Parasitic Nematodes Modulate PIN-Mediated Auxin Transport to Facilitate Infection

Plant-parasitic nematodes are destructive plant pathogens that cause significant yield losses. They induce highly specialized feeding sites (NFS) in infected plant roots from which they withdraw nutrients. In order to establish these NFS, it is thought that the nematodes manipulate the molecular and physiological pathways of their hosts. Evidence is accumulating that the plant signalling molecule auxin is involved in the initiation and development of the feeding sites of sedentary plant-parasitic nematodes. Intercellular transport of auxin is essential for various aspects of plant growth and development. Here, we analysed the spatial and temporal expression of PIN auxin transporters during the early events of NFS establishment using promoter-GUS/GFP fusion lines. Additionally, single and double pin mutants were used in infection studies to analyse the role of the different PIN proteins during cyst nematode infection. Based on our results, we postulate a model in which PIN1-mediated auxin transport is needed to deliver auxin to the initial syncytial cell, whereas PIN3 and PIN4 distribute the accumulated auxin laterally and are involved in the radial expansion of the NFS. Our data demonstrate that cyst nematodes are able to hijack the auxin distribution network in order to facilitate the infection process.     

Activation of geminivirus V‐sense promoters in roots is restricted to nematode feeding sites

Obligate sedentary endoparasitic nematodes, such as the root‐knot and cyst nematodes, elicit the differentiation of specialized nematode nurse or feeding cells [nematode feeding sites (NFS), giant cells and syncytia, respectively]. During NFS differentiation, marked changes in cell cycle progression occur, partly similar to those induced by some geminiviruses. In this work, we describe the activation of V‐sense promoters from the Maize streak virus (MSV) and Wheat dwarf virus (WDV) in NFS formed by root‐knot and cyst nematodes. Both promoters were transiently active in microinjection experiments. In tobacco and Arabidopsis transgenic lines carrying promoter–β‐glucuronidase fusions, the MSV V‐sense promoter was activated in the vascular tissues of aerial plant parts, primarily leaf and cotyledon phloem tissue and some floral structures. Interestingly, in roots, promoter activation was restricted to syncytia and giant cells tested with four different nematode populations, but undetectable in the rest of the root system. As the activity of the promoter in transgenic rootstocks should be restricted to NFS only, the MSV promoter may have utility in engineering grafted crops for nematode control. Therefore, this study represents a step in the provision of some of the much needed additional data on promoters with restricted activation in NFS useful in biotechnological nematode control strategies.     

In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode

Esophageal secretions from endoparasitic sedentary nematodes are thought to play key roles throughout plant parasitism, in particular during the invasion of the root tissue and the initiation and maintenance of the nematode feeding site (NFS) essential for nematode development. The secretion in planta of esophageal cell-wall-degrading enzymes by migratory juveniles has been shown, suggesting a role for these enzymes in the invasion phase. Nevertheless, the secretion of an esophageal gland protein into the NFS by nematode sedentary stages has never been demonstrated. The calreticulin Mi-CRT is a protein synthesized in the esophageal glands of the root-knot nematode Meloidogyne incognita. After three-dimensional modeling of the Mi-CRT protein, a surface peptide was selected to raise specific antibodies. In planta immunolocalization showed that Mi-CRT is secreted by migratory and sedentary stage nematodes, suggesting a role for Mi-CRT throughout parasitism. During the maintenance of the NFS, the secreted Mi-CRT was localized outside the nematode at the tip of the stylet. In addition, Mi-CRT accumulation was observed along the cell wall of the giant cells that compose the feeding site, providing evidence for a nematode esophageal protein secretion into the NFS.     

Do slope orientation and sampling location determine soil biota composition?

