system

Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long‐sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins (“venom interactive proteins” [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum‐based resistance is both population‐ and species‐specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum “resistome” involves broad‐based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co‐evolutionary interactions between species at the molecular level.     

Chimpanzees prey on army ants with specialized tool set

Several populations of chimpanzees have been reported to prey upon Dorylus army ants. The most common tool‐using technique to gather these ants is with “dipping” probes, which vary in length with regard to aggressiveness and lifestyle of the prey species. We report the use of a tool set in army ant predation by chimpanzees in the Goualougo Triangle, Republic of Congo. We recovered 1,060 tools used in this context and collected 25 video recordings of chimpanzee tool‐using behavior at ant nests. Two different types of tools were distinguished based on their form and function. The chimpanzees use a woody sapling to perforate the ant nest, and then a herb stem as a dipping tool to harvest the ants. All of the species of ants preyed upon in Goualougo are present and consumed by chimpanzees at other sites, but there are no other reports of such a regular or widespread use of more than one type of tool to prey upon Dorylus ants. Furthermore, this tool set differs from other types of tool combinations used by chimpanzees at this site for preying upon termites or gathering honey. Therefore, we conclude that these chimpanzees have developed a specialized method for preying upon army ants, which involves the use of an additional tool for opening nests. Further research is needed to determine which specific ecological and social factors may have shaped the emergence and maintenance of this technology. Am. J. Primatol. 72:17–24, 2010. © 2009 Wiley‐Liss, Inc.     

Accuracy of Monoclonal Stool Tests for Determining Cure of Helicobacter pylori Infection After Treatment

Background: Studies comparing new monoclonal fecal tests for evaluating cure of Helicobacter pylori infection after treatment are scarce. The objective was to compare the diagnostic accuracy of three monoclonal stool tests: two rapid in‐office tools –RAPID Hp StAR and ImmunoCard STAT! HpSA – and an EIA test – Amplified IDEIA Hp StAR. Materials and methods: Diagnostic reliability of the three tests was evaluated in 88 patients at least 8 weeks after H. pylori treatment. Readings of immunochromatographic tests were performed by two different observers. Sensitivity, specificity, positive and negative predictive values and 95% confidence intervals were calculated. Results: All tests presented similar performance for post‐eradication testing. Sensitivity for detecting persistent infection was 100% for both Amplified IDEIA and RAPID Hp StAR and 90% for ImmunoCard STAT! HpSA. Respective specificities were 94.9%, 92.3–93.6% and 94.9%. Negative predictive values were very high (100%, 100% and 98.7% respectively). But positive predictive values were lower, ranging from 62.5 to 71.4%. Conclusion: All monoclonal fecal tests in this series presented similar performance in the post‐treatment setting. A negative test after treatment adequately predicted cure of the infection. However, nearly a third of tests were false positive, showing a poor predictive yield for persistent infection.     

Density and structure of Saissetia oleae (Hemiptera: Coccidae) populations on citrus and olives: relative importance of the two annual generations

Saissetia oleae (Olivier) (Hemiptera: Coccidae) populations were studied and compared in citrus (Citrus spp.) and olive (Olea europaea L.) groves to determine the number of generations, crawler emergence periods and changes in population density during the year. Ten citrus and four olive groves were sampled regularly between March 2003 and December 2005 in eastern Spain, covering an area of 10,000 km2. Each sample consisted of 16 branches and 64 leaves. Saissetia oleae populations presented a similar trend in both crops during the three years of study. Populations peaked in July, when crawlers emerged after the egg-laying period, and decreased during several months due to mortality of first instars in summer. A second crawler emergence period, with lower numbers and more variability from year to year, occurred between October and March. Populations did not increase during this period, probably because most eggs and crawlers perished during the winter and also because females that gave rise to this fall-winter generation were half as big and fecund as spring females. No differences were found between the size of mature females that had developed on citrus and on olives during the spring. Considering this population pattern, the best seasonal period to apply pesticides to control S. oleae would be at the end of July, when populations are synchronous, all crawlers have already emerged, and first instars predominate.     

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.     

A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.     

Spatial patterns of white stork (Ciconia ciconia) migratory phenology in the Iberian Peninsula

In contrast to the attention attracted by temporal trends of phenology, the spatial patterns of arrivals, departures or stays of trans-Saharan birds are still nowadays largely unknown in most of their European breeding areas. In the case of the white stork (Ciconia ciconia), some studies have attempted to describe its migratory patterns throughout some European countries but, to our knowledge, no one has related these patterns to some kind of explanatory variable which offers an ecologically-based explanation for the heterogeneous phenology observable among populations. Here, arrivals, departures and stays of this species, recorded in hundreds of Spanish localities, were related to a set of environmental, geographical, biological and spatial predictors, and modeled by multiple regression. The best model for arrival dates accounted for up to 34% of variability of data and pointed towards an earlier arrival in those populations located in south-western Iberia and with higher population densities. This last relationship is probably due to the competition for nest-site fidelity maintenance. However, no variable was able to explain properly the blurred spatial pattern recorded for departure dates. Departure decisions are strongly influenced by social behavior in this species and depend on collective decisions influenced by peculiar local environmental conditions of each year rather than macrogeographical gradients. Environmental, geographical or spatial variables also did not capture much of the observed variability in the length of the stays among populations. However, this variable was strongly related to the arrival and departure dates of populations. White storks stay longer in localities with earlier arrivals and, especially, later departures.     

A conserved sequence immediately N-terminal to the Bateman domains in AMP-activated protein kinase gamma subunits is required for the interaction with the beta subunits

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. alpha, beta, and gamma, with alpha being the catalytic subunit and beta and gamma having regulatory roles. Although several studies have defined different domains in alpha and beta involved in the interaction with the other subunits of the complex, little is known about the regions of the gamma subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the gamma subunit isoforms and studied the interactions with alpha and beta subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in gamma1, gamma2, and gamma3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active alphabetagamma complex. This region is required for the interaction with the beta subunits. The interaction between the alpha and gamma subunits does not require this region and occurs instead within the Bateman domains of the gamma subunit, although the alpha-gamma interaction does appear to stabilize the beta-gamma interaction. In addition, sequential deletions from the C termini of the gamma subunits indicate that deletion of any of the CBS (cystathionine beta-synthase) motifs prevents the formation of a functional complex with the alpha and beta subunits.     

Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H₂O₂ as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.