A current perspective on insect gene transformation

The genetic transformation of non-drosophilid insects is now possible with several systems, with germ-line transformation reported in published and unpublished accounts for about 12 species using four different transposon vectors. For some of these species, transformation can now be considered routine. Other vector systems include viruses and bacterial symbionts that have demonstrated utility in species and applications requiring transient expression, and for some, the potential exists for genomic integration. Many of these findings are quite recent, presenting a dramatic turning point in our ability to study and manipulate agriculturally and medically important insects. This review discusses these findings from the perspective of all the contributions that has made this technology a reality, the research that has yet to be done for its safe and efficient use in a broader range of species, and an overview of the available methodology to effectively utilize these systems.     

Immunocontraception in wild horses (Equus caballus) extends reproductive cycling beyond the normal breeding season

Background

Although the physiological effects of immunocontraceptive treatment with porcine zona pellucida (PZP) have been well studied, little is known about PZP''s effects on the scheduling of reproductive cycling. Recent behavioral research has suggested that recipients of PZP extend the receptive breeding period into what is normally the non-breeding season.

Methodology/Principal Findings

To determine if this is the case, we compiled foaling data from wild horses (Equus caballus) living on Shackleford Banks, North Carolina for 4 years pre- and 8 years post-contraception management with PZP (pre-contraception, n = 65 births from 45 mares; post-contraception, n = 97 births from 46 mares). Gestation lasts approximately 11–12 months in wild horses, placing conception at approximately 11.5 months prior to birth. Since the contraception program began in January 2000, foaling has occurred over a significantly broader range than it had before the contraception program. Foaling in PZP recipients (n = 45 births from 27 mares) has consistently occurred over a broader range than has foaling in non-recipients (n = 52 births from 19 mares). In addition, current recipients of PZP foaled later in the year than did prior recipient and non-recipient mares. Females receiving more consecutive PZP applications gave birth later in the season than did females receiving fewer applications. Finally, the efficacy of PZP declined with increasing consecutive applications before reaching 100% after five consecutive applications.

Conclusions/Significance

For a gregarious species such as the horse, the extension of reproductive cycling into the fall months has important social consequences, including decreased group stability and the extension of male reproductive behavior. In addition, reproductive cycling into the fall months could have long-term effects on foal survivorship. Managers should consider these factors before enacting immunocontraceptive programs in new populations. We suggest minor alterations to management strategies to help alleviate such unintended effects in new populations.     

Artificial insemination in deer and non-domestic bovids

Artificial insemination technology has revolutionized the domestic cattle breeding industry and allowed for the dissemination of valuable genetics worldwide. This technology has been adapted for use in many other taxa for the conservation of threatened and endangered species, but its use for the genetic management of small populations of deer, antelope and other non-domestic bovids has met numerous challenges and limited success. In practice, adaptation of domestic bovine AI protocols to other artiodactylids for genetic management has been limited by: (1) a lack of understanding of species-specific reproductive characteristics; (2) the inability to minimize handling stress; (3) pregnancy losses; and (4) regulatory challenges in semen importation. To date, AI protocols have been developed for seven species of cervid and 14 species of non-domestic bovids; recent developments in this technology has allowed greater use of AI for dissemination of genetics in farmed deer species. However, despite decades of research in the use of assisted reproduction for the conservation of antelope and other non-domestic bovids, even this simplest technique has not been used repeatedly for genetic management.     

Measuring and comparing the accuracy of species distribution models with presence–absence data

Species distribution models have been widely used to predict species distributions for various purposes, including conservation planning, and climate change impact assessment. The success of these applications relies heavily on the accuracy of the models. Various measures have been proposed to assess the accuracy of the models. Rigorous statistical analysis should be incorporated in model accuracy assessment. However, since relevant information about the statistical properties of accuracy measures is scattered across various disciplines, ecologists find it difficult to select the most appropriate ones for their research. In this paper, we review accuracy measures that are currently used in species distribution modelling (SDM), and introduce additional metrics that have potential applications in SDM. For the commonly used measures (which are also intensively studied by statisticians), including overall accuracy, sensitivity, specificity, kappa, and area and partial area under the ROC curves, promising methods to construct confidence intervals and statistically compare the accuracy between two models are given. For other accuracy measures, methods to estimate standard errors are given, which can be used to construct approximate confidence intervals. We also suggest that as general tools, computer‐intensive methods, especially bootstrap and randomization methods can be used in constructing confidence intervals and statistical tests if suitable analytic methods cannot be found. Usually, these computer‐intensive methods provide robust results.     