“Evolution Canyon” is a typical Mediterranean-basin canyon with a summer dry stream at the bottom and large differences in vegetation cover and solar radiation between the north-facing slope (NFS) and the south-facing slope (SFS). It is known to act as an abiotic mediator influencing the community structure of soil fauna. The aim of this study was to determine the spatial dispersion of soil microbial and free-living nematode communities in the open sites (between shrubs) in the upper (0–10 cm) soil layer at four altitudes on both slopes. The combination of relative soil moisture availability and temperature, known to be one of the main triggers for soil biota activity, was explained by slope orientation. The above-mentioned differences were found to significantly affect microbial biomass and CO₂ evolution resulting in temporary stress, corresponding to higher values in metabolic quotient (qCO₂) values. These differences may represent microbial investment in energy in order to overcome stress resulting from the microclimatic differences between the two slopes. Moreover, the degree of substrate limitation (primary production due to the differences in plant cover) of microbial activity was explained by the difference in microbial functional groups. The total abundance of soil free-living nematodes was found to be 2-fold higher on the SFS than on the NFS. Thirty-nine genera, including 12 bacteria-feeders, 5 fungi-feeders, 12 plant-parasites, and 10 omnivore-predators, were found at the study site, with 34 genera on the NFS and 29 on the SFS. The generic diversity of the bacteriafeeding nematodes was higher on the SFS than on the NFS. This study elucidates the importance of slope orientation and its effect on the structural levels of soil microbial and nematode communities.     

Do slope orientation and sampling location determine soil biota composition?

"Evolution Canyon" is a typical Mediterra-nean-basin canyon with a summer dry stream at the bottom and large differences in vegetation cover and solar radiation between the north-facing slope (NFS) and the south-facing slope (SFS). It is known to act as an abiotic mediator influencing the community structure of soil fauna. The aim of this study was to determine the spatial dispersion of soil microbial and free-living nematode communities in the open sites (between shrubs) in the upper (0-10 cm) soil layer at four altitudes on both slopes. The combination of relative soil moisture availability and temperature, known to be one of the main triggers for soil biota activity, was explained by slope orientation. The above-mentioned differences were found to significantly affect microbial biomass and CO2 evolution resulting in temporary stress, corresponding to higher values in metabolic quotient (qCO2) values. These differences may represent microbial investment in energy in order to overcome stress resulting from the microclimatic differ-ences between the two slopes. Moreover, the degree of substrate limitation (primary production due to the differences in plant cover) of microbial activity was explained by the difference in microbial functional groups. The total abundance of soil free-living nematodes was found to be 2-fold higher on the SFS than on the NFS. Thirty-nine genera, including 12 bacteria-feeders, 5 fungi-feeders, 12 plant-parasites, and 10 omnivore-predators, were found at the study site, with 34 genera on the NFS and 29 on the SFS. The generic diversity of the bacteria-feeding nematodes was higher on the SFS than on the NFS. This study elucidates the importance of slope orientation and its effect on the structural levels of soil microbial and nematode communities.     

Activation of a Pollenin Promoter upon Nematode Infection

Three glycine-rich protein genes of Arabidopsis thaliana (Atgrp-6, Atgrp-7, and Atgrp-8) that correspond to putative genes coding for pollenins (AtolnB;2, AtolnB;3, and AtolnB;4, respectively) are expressed predominantly in the anthers and, more specifically, in the tapetum layer. Tapetal cells are responsible for nutrition of developing pollen grains and show some functional similarities to nematode feeding sites (NFS) induced in plant roots by sedentary parasitic nematodes. The aim of this study was to analyze promoter activity of the Atgrp genes in NFS. Transformed Arabidopsis plants containing a promoter-ß-glucuronidase (gus) fusion of the Atgrp-7 gene were inoculated with the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii. GUS assays were performed at different time points after infection. Histochemical analysis revealed an up-regulation of Atgrp-7-gus expression 3 days after inoculation in the feeding sites of both nematodes. Maximal Atgrp-7-gus staining levels in NFS were observed 1 week after nematode infection.     

The L-Cysteine Desulfurase NFS1 Is Localized in the Cytosol where it Provides the Sulfur for Molybdenum Cofactor Biosynthesis in Humans

In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Förster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.     

