Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Development and relations of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> in soil

The development of Fusarium culmorum and Pseudomonas fluorescens in soil, and the relations between them, were studied using membrane filters containing the fungus, the bacterium, or both microorganisms; the filters were incubated in soil. F. culmorum was identified by indirect immunofluorescence; the GUS-labeled strain was used to visualize P. fluorescens. It was found that F. culmorum introduced in soil can develop as a saprotroph, with the formation of mycelium, macroconidia, and a small amount of chlamydospores. Introduction of glucose and cellulose resulted in increased density of the F. culmorum mycelium and macroconidia. P. fluorescens suppressed the development of the F. culmorum mycelium in soil, but stimulated chlamydospore formation. Decreased mycelial density in the presence of P. fluorescens was more pronounced in soil without additions and less pronounced in the case of introduction of glucose or cellulose. F. culmorum had no effect on P. fluorescens growth in soil.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Photosynthetic activity and components of the electron transport chain in the aerobic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter thiooxidans</Emphasis>

Bioenergetics of the aerobic bacteriochlorophyll a-containing (BCl a) bacterium (ABC bacterium) Roseinatronobacter thiooxidans is a combination of photosynthesis, oxygen respiration, and oxidation of sulfur compounds under alkaliphilic conditions. The photosynthetic activity of Rna. thiooxidans cells was established by the photoinhibition of cell respiration and reversible photobleaching discoloration of the BCl a of reaction centers (RC), connected by the chain of electron transfer with cytochrome c ₅₅₁ oxidation. The species under study, like many purple bacteria and some of the known ABC bacteria, possesses a light-harvesting pigment-protein (LHI) complex with the average number of 30 molecules of antenna BCl a per one photosynthetic RC. Under microaerobic growth conditions, the cells contained bc ₁ complex and two terminal oxidases: cbb ₃-cytochrome oxidase and the alternative cytochrome oxidase of the a ₃ type. Besides, Rna. thiooxidans was shown to have several different soluble low- and high-potential cytochromes c, probably associated with the ability of utilizing sulfur compounds as additional electron donors.     

The new alkaliphilic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter monicus</Emphasis> sp. nov. from the hypersaline Soda Mono Lake (California,United States)

Two strains of pink-colored aerobic bacteriochlorophyll a-containing bacteria were isolated from aerobic (strain ROS 10) and anaerobic (strain ROS 35) zones of the water column of Mono Lake (California, United States). Cells of the bacteria were nonmotile oval gram-negative rods multiplying by binary fission by means of a constriction. No intracellular membranes were detected. Polyphosphates and poly-β-hydroxybutyric acid were the storage compounds. Pigments were represented by bacteriochlorophyll a and carotenoids of the spheroidene series. The strains were obligately aerobic, mesophilic (temperature optimum of 25–30°C), alkaliphilic (pH optimum of 8.5–9.5), and moderately halophilic (optimal NaCl concentration of 40 g/l). They were obligately heterotrophic and grew aerobically in the dark and in the light. Respiration was inhibited by light at wavelengths corresponding to the absorption of the cellular pigments. The substrate utilization spectra were strain-specific. In the course of organotrophic growth, the bacteria could oxidize thiosulfate to sulfate; sulfide and polysulfide could also be oxidized. The DNA G+C content was 59.4 mol % in strain ROS 10 and 59 mol % in strain ROS 35. In their phenotypic properties, the new strains were close but not identical to the alkaliphilic bacterium Roseinatronobacter thiooxidans. The distinctions in the nucleotide sequences of the 16S rRNA genes (2%) and low DNA-DNA hybridization level with Rna. thiooxidans (22–25%) allow the new strains to be assigned to a new species of the genus Roseinatronobacter, Roseinatronobacter monicus sp. nov. with the type strain ROS 35^T (=UNIQEM U-251^T = VKM B-2404^T).     

Fine-mapping and identification of a novel locus <Emphasis Type="Italic">Rsc15</Emphasis> underlying soybean resistance to <Emphasis Type="Italic">Soybean mosaic virus</Emphasis>

Key message

Rsc15, a novel locus underlying soybean resistance to SMV, was fine mapped to a 95-kb region on chromosome 6. The Rsc15- mediated resistance is likely attributed to the gene GmPEX14 , the relative expression of which was highly correlated with the accumulation of H ₂ O ₂ along with the activities of POD and CAT during the early stages of SMV infection in RN-9.