Ethnoveterinary medicines used for horses in Trinidad and in British Columbia, Canada

This paper investigates the commonalities in ethnoveterinary medicine used for horses between Trinidad (West Indies) and British Columbia (Canada). These research areas are part of a common market in pharmaceuticals and are both involved in the North American racing circuit. There has been very little research conducted on medicinal plants used for horses although their use is widespread. The data on ethnoveterinary medicines used for horses was obtained through key informant interviews with horse owners, trainers, breeders, jockeys, grooms and animal care specialists in two research areas: Trinidad and British Columbia (BC). A participatory validation workshop was held in BC. An extensive literature review and botanical identification of the plants was also done. In all, 20 plants were found to be used in treating racehorses in Trinidad and 97 in BC. Of these the most-evidently effective plants 19 of the plants used in Trinidad and 66 of those used in BC are described and evaluated in this paper. Aloe vera, Curcuma longaand Ricinus communisare used in both research areas. More research is needed in Trinidad to identify plants that respondents claimed were used in the past. Far more studies have been conducted on the temperate and Chinese medicinal plants used in BC and therefore these ethnoveterinary remedies reflect stronger evidence of efficacy.     

Challenges facing sex preselection of stallion spermatozoa

Since the production of the first live offspring from sex-sorted spermatozoa in 1989, there have been many developments in the fluorescence-activated cell separation (FACS) procedures to preselect X- and Y-chromosome bearing spermatozoa prior to insemination. During this time, FACS technology has been applied to a range of species and has resulted in offspring from rabbits, cattle, sheep, elk and horses. In horses, satisfactory fertility rates have been achieved after hysteroscopic insemination of 20 x 10(6) fresh or stored, sex-sorted spermatozoa. However, many of the sperm processing protocols are still based on the original protocol and components of these procedures may not necessarily be suitable for the stallion. This review examines the details of FACS protocols that have resulted in the production of live offspring and makes comparisons with the published stallion protocols in an attempt to determine how best to improve the fertility of sorted, frozen-thawed stallion spermatozoa.     

Molecular identification methods of fish species: reassessment and possible applications

Fish species identification is traditionally based on external morphological features. Yet, in many cases fishes and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. This work intends to provide an updated and extensive overview on the PCR-methods for fish species identification. Among the ten main methods developed, three PCR-RFLP, PCR-FINS and PCR-specific primers have been the most used. Two other emerging methods, namely real-time PCR and microarray technology, offer new potential for quantification of DNA and simultaneous detection of numerous species, respectively. Almost 500 species have been targeted in the past decade, among which the most studied belong to gadoids, scombroids, and salmonids. The mitochondrial cytochrome b gene was by far the most targeted DNA markers. The most common applications belonged to the forensic, taxonomic, and ecological fields. At last, some key problems, such as the degradation of DNA, the reliability of sequences, and the use of scientific names, likely to be encountered during the development of molecular identification methods are described. In conclusion, the tremendous advances in molecular biology in the past 10 years has rendered possible the study of DNA from virtually any substrates, offering new perspectives for the development of various applications, which will likely continue to increase in the future.     

Overview of sexing sperm

Hundreds of thousands of off springs have been born as a result of AI with sexed sperm. Although this technology has been used for many species, the overwhelming majority of pregnancies have been in cattle, nearly all as a result of sperm that were sexed and subsequently frozen. The technology for sexing sperm has not changed greatly in the past 7 years, but refinements have speeded up the process and reduced damage to sperm. The process of commercialization of sexed sperm has accelerated recently. However, this technology is characterized by high costs, complexity of implementation and lower pregnancy rates than with control sperm. Nevertheless, sexed, frozen bovine sperm are being produced commercially in many countries, although from a limited number of bulls. The main application of sexed sperm to date has been to breed dairy heifers to produce female calves. Because of the slow speed of sexing sperm, fewer sperm are used per insemination dose of sexed than conventional sperm, and pregnancy rates with this product are often only slightly decreased. Successful use of sexed sperm requires excellent management of cattle, careful handling of sperm and use of skilled inseminators. As costs decline, sexed sperm will be used increasingly for cattle breeding, horse breeding and niche applications in other species.     

