Two small heat shock protein genes in Apis cerana cerana: characterization,regulation, and developmental expression

In the present study, we identified and characterized two small heat shock protein genes from Apis cerana cerana, named AccHsp24.2 and AccHsp23.0. An alignment analysis showed that AccHsp24.2 and AccHsp23.0 share high similarity with other members of the α-crystallin/sHSP family, all of which contain the conserved α-crystallin domain. The recombinant AccHsp24.2 and AccHsp23.0 proteins were shown to have molecular chaperone activity by the malate dehydrogenase thermal aggregation assay. Three heat shock elements were detected in the 5′-flanking region of AccHsp24.2 and eleven in AccHsp23.0, and two Drosophila Broad-Complex genes for ecdysone steroid response sites were found in each of the genes. The presence of these elements suggests that the expression of these genes might be regulated by heat shock and ecdysone, which was confirmed by quantitative RT-PCR (RT-qPCR). The results revealed that the expression of the two genes could be induced by cold shock (4 °C) and heat shock (37 °C and 43 °C) in an analogous manner, and AccHsp24.2 was more susceptible than AccHsp23.0. In addition, the expression of the two genes was induced by high concentrations of ecdysone in vitro and in vivo. The accumulation of AccHsp24.2 and AccHsp23.0 mRNA was also detected in different developmental stages and tissues. In spite of the differential expression at the same stage, these genes shared similar developmental patterns, suggesting that they are regulated by similar mechanisms.     

重金属镉胁迫对果蝇dDnmt2、dMBD2/3表达量的影响

【目的】探明重金属镉(Cadmium,Cd)对黑腹果蝇Drosophila melanogaster DNA甲基化修饰相关基因表达的影响,初步分析镉胁迫可能导致果蝇的表观遗传变异及其可遗传性。【方法】收集8 h内羽化未交配的雌、雄果蝇,在添加不同质量浓度(0、0.9375、1.875、3.75、7.5、15.0、30.0、60.0 mg/kg)Cd的培养基中培养,以Real-time PCR定量检测亲代(F0)果蝇生殖系统、去生殖系统体细胞、整体表达量变化趋势及解除胁迫的子代(F1)果蝇DNA甲基化修饰系统相关基因(dDnmt2、dMBD2/3)在mRNA水平的表达变化。【结果】重金属镉胁迫诱导了果蝇卵巢、精巢、去卵巢雌果蝇、去精巢雄果蝇、完整雌果蝇、完整雄果蝇的dDnmt2、dMBD2/3在mRNA水平的表达上调,呈现一定剂量依赖性及雌、雄组织差异性,且这种表达变化持续至子一代。【结论】研究结果揭示了重金属镉胁迫可诱导果蝇dDnmt2、dMBD2/3表达量上调,其可能与果蝇的DNA甲基化修饰过程相关联,导致表观遗传变异并可能向子代传递。     

Female mate choice predicts paternity success in the absence of additive genetic variance for other female paternity bias mechanisms in Drosophila serrata

After choosing a first mate, polyandrous females have access to a range of opportunities to bias paternity, such as repeating matings with the preferred male, facilitating fertilization from the best sperm or differentially investing in offspring according to their sire. Female ability to bias paternity after a first mating has been demonstrated in a few species, but unambiguous evidence remains limited by the access to complex behaviours, sperm storage organs and fertilization processes within females. Even when found at the phenotypic level, the potential evolution of any mechanism allowing females to bias paternity other than mate choice remains little explored. Using a large population of pedigreed females, we developed a simple test to determine whether there is additive genetic variation in female ability to bias paternity after a first, chosen, mating. We applied this method in the highly polyandrous Drosophila serrata, giving females the opportunity to successively mate with two males ad libitum. We found that despite high levels of polyandry (females mated more than once per day), the first mate choice was a significant predictor of male total reproductive success. Importantly, there was no detectable genetic variance in female ability to bias paternity beyond mate choice. Therefore, whether or not females can bias paternity before or after copulation, their role on the evolution of sexual male traits is likely to be limited to their first mate choice in D. serrata.     

SIFamide and SIFamide Receptor Define a Novel Neuropeptide Signaling to Promote Sleep in Drosophila

SIFamide receptor (SIFR) is a Drosophila G protein-coupled receptor for the neuropeptide SIFamide (SIFa). Although the sequence and spatial expression of SIFa are evolutionarily conserved among insect species, the physiological function of SIFa/SIFR signaling remains elusive. Here, we provide genetic evidence that SIFa and SIFR promote sleep in Drosophila. Either genetic ablation of SIFa-expressing neurons in the pars intercerebralis (PI) or pan-neuronal depletion of SIFa expression shortened baseline sleep and reduced sleep-bout length, suggesting that it caused sleep fragmentation. Consistently, RNA interference-mediated knockdown of SIFR expression caused short sleep phenotypes as observed in SIFa-ablated or depleted flies. Using a panel of neuron-specific Gal4 drivers, we further mapped SIFR effects to subsets of PI neurons. Taken together, these results reveal a novel physiological role of the neuropeptide SIFa/SIFR pathway to regulate sleep through sleep-promoting neural circuits in the PI of adult fly brains.     

Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells

We sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.     

Specific functions of Drosophila amyloid precursor‐like protein in the development of nervous system and nonneural tissues

Drosophila amyloid precursor‐like protein (APPL) is expressed extensively in the nervous system soon after neuronal differentiation. By utilizing different transgenic flies, we studied the physiological function of two APPL protein forms, membrane‐bound form (mAPPL) and secreted form (sAPPL), in neural development. We found that neither deletion nor overexpression of APPL protein altered the gross structure of mushroom bodies in the adult brain. No changes were detected in cell types and their relative ration in embryo‐derived cultures from all APPL mutants. However, the neurite length was significantly increased in mutants overexpressing mAPPL. In addition, mutants lacking sAPPL had numerous neurite branches with abnormal lamellate membrane structures (LMSs) and blebs, while no apoptosis was detected in these neurons. The abnormal neurite morphology was most likely due to the disorganization of the cytoskeleton, as shown by double staining of actin filaments and microtubules. Electrophysiologically, A‐type K⁺ current was significantly enhanced, and spontaneous excitatory postsynaptic potentials (sEPSPs) were greatly increased in APPL mutants lacking sAPPL. Moreover, panneural overexpression of different forms of APPL protein generated different defects of wings and cuticle in adult flies. Taken together, our results suggest that both mAPPL and sAPPL play essential roles in the development of the central nervous system and nonneural tissues. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Does Inbreeding and Loss of Genetic Diversity Decrease Disease Resistance?

Inbreeding and loss of genetic diversity are predicted to decrease the resistance of species to disease. However, this issue is controversial and there is limited rigorous scientific evidence available. To test whether inbreeding and loss of genetic diversity affect a host's resistance to disease, Drosophila melanogasterpopulations with different levels of inbreeding and genetic diversity were exposed separately to (a) thuringiensin, an insecticidal toxin produced by some strains of Bacillus thuringiensis, and (b) live Serratia marcescensbacteria. Inbreeding and loss of genetic diversity significantly reduced resistance of D. melanogasterto both the thuringiensin toxin and live Serratia marcescens. For both, the best fitting relationships between resistance and inbreeding were curvilinear. As expected, there was wide variation among replicate inbred populations in disease resistance. Lowered resistances to both the toxin and the pathogen in inbred populations were due to specific resistance alleles, rather than generalized inbreeding effects, as correlations between resistance and population fitness were low or negative. Wildlife managers should strive to minimise inbreeding and loss of genetic diversity within threatened populations and to minimise exposure of inbred populations to disease.