Abstract

Soybean mosaic virus (SMV) causes severe yield losses and seed quality deterioration in soybean [Glycine max (L.) Merr.] worldwide. A series of single dominant SMV resistance genes have been identified on respective soybean chromosomes 2, 13 and 14, while one novel locus, Rsc15, underlying resistance to the virulent SMV strain SC15 from soybean cultivar RN-9 has been recently mapped to a 14.6-cM region on chromosome 6. However, candidate gene has not yet been identified within this region. In the present study, we aimed to fine map the Rsc15 region and identify candidate gene(s) for this invaluable locus. High-resolution fine-mapping revealed that the Rsc15 gene was located in a 95-kb genomic region which was flanked by the two simple sequence repeat (SSR) markers SSR_06_17 and BARCSOYSSR_06_0835. Allelic sequence comparison and expression profile analysis of candidate genes inferred that the gene Glyma.06g182600 (designated as GmPEX14) was the best candidate gene attributing for the resistance of Rsc15, and that genes encoding receptor-like kinase (RLK) (i.e., Glyma.06g175100 and Glyma.06g184400) and serine/threonine kinase (STK) (i.e., Glyma.06g182900 and Glyma.06g183500) were also potential candidates. High correlations were established between the relative expression level of GmPEX14 and the hydrogen peroxide (H₂O₂) concentration and activities of catalase (CAT) and peroxidase (POD) during the early stages of SMV-SC15 infection in RN-9. The results of the present study will be useful in marker-assisted breeding for SMV resistance and will lead to further understanding of the molecular mechanisms of host resistance against SMV.

     

<Emphasis Type="Italic">Alkaliphilus peptidofermentans</Emphasis> sp. nov., a new alkaliphilic bacterial soda lake isolate capable of peptide fermentation and Fe(III) reduction

A novel strain, Z-7036, of anaerobic spore-forming bacteria was isolated from a cellulolytic consortium obtained from the bottom sediments of the low-mineralization soda lake Verkhnee Beloe (Buryatia). The cells of the new strain are short motile gram-positive rods, 1.1–3.0 × 0.25–0.4 μm. The organism is an aerotolerant anaerobe and obligate alkaliphile growing within the pH range of 7.5–9.7 with an optimum at pH 9.1. The strain is mesophilic and halotolerant and grows at NaCl concentrations from 0 to 50 g/l with an optimum at 20 g/l. Carbonates are required. The microorganism ferments peptone, yeast extract, trypticase, tryptone, Bacto Soytone, meat extract, Casamino acids, ornithine, arginine, threonine, and tryptophan. The strain hydrolyzes the bacterial preparations “Gaprin” and “Spirulina”. Acetate and formate are the major fermentation products. The strain reduces amorphous ferric hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), S₂O ₃ ^2? , fumarate, and crotonate. Major fatty acids are C_16:0, C1_6:1ω7c, iso-C₁₇, iso-C₁₅, and iso-C_17:1. The DNA G+C content is 33.8 ± 0.5 mol %. According to the results of the 16S rRNA gene analysis, strain Z-7036 belongs to the genus Alkaliphilus within the cluster XI of low G+C gram-positive bacteria of the family Clostridiaceae. The novel strain is closely related to A. transvaalensis SAGM1^T and A. crotonatoxidans B11-2^T (93.3 and 93.9% 16S rRNA sequence identities, respectively). On the basis of the existing genotypic and phenotypic differences, we propose that strain Z-7036 should be classified as a novel species Alkaliphilus peptidofermentans sp. nov.     

The patterns of utilization and accumulation of polyphosphates in the cytosol of the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under inactivation of exopolyphosphatase genes <Emphasis Type="Italic">PPX1</Emphasis> and <Emphasis Type="Italic">PPN1</Emphasis>

The effect of inactivation of the PPX1 and PPN1 genes encoding the yeast exopolyphosphatases on the activities of these enzymes and polyphosphate content in the cytosol of Saccharomyces cerevisiae was studied under P_i deficit and P_i excess in the cultivation medium. Under P_i deficit, exopolyphosphatase activity in strain CRN (with inactivated PPN1 gene) and in the parent strain CRY increased 3- and 1.5-fold, respectively. In the strain CRX (with inactivated PPX1 gene), exopolyphosphatase activity did not change under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by an ～1.7-fold increase of exopolyphosphatase activities in the cytosol preparations of strains CRY, CRX, and CRN. In the cytosol of the double mutant, exopolyphosphatase activity was practically absent under all of the above cultivation conditions. The content of polyphosphates in the cytosol preparations of all strains under study substantially decreased under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by polyphosphate over-accumulation only in the cytosol preparations of stains CRX and CNX, where their levels increased ～1.3 and 3.5-fold, respectively. No over-accumulation was observed in the parent strain CRY and in the PPN1-deficient strain CRN. These data suggest that the exopolyphosphatases encoded by the PPX1 and PPN1 genes are not involved in polyphosphate synthesis.     