Molecular approaches: advantages and artifacts in assessing bacterial diversity

Bacteria account for a major proportion of Earth’s biological diversity. They play essential roles in quite diverse environments and there has been an increasing interest in bacterial biodiversity. Research using novel and efficient tools to identify and characterize bacterial communities has been the key for elucidating biological activities with potential for industrial application. The current approach used for defining bacterial species is based on phenotypic and genomic properties. Traditional and novel DNA-based molecular methods are improving our knowledge of bacterial diversity in nature. Advances in molecular biology have been important for studies of diversity, considerably improving our knowledge of morphological, physiological, and ecological features of bacterial taxa. DNA–DNA hybridization, which has been used for many years, is still considered the golden standard for bacteria species identification. PCR-based methods investigating 16S rRNA gene sequences, and other approaches, such as the metagenome, have been used to study the physiology and diversity of bacteria and to identify novel genes with potential pharmaceutical and other biotechnological applications. We examined the advantages and limitations of molecular methods currently used to analyze bacterial diversity; these are mainly based on the 16S rRNA gene. These methods have allowed us to examine microorganisms that cannot be cultivated by routine methods and have also been useful for phylogenetic studies. We also considered the importance of improvements in microbe culture techniques and how we can combine different methods to allow a more appropriate assessment of bacterial diversity and to determine their real potential for industrial applications.     

Criteria for dynamic similarity in bouncing gaits

Animals of different sizes tend to move in a dynamically similar manner when travelling at speeds corresponding to equal values of a dimensionless parameter (DP) called the Froude number. Consequently, the Froude number has been widely used for defining equivalent speeds and predicting speeds of locomotion by extinct species and on other planets. However, experiments using simulated reduced gravity have demonstrated that equality of the Froude number does not guarantee dynamic similarity. This has cast doubt upon the usefulness of the Froude number in locomotion research. Here we use dimensional analysis of the planar spring-mass model, combined with Buckingham's Pi-Theorem, to demonstrate that four DPs must be equal for dynamic similarity in bouncing gaits such as trotting, hopping and bipedal running. This can be reduced to three DPs by applying the constraint of maintaining a constant average speed of locomotion. Sensitivity analysis indicates that all of these DPs are important for predicting dynamic similarity. We show that the reason humans do not run in a dynamically similar manner at equal Froude number in different levels of simulated reduced gravity is that dimensionless leg stiffness decreases as gravity increases. The reason that the Froude number can predict dynamic similarity in Earth gravity is that dimensionless leg stiffness and dimensionless vertical landing speed are both independent of size. In conclusion, although equal Froude number is not sufficient for dynamic similarity, it is a necessary condition. Therefore, to detect fundamental differences in locomotion, animals of different sizes should be compared at equal Froude number, so that they can be as close to dynamic similarity as possible. More generally, the concept of dynamic similarity provides a powerful framework within which similarities and differences in locomotion can be interpreted.     

Systematics of malaria vectors with particular reference to the Anopheles punctulatus group

The appearance of groups and complexes of closely related cryptic or sibling species in many of the anopheline taxa has impeded studies on malaria transmission and the evaluation of control strategies which have relied on morphological characters to identify the vector species involved. The advantages of morphological identification are low cost, speed and simplicity, which allow large numbers of specimens to be processed rapidly in the field. The need for accurate identification is crucial, as time and money may be wasted in studying and controlling species of no medical importance. Various techniques such as cross-mating, chromosome studies and allozyme analysis have been developed to resolve problems of identifying sibling species, though none, as yet, can match the speed and simplicity afforded by morphology markers. The latest of these identification methods comes from advances that have been made in DNA-based technology. Although costly and requiring fairly sophisticated laboratory support, methods such as DNA probe hybridisation and PCR are the quickest and most user-friendly to date. The use of DNA has other advantages in the study of intraspecific differences and in providing characters for phylogenetic studies. This review looks at the development of DNA-based techniques for taxonomic and systematic studies of anopheline mosquitoes. The Anopheles punctulatus group of the southwest Pacific is featured as an example of how this technology has been applied and how it has progressed.     