Native flowering shrubs promote beneficial insects in avocado orchards

1. Flower strips can promote and conserve beneficial insects in agroecosystems. Knowing which groups are favoured and which plant traits affect visitation rates by beneficial insects is important for the design of plant strips.
2. We established 21 Native Flower Strips (NFS) in avocado orchards in Central Chile. NFS contained 7–11 plant species, with variable corolla length and flowering period, to promote beneficial insects. We assessed flight activity of ladybirds (Coccinellidae) and bees (Apiformes) in sites adjacent to and far from NFS within avocado orchards. Additionally, we evaluated flower visitation for the main flower visitor groups (Apiformes, Lepidoptera, Diptera, and Coleoptera) to the plant species in NFS according to season and corolla length.
3. We found almost six times greater flight activity of coccinellids and bees in NFS than sites far from NFS within avocado orchards. Visitation rates of pollinator groups varied according to corolla length and season. Diptera, Coleoptera, and Lepidoptera consistently visited short corolla flowers. NFS were highly visited in summer and autumn, when avocados were not in bloom.
4. NFS in orchards should be encouraged by policymakers because they support beneficial insects that could deliver ecosystem services and contribute to local biological conservation.

     

Role of splenin and its active factor in regulating liver monooxygenase system in experimental immunodeficiency and hepatosohepatitis

An influence of splenin and its non-peptide factor of splenin (NFS) on the state of cytochrome P-450 dependent monooxygenase system (MOS) of liver microsomes in healthy animals under immunodeficiency (splenectomy, administration of gamma-aminobutyric acid (GABA) and toxic hepatosohepatitis was studied. The stimulating action of splenin and NFS on cytochrome P-450 content and MOS activity of liver microsomes in healthy animals has been established. The indices studied markedly decreased after splenectomy. The splenin or NFS administrations promote the recovery of these indices up to starting level in asplenic animals. A decrease in thymic mass dependent in GABA administration is prevented by NFS pretreatment of animals; there is no any effect of mediator acid on cytochrome P-450 content and MOS activity was noted. The preliminary administration NFS potentiates hepatotoxic effect of carbon tetrachloride and increases its inhibitory effect on P-450 dependent MOS of liver microsomes. Under the NFS action the effect in activity of the last is caused by the factor influence on the reparative processes in the liver.     

Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration

We made a three-dimensional (3-D) nanofibrous fibroin scaffold (NFS) with high porosity (94%) and examined its feasibility in bone regeneration. Under scanning electron microscopy, MC3T3-E1 preosteoblasts on the scaffold showed more spread on the first day after seeding compared with a 2-D scaffold. MTT assay showed significantly increased proliferation in 3-D NFS compared with 2-D NFS 7 days after seeding (P < 0.05). Western immunoblotting for activated paxillin, FAK, AKT, C-Src, and ERK1/2 antibodies showed signals from the extracellular matrix were significantly increased in 3-D NFS. Newly developed 3-D electrospun NFS may be a good candidate for use in bone regeneration. Ki and Park equally contributed in this paper.     

Cost-Effectiveness Analysis: Risk Stratification of Nonalcoholic Fatty Liver Disease (NAFLD) by the Primary Care Physician Using the NAFLD Fibrosis Score

Background

The complications of Nonalcoholic Fatty Liver Disease (NAFLD) are dependent on the presence of advanced fibrosis. Given the high prevalence of NAFLD in the US, the optimal evaluation of NAFLD likely involves triage by a primary care physician (PCP) with advanced disease managed by gastroenterologists.

Methods

We compared the cost-effectiveness of fibrosis risk-assessment strategies in a cohort of 10,000 simulated American patients with NAFLD performed in either PCP or referral clinics using a decision analytical microsimulation state-transition model. The strategies included use of vibration-controlled transient elastography (VCTE), the NAFLD fibrosis score (NFS), combination testing with NFS and VCTE, and liver biopsy (usual care by a specialist only). NFS and VCTE performance was obtained from a prospective cohort of 164 patients with NAFLD. Outcomes included cost per quality adjusted life year (QALY) and correct classification of fibrosis.