A new <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant <Emphasis Type="Italic">apetala1-20</Emphasis>

A new deletion allele of the APETALA1 (AP1) gene encoding a type II MADS-box protein with the key role in the initiation of flowering and development of perianth organs has been identified in A. thaliana. The deletion of seven amino acids in the conserved region of the K domain in the ap1-20 mutant considerably delayed flowering and led to a less pronounced abnormality in the corolla development compared to the weak ap1-3 and intermediate ap1-6 alleles. At the same time, a considerable stamen reduction has been revealed in ap1-20 as distinct from ap1-3 and ap1-6 alleles. These data indicate that the K domain of AP1 can be crucial for the initiation of flowering and expression regulation of B-class genes controlling stamen development.     

Cytochromes <Emphasis Type="Italic">c</Emphasis> of the anaerobic methacrylate reducer <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> AM-1

Cytochromes c were found in the cells of the bacterium Geobacter sulfurreducens AM-1 grown on acetate and methacrylate. The periplasmic extract of G. sulfurreducens AM-1 contained about 88% of the total content of cytochromes c of intact cells. The analysis of cytochromes c from the native cells of G. sulfurreducens AM-1, from the periplasmic extract and from the cells treated by an alkaline solution showed the presence of nine proteins containing heme c. The molecular masses of cytochromes c from G. sulfurreducens AM-1 were 12.5, 15.5, 25.7, 29.5, 34.7, 41.7, 50.1, 63.1, and 67.6 kDa; localization of each cytochrome c was determined. Three heme-containing proteins (15.5 kDa, 25.7 kDa, and 29.5 kDa with the most intensive staining) were present mainly in the periplasm of the bacterium. The other two (50.1 and 67.6 kDa) were supposedly localized in the cell membrane. Cytochromes c with the molecular masses of 12.5, 15.5, and 67.6 kDa are considered as possible components of the methacrylate redox system of G. sulfurreducens AM-1.     

Polymorphism of <Emphasis Type="Italic">yellow</Emphasis> locus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mutants from natural populations

We have studied the molecular characteristics of the yellow locus (y; 1–0.0), which determines the body color of phenotypically wild-type and mutant alleles isolated in different years from geographically distant populations of Drosophila melanogaster. According to the Southern blot, data restriction maps of the yellow locus of all examined strains differ from one another, as well as from Oregon stock. FISH analysis shows that, in the neighborhood of the yellow locus in the X chromosome, neither P nor hobo elements are found in y^1–775 stock, while only hobo is found in these region in y^1–859 and y^1–866 stocks, only the P element is found in y⁺sn⁸⁴⁹ stock, and both elements are found in y^1–719 stock. Thus, all yellow mutants studied are of independent origin. Locus yellow located on the end of X chromosome (region 1A5–8 on the cytologic map) carries significantly more transposon than retrotransposon induced mutations compared to the white locus (region 3C2). It is possible that, at the ends of Drosophila melanogaster chromosomes, transposons are more active than retrotransposons.     

Interactions between the grass shrimp <Emphasis Type="Italic">Palaemonetes pugio</Emphasis> and the salt marsh grasses <Emphasis Type="Italic">Phragmites australis</Emphasis> and <Emphasis Type="Italic">Spartina alterniflora</Emphasis>

The grass shrimp Palaemonetes pugio, a species common to Spartina alterniflora-dominated marshes, may be sensitive to the invasion of the common reed Phragmites australis in northeastern US salt marshes. We examined two questions: (1) Do grass shrimp have a preference for the native plant over the non-native plant? (2) Are grass shrimp more effective foragers on P. australis? We tested the first hypothesis by comparing the amount of time shrimp spend in physical contact with the plant types over a 1-h period. Shrimp were observed under different arrangements of vegetation to control for differences in conspicuous structural features. Additionally, the amount of time shrimp spent foraging on S. alterniflora and P. australis shoots was compared to determine if shrimp graze more often on S. alterniflora. Shrimp spent significantly more time in contact with S. alterniflora only when plant types were grouped at opposite ends of aquaria, but did not exhibit a foraging preference for this plant type. To address our second question, we investigated the effects of shrimp foraging on stem epifauna, an assemblage of semi-aquatic invertebrates associated with macrophyte shoots. Previous research showed that P. australis supports a lower density of stem-dwelling epifauna relative to S. alterniflora. We hypothesized that the primary grazer of this community, P. pugio, can forage on P. australis stems more effectively due to structural differences between the two plants, causing the lower abundance of epifauna through top-down effects. We exposed individual shoots inhabited by epifauna to shrimp and compared faunal densities on exposed shoots to densities on control shoots after 18 h. The reduction of epifauna by predation was proportional on the two plant types. Therefore, top-down effects can be ruled out as an explanation for the patchy distribution of epifauna observed in P. australis–S. alterniflora marshes.     

