Evaluation of marking and tagging methods for genetic studies in carp

A variety of marking and tagging methods were tested on common carp (Cyprinus carpio L.) with the aim of identifying suitable methods for genetic studies in this and other species of carp. Elastomer and Alcian blue dye marking; Cold and Silver nitrate branding; Floy, Fingerling, Carlin disc and visible implant tags; and fin clipping were all tested on a range of sizes of common carp (from mean weights of 10–25 g up to 600–800 g). The branding and tagging methods tested did not give satisfactory retention rates. A combination of elastomer marking and fin clipping was then tested as a method for strain identification in a growth comparison trial on catla (Catla catla Hamilton) and found to be satisfactory for this purpose. Passive integrated transponders (PIT) tags were used to individually identify catla of wild or hatchery origin being grown for use as broodstock. These had almost 100% (98.8%) retention rates, but are expensive compared to most other tagging methods.     

Biotin tagging coupled with amino acid‐coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade‐offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid‐coded mass tagging, for highly sensitive and accurate screening of mammalian protein–protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin‐tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled‐down complex amino acid‐coded mass tagging serves as “in‐spectra” quantitative markers to distinguish those bait‐specific interactors from non‐specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid‐coded mass tagging approach, we first biotin‐tagged in vivo a multi‐functional protein family member, 14‐3‐3ε, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14‐3‐3ε were identified in high confidence.     

Clipping defoliation and nitrogen addition shift competition between a C3 grass (Leymus chinensis) and a C4 grass (Hemarthria altissima)

• Human‐induced disturbances, including grazing and clipping, that cause defoliation are common in natural grasslands. Plant functional type differences in the ability to compensate for this tissue loss may influence interspecific competition.
• To explore the effects of different intensities of clipping and nitrogen (N) addition on compensatory growth and interspecific competition, we measured accumulated aboveground biomass (AGB), belowground biomass (BGB), tiller number, non‐structural carbohydrates concentrations and leaf gas exchange parameters in two locally co‐occurring species (the C₃ grass Leymus chinensis and the C₄ grass Hemarthria altissima) growing in monoculture and in mixture.
• For both grasses, the clipping treatment had significant impacts on the accumulated AGB, and the 40% clipping treatment had the largest effect. BGB gradually decreased with increasing defoliation intensity. Severe defoliation caused a significant increase in tiller number. Stored carbohydrates in the belowground biomass were mobilised and transported aboveground for the growth of new leaves to compensate for clipping‐induced injury. The net CO₂ assimilation rate (A) of the remaining leaves increased with clipping intensity and peaked under clipping intensities of 20% or 40%. Nitrogen addition, at a rate of 10 g·N·m^?2·year^?1, enhanced A of the remaining leaves and non‐structural carbohydrate concentrations, which benefited plant compensatory growth, especially for the C₃ grass. Under the mixed planting conditions, the clipping and N addition treatments lowered the competitive advantage of the C₄ grass.
• The results suggest that a combination of defoliation and N deposition have the potential to benefit the coexistence of C₃ and C₄ grasses.

     

Photo‐identification as a simple tool for studying invasive lionfish Pterois volitans populations

Photo‐tagging, i.e. using a specific software to match colour patterns on photographs, was tested as a means to identify individual Indo‐Pacific Pterois volitans to assist with population and movement studies of this invasive species. The stripe pattern on the flank of adult P. volitans (n = 48) was the most individually distinctive of three body regions tested, leading to correct individual identification on 68 and 82% of tests with a single and two images of the reference individual, respectively. Photo‐tagging is inexpensive, logistically simple and can involve citizen scientists, making it a viable alternative to traditional tagging to provide information on P. volitans distribution, movement patterns and recolonization rates after removals.     

Association study of FOXO3A SNPs and aging phenotypes in Danish oldest‐old individuals

FOXO3A variation has repeatedly been reported to associate with human longevity, yet only few studies have investigated whether FOXO3A variation also associates with aging‐related traits. Here, we investigate the association of 15 FOXO3A tagging single nucleotide polymorphisms (SNPs) in 1088 oldest‐old Danes (age 92–93) with 4 phenotypes known to predict their survival: cognitive function, hand grip strength, activity of daily living (ADL), and self‐rated health. Based on previous studies in humans and foxo animal models, we also explore self‐reported diabetes, cancer, cardiovascular disease, osteoporosis, and bone (femur/spine/hip/wrist) fracture. Gene‐based testing revealed significant associations of FOXO3A variation with ADL (P = 0.044) and bone fracture (P = 0.006). The single‐SNP statistics behind the gene‐based analysis indicated increased ADL (decreased disability) and reduced bone fracture risk for carriers of the minor alleles of 8 and 10 SNPs, respectively. These positive directions of effects are in agreement with the positive effects on longevity previously reported for these SNPs. However, when correcting for the test of 9 phenotypes by Bonferroni correction, bone fracture showed borderline significance (P = 0.054), while ADL did not (P = 0.396). Although the single‐SNP associations did not formally replicate in another study population of oldest‐old Danes (n = 1279, age 94–100), the estimates were of similar direction of effect as observed in the Discovery sample. A pooled analysis of both study populations displayed similar or decreased sized P‐values for most associations, hereby supporting the initial findings. Nevertheless, confirmation in additional study populations is needed.     

