Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO₂ anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO₂ in Drosophila melanogaster. The impact of CO₂ anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.     

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson’s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 MΩ was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.     

Global Invader Impact Network (GIIN): toward standardized evaluation of the ecological impacts of invasive plants

Terrestrial invasive plants are a global problem and are becoming ubiquitous components of most ecosystems. They are implicated in altering disturbance regimes, reducing biodiversity, and changing ecosystem function, sometimes in profound and irreversible ways. However, the ecological impacts of most invasive plants have not been studied experimentally, and most research to date focuses on few types of impacts, which can vary greatly among studies. Thus, our knowledge of existing ecological impacts ascribed to invasive plants is surprisingly limited in both breadth and depth. Our aim was to propose a standard methodology for quantifying baseline ecological impact that, in theory, is scalable to any terrestrial plant invader (e.g., annual grasses to trees) and any invaded system (e.g., grassland to forest). The Global Invader Impact Network (GIIN) is a coordinated distributed experiment composed of an observational and manipulative methodology. The protocol consists of a series of plots located in (1) an invaded area; (2) an adjacent removal treatment within the invaded area; and (3) a spatially separate uninvaded area thought to be similar to pre-invasion conditions of the invaded area. A standardized and inexpensive suite of community, soil, and ecosystem metrics are collected allowing broad comparisons among measurements, populations, and species. The method allows for one-time comparisons and for long-term monitoring enabling one to derive information about change due to invasion over time. Invader removal plots will also allow for quantification of legacy effects and their return rates, which will be monitored for several years. GIIN uses a nested hierarchical scale approach encompassing multiple sites, regions, and continents. Currently, GIIN has network members in six countries, with new members encouraged. To date, study species include representatives of annual and perennial grasses; annual and perennial forbs; shrubs; and trees. The goal of the GIIN framework is to create a standard yet flexible platform for understanding the ecological impacts of invasive plants, allowing both individual and synthetic analyses across a range of taxa and ecosystems. If broadly adopted, this standard approach will offer unique insight into the ecological impacts of invasive plants at local, regional, and global scales.     

Indirect selection of thermal tolerance during experimental evolution of Drosophila melanogaster

Natural selection alters the distribution of a trait in a population and indirectly alters the distribution of genetically correlated traits. Long‐standing models of thermal adaptation assume that trade‐offs exist between fitness at different temperatures; however, experimental evolution often fails to reveal such trade‐offs. Here, we show that adaptation to benign temperatures in experimental populations of Drosophila melanogaster resulted in correlated responses at the boundaries of the thermal niche. Specifically, adaptation to fluctuating temperatures (16–25°C) decreased tolerance of extreme heat. Surprisingly, flies adapted to a constant temperature of 25°C had greater cold tolerance than did flies adapted to other thermal conditions, including a constant temperature of 16°C. As our populations were never exposed to extreme temperatures during selection, divergence of thermal tolerance likely reflects indirect selection of standing genetic variation via linkage or pleiotropy. We found no relationship between heat and cold tolerances in these populations. Our results show that the thermal niche evolves by direct and indirect selection, in ways that are more complicated than assumed by theoretical models.     

Effects of sampling method on foliar δ13C of Leymus chinensis at different scales

Stable carbon isotope composition (δ¹³C) usually shows a negative relationship with precipitation at a large scale. We hypothesized that sampling method affects foliar δ¹³C and its response pattern to precipitation. We selected 11 sites along a precipitation gradient in Inner Mongolia and collected leaves of Leymus chinensis with five or six replications repeatedly in each site from 2009 to 2011. Additionally, we collected leaves of L. chinensis separately from two types of grassland (grazed and fenced) in 2011. Foliar δ¹³C values of all samples were measured. We compared the patterns that foliar δ¹³C to precipitation among different years or different sample sizes, the differences of foliar δ¹³C between grazed and fenced grassland. Whether actual annual precipitation (AAP) or mean annual precipitation (MAP), it was strongly correlated with foliar δ¹³C every year. Significant difference was found between the slopes of foliar δ¹³C to AAP and MAP every year, among the slopes of foliar δ¹³C to AAP from 2009 to 2011. The more samples used at each site the lower and convergent P‐values of the linear regression test between foliar δ¹³C and precipitation. Furthermore, there was significant lower foliar δ¹³C value in presence of grazed type than fenced type grassland. These findings provide evidence that there is significant effect of sampling method to foliar δ¹³C and its response pattern to precipitation of L. chinensis. Our results have valuable implications in methodology for future field sampling studies.     

Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

木瓜籽毛油精炼条件筛选及其理化指标分析

以磷脂含量为指标对木瓜〔Chaenomeles sinensis ( Thouin) Koehne〕籽毛油水化脱胶过程中脱胶剂种类、脱胶剂添加量、脱胶时间、加水量和脱胶温度进行单因素实验,并在此基础上对脱胶时间、加水量和脱胶温度进行L9(33)正交实验;以酸价为指标对碱炼脱酸过程中的碱液(NaOH溶液)浓度、碱炼温度和超碱用量进行单因素实验和L9(33)正交实验;并比较了毛油、脱胶油、脱酸油和精炼油的主要理化指标变化。单因素实验和正交实验结果表明：在木瓜籽毛油水化脱胶过程中采用不同的脱胶剂种类(包括柠檬酸、草酸和蒸馏水)、脱胶剂添加量(质量分数0.1%~0.5%)、脱胶时间(10~70 min)、加水量(质量分数1%~6%)和脱胶温度(65℃~85℃),毛油中的磷脂含量均有明显差异;而碱炼脱酸过程中采用不同的碱液浓度(质量分数6%~14%)、碱炼温度(40℃~80℃)和超碱用量(质量分数0.15%~0.40%),毛油酸价也有明显变化。总体上看,木瓜籽毛油水化脱胶的适宜条件为添加质量分数0.2%柠檬酸为脱胶剂、脱胶温度75℃、加水量为质量分数4%、脱胶时间50 min;碱炼脱酸的适宜条件为碱液浓度为质量分数12%、碱炼温度80℃、超碱用量为质量分数0.30%。理化指标的测定结果表明：与毛油相比,脱胶油、脱酸油和精炼油的碘值略升高但差异不明显、过氧化值明显升高、磷脂含量和皂化值均明显下降,而脱酸油和精炼油的酸价也明显下降。研究结果显示：经过脱胶、脱酸、水洗干燥一系列过程后获得的木瓜籽精炼油的理化指标基本符合国家食用植物油卫生标准。     

Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1 M dimethyl sulfoxide (Me₂SO), 1 M ethylene glycol (EG), 1 M methanol (MeOH) and 0.1 M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and −2 °C for 5 min, 1 h and 3 h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and −2 °C at 1 h and 3 h exposure in each treatment. Heartbeat stage was more tolerant against chilling at −2 °C for 3 h exposure in Me₂SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.     

Quantification methods for Bacillus cereus vegetative cells and spores in the gastrointestinal environment

There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments.