Artificial and factitious foods support the development and reproduction of the predatory mite Amblyseius swirskii

The generalist predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) was reared on Ephestia kuehniella Zeller eggs (Lepidoptera: Pyralidae), decapsulated dry cysts of the brine shrimp Artemia franciscana Kellogg (Anostraca: Artemiidae), and on meridic artificial diets (composed of honey, sucrose, tryptone, yeast extract, and egg yolk) supplemented with pupal hemolymph of the Chinese oak silkworm Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae) (AD1), with E. kuehniella eggs (AD2) or with A. franciscana cysts (AD3). Development, reproduction and predation capacity of the predatory mites were assessed in the first (G1) and sixth generation (G6) of rearing on the different diets. Immature survival rates in G1 were similar on all diets (96.8–100 %). After six generations, however, survival of A. swirskii was significantly reduced on all diets except on A. franciscana cysts. Oviposition rates did not differ between generations when females were fed on E. kuehniella, AD2 or AD3. The total number of deposited eggs was similar among diets except in G6 where the females fed on A. franciscana cysts produced more eggs than those maintained on E. kuehniella eggs. On most diets the intrinsic rates of increase in G1 were superior to those in G6, except for predators supplied with A. franciscana cysts where no differences were observed among generations. Female mites did not lose their capacity to kill first instar Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) after six generations on the different diets, but predation rates in G6 on E. kuehniella were lower than in G1. In conclusion, the different factitious and artificial diets tested in the present study supported the development and reproduction of A. swirskii for a single generation but fitness losses occurred to a varying degree after several generations on E. kuehniella eggs or the artificial diets. Artificial diet enriched with A. franciscana cysts yielded better results than the other artificial diets. Amblyseius swirskii performed best on decapsulated Artemia cysts indicating their potential for use in the mass production of the predator or to sustain its populations in the crop after release.     

Detection of tetrodotoxin-producing Providencia rettgeri T892 in Lagocephalus pufferfish

Tetrodotoxin (TTX) is a potent toxin but it could be used in pharmaceutical field. Identification of TTX producing bacteria in pufferfish is necessary for TTX yield and the pufferfish conservation. In this study, Lagocephalus was collected from Cam Ranh Sea, a central part of Vietnam during spring season. The liver and intestine were incubated in 0.9 % NaCl for TTX detection in pufferfish. To be benefited from the isolation of new TTX producing bacteria, the liver and intestine were incubated in 6.5 % NaCl. The cultures were used to test the toxin and to isolate the bacterial community that could yield TTX. Surprisingly, Providencia rettgeri T892 in intestine could produce TTX identified by biochemical test and 16S rRNA sequencing. This strain was used to test the production of TTX, based on thin layer chromatography (TLC), mouse bioassay and high performance liquid chromatography (HPLC) analysis. The bacterium was optimized for TTX production in media prepared from the meat-washing water of Auxis thazard, Megalaspis cordyla and Decapterus maruadsi. Interestingly, the TTX obtained 0.106 mg/mL and 0.055 mg/mL in medium prepared from A. thazard and M. cordyla, respectively while there was no TTX production detected in medium prepared from D. maruadsi. This paper could contribute to warn to the human health care system about a possible TTX poisoning in some cases related to eating fishes.     

The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

Developmental genetic studies of aminopeptidases in Drosophila melanogaster

Summary TheLap-D locus ofDrosophila melanogaster was investigated. The mode of inheritance of the alleles at this locus was confirmed and a new allele was discovered. The enzymes are not exclusively pupal enzymes but are found throughout the life cycle. Amino acid naphthylamides other than leucine naphthylamide were found to be effective substrates for these enzymes. It is suggested, therefore, that the restrictive designation leucine aminopeptidase be abandoned in favor of the more general amino acid naphthylamidase or aminopeptidase.This work was supported by the U. S. Atomic Energy Commission under contract No. AT (11-1) 1338.     

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.     

The application of nanosecond fluorometry to an allosteric protein

The fluorescent probe 1-anilino-8-naphthalene sulfonate, when bound by the inactive form of phosphorylase b, behaves as if distributed between two different binding sites, whose bound dye emits fluorescence with decay times of 19 and 8 nanosecs. In 0.1 M glycylglycine, pH 7.0, the addition of the allosteric activator adenosine-5′-phosphate or the substrate glucose-1-phosphate results in the progressive loss of the component of longer decay time. At intermediate levels of activator the decay curves could be represented as the sum of the weighted contributions of two species, corresponding to those prevailing in the absence of activator and in excess activator.The activity of glycogen phosphorylase b, which is one of the most extensively studied allosteric enzymes, is controlled by the allosteric activator AMP (1, 2). Binding studies have shown that the binding of AMP in the absence of substrate is strongly cooperative, with a value of the Hill coefficient of 1.7 (2). Binding and kinetic studies have indicated that strong heterotropic cooperativity exists between the combination of the enzyme with AMP and either of the substrates glucose-1-phosphate and inorganic phosphate (1, 2, 3, 4).Phosphorylase b (molecular weight 185,000) consists of two equivalent subunits of molecular weight 92,500. Each subunit contains a single strong binding site for AMP(1, 2, 3).The AMP-induced activation of phosphorylase b has usually been attributed to a conformational transition, AMP being preferentially bound by the active form (1). The high degree of cooperativity of AMP binding has been attributed to the simultaneous conversion of both subunits to the active conformation upon the binding of a molecule of AMP by one subunit (1, 2).The existing kinetic studies have been interpreted as indicating that the binding of substrate likewise favors the transition to the active form, although differences of opinion exist as to the number and equivalence of the active states (1, 2, 5). However, a basic weakness of the model described above is the paucity of direct physical evidence for a conformational change.In recent years a rapid evolution of technique has made it possible to monitor the time decay of fluorescence directly (6). The technique of nanosecond fluorometry has been applied to a number of systems with the objective of analyzing complex decay curves or detecting excited state reactions (7).With the development of programs for analyzing multicomponent decay curves in terms of the lifetimes and amplitudes of the individual emitting species (8) it has become possible to apply nanosecond fluorometry to the problem of monitoring conformational transitions of proteins, and in particular, to the activator - or substrate - induced transformations of allosteric proteins.In general, when emission occurs from a collection of sources with different decay times, we have for the fluorescence intensity i (t) as a function of time, t:

(1)$\text{i(t) =}\text{∑}\text{j}\text{α}{}_{\mathrm{j}}\text{,}\text{e}\text{,}{}^{\mathrm{<mtext>-t</mtext><mtext>\tau </mtext>}}\text{j}$

where α_j and τ_j are the amplitude and decay time, respectively, corresponding to the jth component. If the number of components is small, the experimental curves of i(t) versus t may, in principle, be analyzed by the method of moments to yield the set of values of α_j and τ_j.In this report, we shall describe the results of the application of nanosecond fluorometry to the phosphorylase b system, using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). This probe, which is virtually non-fluorescent in aqueous solution, acquires an intense fluorescence when bound by a non-polar site on a protein (9, 10).In an earlier report, Seery and Anderson have found that the fluorescence intensity of ANS bound to phosphorylase b is sharply reduced in the presence of AMP or substrates. From parallel equilibrium dialysis studies, it was concluded that the greater part of the effect arose from the dissociation of ANS, although it was not possible to demonstrate a quantitative correlation (11). For this reason, it was not possible to make any statement as to the quantum yield or excited lifetime of the residual bound dye.