Babesia divergens: identification and characterization of BdHSP-20, a small heat shock protein

This study describes the identification and characterization of the Babesia divergens α-crystallin/small heat shock protein 20 (BdHSP-20). BdHSP-20 was recognized by the DG7 monoclonal antibody (DG7 mAb) originally produced by Precigout et al. [Precigout, E., Valentin, A., Carcy, B., Gorenflot, A., Nakamura, K., Aikawa, M., Schrevel, J. 1993. Babesia divergens: characterization of a 17-kDa merozoite membrane protein. Experimental Parasitology 77, 425-434] against B. divergens merozoites. We used DG7 mAb to immunoscreen a B. divergens cDNA library to clone the gene encoding the small heat shock protein. Bdhsp-20 is a single copy gene interrupted by one intron. The deduced gene product (BdHSP-20) clearly belongs to the α-crystallin family and shows significant homology to Babesia bovis, Plasmodiumfalciparum and Toxoplasma gondii sHSPs, with the highest degree of sequence identity around the catalytic domain. Nutritient stress (serum depletion) treatment of the parasites induced the upregulation of BdHSP-20 gene expression observed by semi-quantitative PCR and immunoprecipitation. This regulation pattern suggests that BdHSP-20 could probably be of importance for parasite survival in the case of environmental stress. BdHSP-20 has previously been shown to be highly conserved among different strains and antibodies against the protein drastically reduce parasitemia in vitro.     

Effects of exposure to short-term heat stress on male reproductive fitness in a soil arthropod

Ambient temperature is a key environmental factor influencing a variety of aspects of the ecology and evolution of ectotherms. Reproductive traits have been suggested to be more sensitive to thermal stress than other life history traits. This study investigated the direct and indirect effects of heat shock on male reproductive success in the widespread springtail Orchesella cincta. Male springtails were exposed to four temperature treatments: heat hardening (35.2 °C for 1 h), heat shock (37.2 °C for 1 h), heat hardening + heat shock (35.2 °C for 1 h, followed 15 h later by 37.2 °C for 1 h), and control (20 °C). The heat shock gene Hsp70 showed high expression in all the heat treatments, indicating that the treatments indeed induced thermal stress. Significant mortality was only found in the treatment with heat shock, both with and without heat hardening. A direct effect of heat treatment was found on time to first reproduction, which was significantly longer after heat shock (with or without heat hardening) than in the control treatment. There was no difference among treatments in the number of spermatophores produced in the first reproductive instar. Heat treatment also had indirect effects on male reproductive success. Females chose significantly more spermatophores from control males than from males that received heat shock, heat hardening or both. A high percentage of spermatophores produced by heat shocked males caused reproductive failure in females, but no significant differences among treatments were found.Our results suggest that not all traits were equally affected by the heat stress. Heat hardening did not protect reproductive traits against the negative effects of heat shock. The indirect effects of heat shock on reproduction may be equally important as the direct effects.     

Structural and Mechanical Hierarchies in the α-Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.     

Differential expression of two small Hsps during diapause in the corn stalk borer Sesamia nonagrioides (Lef.)

We isolated and characterized two members of the α-crystallin/sHsp family, SnoHsp19.5 and SnoHsp20.8 from Sesamia nonagrioides (Lepidoptera: Noctuidae). The cDNAs encoded proteins of 174 and 185 amino acids, with calculated molecular weights of 19.5 and 20.8 kDa, respectively. The deduced amino acid sequences of SnoHsp19.5 and SnoHsp20.8 showed highest homology to Hsp19.7 of Mamestra brassicae and to Bombyx mori Hsp20.4, respectively. Expression patterns of SnoHsp19.5 and SnoHsp20.8 in non-diapausing individuals under different environmental conditions (heat or cold) showed different accumulation profiles for the two genes after heat and cold treatment. SnoHsp19.5 was consistently expressed, while SnoHsp20.8 gene was down-regulated in deep diapause and was up-regulated at the termination of diapause. Our results suggest that these two genes play distinctive roles in the regulation of diapause.     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Thermal tolerance,net CO2 exchange and growth of a tropical tree species, Ficus insipida, cultivated at elevated daytime and nighttime temperatures

Global warming and associated increases in the frequency and amplitude of extreme weather events, such as heat waves, may adversely affect tropical rainforest plants via significantly increased tissue temperatures. In this study, the response to two temperature regimes was assessed in seedlings of the neotropical pioneer tree species, Ficus insipida. Plants were cultivated in growth chambers at strongly elevated daytime temperature (39 °C), combined with either close to natural (22 °C) or elevated (32 °C) nighttime temperatures. Under both growth regimes, the critical temperature for irreversible leaf damage, determined by changes in chlorophyll a fluorescence, was approximately 51 °C. This is comparable to values found in F. insipida growing under natural ambient conditions and indicates a limited potential for heat tolerance acclimation of this tropical forest tree species. Yet, under high nighttime temperature, growth was strongly enhanced, accompanied by increased rates of net photosynthetic CO₂ uptake and diminished temperature dependence of leaf-level dark respiration, consistent with thermal acclimation of these key physiological parameters.     

A novel 1-Cys thioredoxin peroxidase gene in Apis cerana cerana: characterization of AccTpx4 and its role in oxidative stresses

Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H₂O₂, and HgCl₂. The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H₂O₂ in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.     

Hemolymph proteome changes during worker brood development match the biological divergences between western honey bees (Apis mellifera) and eastern honey bees (Apis cerana)

Background

Hemolymph plays key roles in honey bee molecule transport, immune defense, and in monitoring the physiological condition. There is a lack of knowledge regarding how the proteome achieves these biological missions for both the western and eastern honey bees (Apis mellifera and Apis cerana). A time-resolved proteome was compared using two-dimensional electrophoresis-based proteomics to reveal the mechanistic differences by analysis of hemolymph proteome changes between the worker bees of two bee species during the larval to pupal stages.

Results

The brood body weight of Apis mellifera was significantly heavier than that of Apis cerana at each developmental stage. Significantly, different protein expression patterns and metabolic pathways were observed in 74 proteins (166 spots) that were differentially abundant between the two bee species. The function of hemolymph in energy storage, odor communication, and antioxidation is of equal importance for the western and eastern bees, indicated by the enhanced expression of different protein species. However, stronger expression of protein folding, cytoskeletal and developmental proteins, and more highly activated energy producing pathways in western bees suggests that the different bee species have developed unique strategies to match their specific physiology using hemolymph to deliver nutrients and in immune defense.

Conclusions

Our disparate findings constitute a proof-of-concept of molecular details that the ecologically shaped different physiological conditions of different bee species match with the hemolymph proteome during the brood stage. This also provides a starting point for future research on the specific hemolymph proteins or pathways related to the differential phenotypes or physiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-563) contains supplementary material, which is available to authorized users.