Label-free detection methods for protein microarrays

With the growth of the "-omics" such as functional genomics and proteomics, one of the foremost challenges in biotechnologies has become the development of novel methods to monitor biological process and acquire the information of biomolecular interactions in a systematic manner. To fully understand the roles of newly discovered genes or proteins, it is necessary to elucidate the functions of these molecules in their interaction network. Microarray technology is becoming the method of choice for such a task. Although protein microarray can provide a high throughput analytical platform for protein profiling and protein-protein interaction, most of the current reports are limited to labeled detection using fluorescence or radioisotope techniques. These limitations deflate the potential of the method and prevent the technology from being adapted in a broader range of proteomics applications. In recent years, label-free analytical approaches have gone through intensified development and have been coupled successfully with protein microarray. In many examples of label-free study, the microarray has not only offered the high throughput detection in real time, but also provided kinetics information as well as in situ identification. This article reviews the most significant label-free detection methods for microarray technology, including surface plasmon resonance imaging, atomic force microscope, electrochemical impedance spectroscopy and MS and their applications in proteomics research.     

Practical applications of the microphotometric quantification of histoenzyme reactions

Summary There are many histochemical reactions which can be used for the accurate topographic detection of enzymes in tissues. There are also many reports of applications of these techniques in which the results have been evaluated subjectively. However, only a few of these methods have been the subject of quantification, using different methodologies, most of them based on microphotometry. A critical analysis of these procedures has generally shown that they are valid either relatively or at an absolute level. Nevertheless, the routine application of these methods is not as widespread as one might expect.In this paper, the methodological developments that have taken place in the histochemical assay of some enzymes by microspectrophotometry are reviewed briefly. The enzymes include acid phosphatase, non-specific esterase, succinate dehydrogenase and cytochrome oxidase. These methods have given consistent and useful results when applied to our experimental models of irradiated skin, and thus seem useful for various other applications.The reasons why there have been so few applications of quantitative histochemistry are also discussed together with possible ways of making its use more general.     

The Parallel Worm Tracker: a platform for measuring average speed and drug-induced paralysis in nematodes

Background

Caenorhabditis elegans locomotion is a simple behavior that has been widely used to dissect genetic components of behavior, synaptic transmission, and muscle function. Many of the paradigms that have been created to study C. elegans locomotion rely on qualitative experimenter observation. Here we report the implementation of an automated tracking system developed to quantify the locomotion of multiple individual worms in parallel.

Methodology/Principal Findings

Our tracking system generates a consistent measurement of locomotion that allows direct comparison of results across experiments and experimenters and provides a standard method to share data between laboratories. The tracker utilizes a video camera attached to a zoom lens and a software package implemented in MATLAB®. We demonstrate several proof-of-principle applications for the tracker including measuring speed in the absence and presence of food and in the presence of serotonin. We further use the tracker to automatically quantify the time course of paralysis of worms exposed to aldicarb and levamisole and show that tracker performance compares favorably to data generated using a hand-scored metric.

Conclusions/Signficance

Although this is not the first automated tracking system developed to measure C. elegans locomotion, our tracking software package is freely available and provides a simple interface that includes tools for rapid data collection and analysis. By contrast with other tools, it is not dependent on a specific set of hardware. We propose that the tracker may be used for a broad range of additional worm locomotion applications including genetic and chemical screening.     

Quantification of human motion: gait analysis-benefits and limitations to its application to clinical problems

The technology supporting the analysis of human motion has advanced dramatically. Past decades of locomotion research have provided us with significant knowledge about the accuracy of tests performed, the understanding of the process of human locomotion, and how clinical testing can be used to evaluate medical disorders and affect their treatment. Gait analysis is now recognized as clinically useful and financially reimbursable for some medical conditions. Yet, the routine clinical use of gait analysis has seen very limited growth. The issue of its clinical value is related to many factors, including the applicability of existing technology to addressing clinical problems; the limited use of such tests to address a wide variety of medical disorders; the manner in which gait laboratories are organized, tests are performed, and reports generated; and the clinical understanding and expectations of laboratory results. Clinical use is most hampered by the length of time and costs required for performing a study and interpreting it. A “gait” report is lengthy, its data are not well understood, and it includes a clinical interpretation, all of which do not occur with other clinical tests. Current biotechnology research is seeking to address these problems by creating techniques to capture data rapidly, accurately, and efficiently, and to interpret such data by an assortment of modeling, statistical, wave interpretation, and artificial intelligence methodologies. The success of such efforts rests on both our technical abilities and communication between engineers and clinicians.     