Results

Risk-stratification by the PCP using the NFS alone costs $5,985 per QALY while usual care costs $7,229/QALY. In the microsimulation, at a willingness-to-pay threshold of $100,000, the NFS alone in PCP clinic was the most cost-effective strategy in 94.2% of samples, followed by combination NFS/VCTE in the PCP clinic (5.6%) and usual care in 0.2%. The NFS based strategies yield the best biopsy-correct classification ratios (3.5) while the NFS/VCTE and usual care strategies yield more correct-classifications of advanced fibrosis at the cost of 3 and 37 additional biopsies per classification.

Conclusion

Risk-stratification of patients with NAFLD primary care clinic is a cost-effective strategy that should be formally explored in clinical practice.     

Native flower strips increase visitation by non-bee insects to avocado flowers and promote yield

Pollination is an essential ecosystem service for pollinator-dependent crops and plant communities. Apis mellifera L. is by far the most commonly used species to obtain this service in agriculture. However, there is growing evidence of the importance for crop yields of the service provided by wild bees and non-bee insect pollinators. Establishing flower resources in agricultural landscapes is a management practice that can increase insect pollinator populations and improve crop yields. We established perennial native flower strips (NFS) in four avocado orchards in central Chile during autumn 2017. We monitored flower visitors and counted newly formed fruits in avocados near and far from NFS in spring 2019, to assess flower visitor groups, flower visitation rates and fruit numbers. Only A. mellifera visited avocado flowers within bees, whereas both the managed A. mellifera and wild bees were the main visitors to the NFS. NFS increased visitation rates to adjacent avocado of flies, and with a trend for the sum of all non-managed flower visitors (i.e. excluding A. mellifera). However, there were no differences in the rates of A. mellifera visitation to avocados near and far from NFS. Avocado fruit numbers were higher among avocados near NFS than among those farther away. This difference could be due to better pollination by the increased visits to avocado flowers by flies or other wild insects. Therefore, NFS could contribute to crop fruit number, as well as conservation of native flora, wild bees and non-bee pollinators on fruit farms in the “Central Chile” biodiversity hotspot.     

IL-2 production, IL-2 receptor expression, and IL-2 responsiveness of spleen and mesenteric lymph node cells from inbred mice infected with Trichinella spiralis

The in vitro production of IL-2 and IL-2R expression by lymphoid cells of inbred mice of strong (NFS), intermediate (C3H), or weak (B10.BR) in phenotype of Trichinella spiralis (TS) rejection was measured during a primary infection. Maximum production of IL-2 by spleen and mesenteric lymph node (MLN) cells occurred at 5 days postinfection. Cell depletion experiments demonstrated that Lyt-1.2+ T cells were predominantly responsible for in vitro IL-2 production. Cells from strong-responder NFS mice produced more IL-2 than cells from intermediate-responder C3H or weak-responder B10.BR mice. Similarly, after TS infection, NFS mice had significantly more IL-2R expressing MLN cells than B10.BR or C3H MLN cells. All mouse strains displayed a dose-dependent increase in in vitro IL-2 production after infection with 100 to 800 TS. This effect was most pronounced in NFS mice. Limiting dilution analysis of day 5 infected MLN cells demonstrated that the frequency of TS-reactive CD4+ cells was threefold higher in NFS mice than B10.BR and fourfold higher than in C3H mice. Finally, MLN cells taken from infected NFS mice responded to an exogenous source of IL-2, whereas MLN cells from infected C3H or B10.BR mice were unable to do so. We conclude that strong responsiveness in parasite rejection may be related to the amount of IL-2 produced as well as to the capacity of the lymphocytes of each mouse strain to respond to IL-2. Although these differences help explain the strong rejection phenotype of NFS mice, they fail to separate C3H and B10.BR mice where TS-responsive CD4+ precursors, IL-2 production, and dose responsiveness are all lower for the intermediate phenotype (worm rejection) C3H than the weak phenotype B10.BR mice.     

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability: IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER,COXPD19*

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.     

Interspersion of the VHQ52 and VH7183 gene families in the NFS/N mouse

Deletion mapping analysis has shown that members of the VH7183 and VHQ52 gene families are interspersed in the NFS/N mouse. To obtain direct evidence that members of these gene families are physically linked, an NFS/N liver library was constructed and genomic clones were analyzed for hybridization to both VHQ52 and VH7183 gene probes. Four clones were identified which contained both VHQ52 and VH7183 hybridizable restriction fragments. Two clones containing rearranged VHQ52 genes were also found to hybridize with the VH7183 gene probe. Sequence analysis of three of the VH7183-containing restriction fragments indicate that all are pseudogenes which contain interruptions at either the 5' and/or 3' ends of the VH coding region. Given the D-proximal location of at least a portion of the VHQ52 gene family relative to VH7183 in NFS/N mice, and the known correlation between D proximity and the frequency of VH gene utilization, 22 NFS/N-derived pre-B cell lines were analyzed for VHQ52 gene utilization. More than 40% of the identified H chain (VHDJH) rearrangements in this survey used members of this gene family. Furthermore, analysis of poly(A)+ RNA from NFS/N fetal liver and adult spleen also indicates preferential utilization of VHQ52 family in fetal liver. Kinetic studies show, however, that there are no changes in relative utilization throughout fetal ontogeny. The implications of these findings for the expression and randomization of the VH repertoire are discussed.     

Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum

Genetic diversity was studied in six subpopulations (a total of 60 individuals) of wild barley, Hordeum spontaneum , the progenitor of cultivated barley, sampled from six stations located along a transect of 300 m across the two opposing slopes of 'Evolution Canyon', a Mediterranean microsite at Lower Nahal Oren, Mt Carmel. The two opposing slopes are separated by between 100 and 400 m and designated SFS (South-Facing Slope) and NFS (North-Facing Slope) with each having three equidistant test stations. The SFS, which receives up to 300% more solar radiation, is drier, ecologically more heterogeneous, fluctuating, and more stressful than the NFS. Analysis of 12 RAPD primers, representing a total of 51 putative loci, revealed a significant inter- and intraslope variation in RAPD band polymorphism. A significantly higher proportion of polymorphic RAPD loci was found amongst the subpopulations on the SFS (mean P = 0.909) than on the NFS (mean P = 0.682), on the basis of the presence and absence of 22 strong bands. Polymorphism generally increased upwards from the bottom to the top of the SFS (0.636, 0.773, 0.955) and NFS (0.409, 0.500, 0.545), respectively. Gametic phase disequilibria estimates, D, revealed SFS and NFS unique predominant combinations which sharply differentiated the two slopes and indicated that there is differential interslope selection favouring slope-specific multilocus combinations of alleles, or blocks of genes over tens to hundreds of meters. This suggests that selection overrides migration. RAPD polymorphism appears to parallel allozyme diversity which is climatically adaptive and driven by natural selection in the same subpopulations at the microsite.     

Human Mitochondrial Chaperone (mtHSP70) and Cysteine Desulfurase (NFS1) Bind Preferentially to the Disordered Conformation,Whereas Co-chaperone (HSC20) Binds to the Structured Conformation of the Iron-Sulfur Cluster Scaffold Protein (ISCU)