Isolation and characterization of nitrogen-fixing bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis> from the soil of a <Emphasis Type="Italic">Sphagnum</Emphasis> peat bog

he presence of nitrogen-fixing bacteria of the genus Azospirillum in the soils of acidic raised Sphagnum bogs is revealed for the first time. Three Azospirillum strains, B2, B21, and B22, were isolated as a component of methane-oxidizing enrichment cultures, whereas attempts to isolate them directly from peat samples have failed. The results of comparative analysis of the nucleotide sequences of 16S rRNA genes, DNA-DNA hybridization, and the analysis of the sequences of the functional genes encoding nitrogenase and ribulose-1, 5-bisphosphate carboxylase reveal that all the newly obtained strains can be classified as Azospirillum lipoferum. Yet, unlike A. lipoferum, the isolates do not require biotin and utilize sucrose, inositol, and glycerol for growth. The cell morphology of strain B2 differs from that of the type strain and strains B21 and B22. The results obtained indicate the variability of morphological, physiological, and biochemical properties in closely related Azospirillum strains and suggest the existence of metabolic relationships between methanotrophic bacteria and the representatives of the genus Azospirillum under peat bog conditions.     

Chromosomal differentiation of the sibling species <Emphasis Type="Italic">Pichia membranifaciens</Emphasis> and <Emphasis Type="Italic">Pichia manshurica</Emphasis>

The molecular karyotyping analysis of 21 strains within the taxonomic complex Pichia membranifaciens allowed the sibling species P. membranifaciens and P. manshurica, as well as P. deserticola and P. punctispora, to be differentiated. Heterogeneity of the species P. membranifaciens at the variety level is discussed.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

<Emphasis Type="Italic">Rhodobaca barguzinensis</Emphasis> sp. nov., a new alkaliphilic purple nonsulfur bacterium isolated from a soda lake of the Barguzin Valley (Buryat Republic,Eastern Siberia)

A novel strain, alga-05, of alkaliphilic purple nonsulfur bacteria was isolated from sediments of a small saline (60 g/l) soda lake near Lake Algin (Barguzin Valley, Buryat Republic, Russia). These bacteria contain bacteriochlorophyll a and carotenoids of the alternative spirilloxanthin group with predominating demethylspheroidenone. They are facultative anaerobes; their photosynthetic structures are of the vesicular type and arranged along the cell periphery. Growth of this strain is possible in a salinity range of 5–80 g/l NaCl, with an optimum at 20 g/l NaCl. Best growth occurred at 20–35°C. Analysis of the 16S rRNA gene sequences demonstrated that the studied isolate is closely related to the alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis (99% similarity) isolated from soda lakes of the African Rift Zone. According to the results of DNA-DNA hybridization, strain alga-05 has a 52% similarity with the type species of the genus Rhodobaca. On the basis of the obtained genotypic data and some phenotypic properties (dwelling in a hypersaline soda lake of Siberia, moderate halophily, ability to grow at relatively low temperatures, etc.), the isolated strain of purple bacteria was described as a new species of the genus Rhodobaca, Rca. barguzinensis sp. nov.     

Anti-tremellomycetes activity of <Emphasis Type="Italic">Cryptococcus pinus</Emphasis> mycocin

The mycocin with a molecular mass of at least 15 kDa secreted by Cryptococcus pinus exhibited fungicidal activity at pH values below 6.5. It was thermolabile and resistant to proteases. In the class Tremellomycetes the species of the orders Filobasidiales and Tremellales are sensitive to this mycocin.     

Thermotolerance and place memory in adult <Emphasis Type="Italic">Drosophila</Emphasis> are independent of natural variation at the <Emphasis Type="Italic">foraging</Emphasis> locus

Natural polymorphisms at the foraging (for) gene influence several behaviors. However, it is seldom clear how different for alleles could be selected. In one case, Drosophila with the rover allele (for ^r ) have higher locomotor activity in the presence of food than animals with the sitter allele (for ^s ), suggesting a complementary feeding strategy. There are, in addition, differences between for ^r and for ^s Drosophila in some tests of short-term memory (for ^r animals generally perform at higher levels) and thermotolerance (for ^s larvae are more resistant to the effects of high-temperature). We asked whether there could be a direct compensating advantages in adult for ^s flies that could maintain the natural for variants. First, are adult for ^s flies more thermotolerant? Second, do for ^r flies have a higher short-term place memory? Third, as an alternative, might for ^s flies have higher place memory? Our results do not confirm these possibilities. Thus, a thermotolerance advantage of for ^s flies does not compensate for a potential for ^r short-term memory advantage; for ^r flies do not have a universal advantage in short-term memory; and for ^s flies do not have an advantage in place memory that could compensate for for ^r advantages in other learning contexts.