How do ecologists select and use indicator species to monitor ecological change? Insights from 14 years of publication in Ecological Indicators

Indicator species (IS) are used to monitor environmental changes, assess the efficacy of management, and provide warning signals for impending ecological shifts. Though widely adopted in recent years by ecologists, conservation biologists, and environmental practitioners, the use of IS has been criticized for several reasons, notably the lack of justification behind the choice of any given indicator. In this review, we assess how ecologists have selected, used, and evaluated the performance of the indicator species. We reviewed all articles published in Ecological Indicators (EI) between January 2001 and December 2014, focusing on the number of indicators used (one or more); common taxa employed; terminology, application, and rationale behind selection criteria; and performance assessment methods. Over the last 14 years, 1914 scientific papers were published in EI, describing studies conducted in 53 countries on six continents; of these, 817 (43%) used biological organisms as indicators. Terms used to describe organisms in IS research included “ecological index”, “environmental index”, “indicator species”, “bioindicator”, and “biomonitor,” but these and other terms often were not clearly defined. Twenty percent of IS publications used only a single species as an indicator; the remainder used groups of species as indicators. Nearly 50% of the taxa used as indicators were animals, 70% of which were invertebrates. The most common applications behind the use of IS were to: monitor ecosystem or environmental health and integrity (42%); assess habitat restoration (18%); and assess effects of pollution and contamination (18%). Indicators were chosen most frequently based on previously cited research (40%), local abundance (5%), ecological significance and/or conservation status (13%), or a combination of two or more of these reasons (25%). Surprisingly, 17% of the reviewed papers cited no clear justification for their choice of indicator. The vast majority (99%) of publications used statistical methods to assess the performance of the selected indicators. This review not only improves our understanding of the current uses and applications of IS, but will also inform practitioners about how to better select and evaluate ecological indicators when conducting future IS research.     

The challenges of implementing pathogen control strategies for fishes used in biomedical research

Over the past several decades, a number of fish species, including the zebrafish, medaka, and platyfish/swordtail, have become important models for human health and disease. Despite the increasing prevalence of these and other fish species in research, methods for health maintenance and the management of diseases in laboratory populations of these animals are underdeveloped. There is a growing realization that this trend must change, especially as the use of these species expands beyond developmental biology and more towards experimental applications where the presence of underlying disease may affect the physiology animals used in experiments and potentially compromise research results. Therefore, there is a critical need to develop, improve, and implement strategies for managing health and disease in aquatic research facilities. The purpose of this review is to report the proceedings of a workshop entitled "Animal Health and Disease Management in Research Animals" that was recently held at the 5th Aquatic Animal Models for Human Disease in September 2010 at Corvallis, Oregon to discuss the challenges involved with moving the field forward on this front.     

Multiplex-fluorescence in situ hybridization for chromosome karyotyping

Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by multichannel image-analysis methods. Advantages of M-FISH include rapid analysis of metaphase spreads, even in complex cases with multiple chromosomal rearrangements, and identification of marker chromosomes. The M-FISH technology has been extended to other species, such as the mouse. Furthermore, in addition to painting probes, the method has been used with a variety of region-specific probes. M-FISH has even recently been used for 3D studies to analyze the distribution of human chromosomes in intact and preserved interphase nuclei. Hence, M-FISH has evolved into an essential tool for both clinical diagnostics and basic research. In this protocol, we describe how to use M-FISH to karyotype chromosomes, a procedure that takes approximately 14 d if new M-FISH probes have to be generated and 3 d if the M-FISH probes are ready to use.     

Stochastic properties of generalised Yule models, with biodiversity applications

The Yule model is a widely used speciation model in evolutionary biology. Despite its simplicity many aspects of the Yule model have not been explored mathematically. In this paper, we formalise two analytic approaches for obtaining probability densities of individual branch lengths of phylogenetic trees generated by the Yule model. These methods are flexible and permit various aspects of the trees produced by Yule models to be investigated. One of our methods is applicable to a broader class of evolutionary processes, namely the Bellman-Harris models. Our methods have many practical applications including biodiversity and conservation related problems. In this setting the methods can be used to characterise the expected rate of biodiversity loss for Yule trees, as well as the expected gain of including the phylogeny in conservation management. We briefly explore these applications.