Human ISCU is the scaffold protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis and transfer. NMR spectra have revealed that ISCU populates two conformational states; that is, a more structured state (S) and a partially disordered state (D). We identified two single amino acid substitutions (D39V and N90A) that stabilize the S-state and two (D39A and H105A) that stabilize the D-state. We isolated the two constituent proteins of the human cysteine desulfurase complex (NFS1 and ISD11) separately and used NMR spectroscopy to investigate their interaction with ISCU. We found that ISD11 does not interact directly with ISCU. By contrast, NFS1 binds preferentially to the D-state of ISCU as does the NFS1-ISD11 complex. An in vitro Fe-S cluster assembly assay showed that [2Fe-2S] and [4Fe-4S] clusters are assembled on ISCU when catalyzed by NFS1 alone and at a higher rate when catalyzed by the NFS1-ISD11 complex. The DnaK-type chaperone (mtHSP70) and DnaJ-type co-chaperone (HSC20) are involved in the transfer of clusters bound to ISCU to acceptor proteins in an ATP-dependent reaction. We found that the ATPase activity of mtHSP70 is accelerated by HSC20 and further accelerated by HSC20 plus ISCU. NMR studies have shown that mtHSP70 binds preferentially to the D-state of ISCU and that HSC20 binds preferentially to the S-state of ISCU.     

Carbon source utilization patterns of Bacillus simplex ecotypes do not reflect their adaptation to ecologically divergent slopes in 'Evolution Canyon', Israel

The 'Evolution Canyons' I and II in Israel are model habitats to study adaptation and speciation of bacteria in the environment. These canyons represent similar ecological replicates, separated by 40 km, with a strongly sun-exposed and hot 'African' south-facing slope (SFS) vs. a cooler and mesic-lush 'European' north-facing slope (NFS). Previously, among 131 Bacillus simplex isolates, distinct genetic lineages (ecotypes), each specific for either SFS or NFS, were identified, suggesting a temperature-driven slope-specific adaptation. Here, we asked whether the ecological heterogeneity of SFS vs. NFS also affected carbon utilization abilities, as determined using the Biolog assay. Contrary to expectation, a correlation between substrate utilization patterns and the ecological origin of strains was not found. Rather, the patterns split according to the two major phylogenetic lineages each of which contain SFS and NFS ecotypes. We conclude that traits related to the general energy metabolism, as far as assessed here, are neither shaped by the major abiotic features of 'Evolution Canyon', namely solar radiation, temperature, and drought, nor by the soil characteristics. We further conclude that some traits diverge rather neutrally from each other, whereas other, more environmentally related traits are shaped by natural selection and show evolutionary convergence.     

Microclimate,developmental plasticity and community structure in artificial temporary pools

Solar radiation, which influences temperature and hydroperiod, should strongly influence colonization, development rate, development strategies and survival in temporary pools. We assessed these hypotheses at two spatial microscales with varying microclimate. One scale, ca. 200 m, was opposing slopes of a canyon on MT Carmel, Israel, referred to as 'Evolution Canyon' (Nevo, 1995). Due primarily to differential solar radiation, the warmer and drier South-facing slope (SFS) supports a savanna-like open park forest while the cooler, moister North-facing slope (NFS) is heavily forested, primarily with live oak. Using artificial pools, we assessed how these close but contrasting environments may influence developmental plasticity in the frog, Hyla savignyi, and colonization of insects. As expected, hydroperiod was considerably longer on the NFS. Hyla tadpole development was significantly faster on the SFS, while size at metamorphosis was greater on the NFS. Insect species richness was higher in NFS pools. The most common species, Culex pipiens, oviposited significantly more in NFS pools than in SFS pools. In the second scale, we placed pools only meters apart, but varying in shade and thus maximum temperature at Haifa University Campus, MT Carmel. Species richness and maximum temperature among pools were not correlated during winter and early spring (cool to mild temperatures), but were strongly and negatively correlated during early summer (hot temperatures). Thermal tolerance in the laboratory of selected species matched their thermal spatial distribution in the field. These results indicate that temperature can play a strong role in affecting developmental strategies, species′ distributions and community structure in temporary pools over a very small spatial scale. This revised version was published online in July 2006 with corrections to the Cover Date.