Radio‐transmitters do not affect seasonal productivity of female Golden‐winged Warblers

Investigating the potential effects of handling and marking techniques on study animals is important for correct interpretation of research results and to effect progress in data‐collection methods. Few investigators have compared the reproductive output of radio‐tagged and non‐radio‐tagged songbirds, and no one to date has examined the possible effect of radio‐tagging adult songbirds on the survival of their fledglings. In 2011 and 2012, we compared several parameters of reproductive output of two groups of female Golden‐winged Warblers (Vermivora chrysoptera) breeding in Minnesota, including 45 females with radio‐transmitters and 73 females we did not capture, handle, or mark. We found no difference between groups in clutch sizes, hatching success, brood sizes, length of incubation and nestling stages, fledging success, number of fledglings, or survival of fledglings to independence. Thus, radio‐tags had no measurable impact on the productivity of female Golden‐winged Warblers. Our results build upon previous studies where investigators have reported no effects of radio‐tagging on the breeding parameters of songbirds by also demonstrating no effect of radio‐tagging through the post‐fledging period and, therefore, the entire breeding season.     

Extirpation or Coexistence? Management of a Persistent Introduced Grass in a Prairie Restoration

Abstract Introduced perennial grasses are one of the greatest constraints to prairie restoration. Herbicides suppress but do not eliminate introduced grasses, so we explored the interaction of herbicide with two additional controls: heavy clipping (to simulate grazing) and competition from native species. A 50‐year‐old stand of the introduced perennial grass Agropyron cristatum (crested wheatgrass) in the northern Great Plains was seeded with native grasses and treated with herbicide annually for 7 years in a factorial experiment. Clipping was applied as a subplot treatment in the final 3 years. Both herbicide and clipping significantly reduced the cover of A. cristatum, but clipping produced an immediate and consistent decrease, whereas herbicide control varied among years. The cover of A. cristatum decreased significantly with increasing cover of a seeded native grass, Bouteloua gracilis (blue grama), suggesting that both top‐down (i.e., grazing) and bottom‐up (i.e., resource competition) strategies can contribute to A. cristatum control. No treatment had any effect on the seed bank of A. cristatum. Even in the most effective control treatments, A. cristatum persisted at low amounts (approximately 5% cover) throughout the experiment. The cover of B. gracilis increased significantly with seed addition and herbicide, and, after 7 years, was similar to that in undisturbed prairie. The total cover of native species increased significantly with clipping and herbicide, and species richness was significantly higher in plots receiving herbicide. Clipping season had no effect on any variable. In summary, no method extirpated A. cristatum, but clipping reduced its cover by 90% and doubled the cover of native species. Extirpation might not be a realistic goal, but relatively simple management allowed coexistence of native species.     

Drought mildly reduces plant dominance in a temperate prairie ecosystem across years

1. Shifts in dominance and species reordering can occur in response to global change. However, it is not clear how altered precipitation and disturbance regimes interact to affect species composition and dominance.
2. We explored community‐level diversity and compositional similarity responses, both across and within years, to a manipulated precipitation gradient and annual clipping in a mixed‐grass prairie in Oklahoma, USA. We imposed seven precipitation treatments (five water exclusion levels [?20%, ?40%, ?60%, ?80%, and ?100%], water addition [+50%], and control [0% change in precipitation]) year‐round from 2016 to 2018 using fixed interception shelters. These treatments were crossed with annual clipping to mimic hay harvest.
3. We found that community‐level responses were influenced by precipitation across time. For instance, plant evenness was enhanced by extreme drought treatments, while plant richness was marginally promoted under increased precipitation.
4. Clipping promoted species gain resulting in greater richness within each experimental year. Across years, clipping effects further reduced the precipitation effects on community‐level responses (richness and evenness) at both extreme drought and added precipitation treatments.
5. Synthesis: Our results highlight the importance of studying interactive drivers of change both within versus across time. For instance, clipping attenuated community‐level responses to a gradient in precipitation, suggesting that management could buffer community‐level responses to drought. However, precipitation effects were mild and likely to accentuate over time to produce further community change.

     

Two proteolytic modules are involved in regulated intramembrane proteolysis of Bacillus subtilis RsiW

Stress‐induced degradation of the Bacillus subtilis anti‐sigma factor RsiW results in the induction of genes controlled by the extracytoplasmic function sigma factor σ^W. RsiW is cleaved by the mechanism of regulated intramembrane proteolysis at site‐1 and ‐2 by PrsW and RasP respectively, and is then further degraded by cytoplasmic Clp peptidases. In a reconstituted Escherichia coli system, PrsW removes 40 amino acids from RsiW by cleaving between Ala168 and Ser169 of the extracytoplasmic domain, thereby generating RsiW‐S1. Further trimming of RsiW‐S1's C‐terminus by the periplasmic tail‐specific protease Tsp is crucial for subsequent RasP‐catalysed clipping. In B. subtilis, mutation of RsiW at Ala168 severely impairs site‐1 processing. RsiW‐S1 is undetectable in wild‐type B. subtilis and knockout strains lacking various extracytoplasmic proteases. While it can be stabilized by C‐terminal tagging, even this fusion protein is still attacked. Thus, several peptidases seem to be involved in trimming of RsiW downstream of PrsW and upstream of RasP in B. subtilis. Overall, the RsiW degradation pathway can be subdivided into two modules each consisting of a site‐specific peptidase that prepares RsiW for further degradation by downstream proteases.     

Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide‐1‐MIMETIBODYTM: Factors that influence productivity and product quality

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide‐1‐antibody fusion protein (also known as a Glucagon like peptide‐1 MIMETIBODY^TM), we have noted that the N‐terminal GLP‐1 portion of the MIMETIBODY^TM was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O‐linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N‐terminal clipping had increased level of O‐linked glycosylation in the linker region, giving credence to the hypothesis that O‐linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N‐terminal clipping had longer in vivo serum half‐life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules. Biotechnol. Bioeng. 2009;103: 162–176. © 2008 Wiley Periodicals, Inc.     

Local pre‐adaptation to disturbance and inbreeding–environment interactions affect colonisation abilities of diploid and tetraploid Centaurea stoebe

• Primary colonisation in invasive ranges most commonly occurs in disturbed habitats, where anthropogenic disturbance may cause physical damage to plants. The tolerance to such damage may differ between cytotypes and among populations as a result of differing population histories (adaptive differentiation between ruderal verus natural habitats). Moreover, founder populations often experience inbreeding depression, the effects of which may increase through physical damage due to inbreeding–environment interactions. We aimed to understand how such colonisation processes differ between diploid and tetraploid Centaurea stoebe populations, with a view to understanding why only tetraploids are invasive.
• We conducted a clipping experiment (frequency: zero, once or twice in the growing season) on inbred versus outbred offspring originating from 37 C. stoebe populations of varying cytotype, range and habitat type (natural versus ruderal). Aboveground biomass was harvested at the end of the vegetation period, while re‐sprouting success was recorded in the following spring.
• Clipping reduced re‐sprouting success and biomass, which was significantly more pronounced in natural than in ruderal populations. Inbreeding depression was not detected under benign conditions, but became increasingly apparent in biomass when plants were clipped. The effects of clipping and inbreeding did not differ between cytotypes.
• Adaptive differentiation in disturbance tolerance was higher among populations than between cytotypes, which highlights the potential of pre‐adaptation in ruderal populations during early colonisation on anthropogenically disturbed sites. While the consequences of inbreeding increased through clipping‐mediated stress, they were comparable between cytotypes, and consequently do not contribute to understanding the cytotype shift in the invasive range.

     

Effects of double bands on Magellanic Penguins

ABSTRACT Banding penguins is controversial because bands can alter the survival, reproduction, and behavior of marked individuals. The effects of bands are not consistent among band types and, although stainless steel is thought to be better than other materials, tests of the long‐term impact of bands on tag‐loss rates and the reproduction and survival of individuals are needed. We tested three types of external tags on Magellanic Penguins (Spheniscus magellanicus) to measure band effects and tag‐loss rates. In 1993, we double‐tagged 300 penguins with aluminum flipper bands, stainless‐steel flipper bands, or small (2 mm × 10 mm) metal tags attached to foot webbing. We searched for double‐tagged birds for 13 of 15 yrs (1994–2008). Aluminum bands deformed, caused feather wear, injured and killed some penguins, and were lost more often than stainless‐steel bands or web tags. During the first 2 yrs of our study, at least nine penguins lost one aluminum band (N= 71 penguins resighted), but no penguins lost a stainless‐steel band (N= 84) or a web tag (N= 88). During the next 13 yrs, five penguins lost one of their two web tags (N= 89), but none lost a stainless‐steel band (N= 84). Females laid eggs of similar size before they carried a band and in the year following tagging (P= 0.09). The type of tags a female carried did not significantly change egg size (P > 0.22). During the first breeding season after tagging, penguins with aluminum bands had lower reproductive success than penguins with stainless‐steel bands or web‐tags (P= 0.04). The annual survival of females with two stainless‐steel bands was lower (0.79) than that of males with two stainless‐steel bands or males and females with two web‐tags (0.87). Aluminum bands injured Magellanic Penguins, were lost at high rates, and should not be used. Double stainless‐steel bands had no apparent effects on adult male Magellanic Penguins, but reduced survival rates of adult females. A single stainless‐steel band would likely have less impact than two bands, and our results suggest that the impact of a single band would be difficult to measure.     

Assessment of flipper tag site healing in gray seal pups using thermography

Infrared thermography was used to monitor the healing process at flipper tag sites in gray seal (Halichoerus grypus) pups. We tested the hypothesis that tagging would result in a rise in surface temperature associated with tag site healing processes compared with adjacent untagged areas of the flipper. Prior to tagging thermal images were recorded of the dorsal side of hind flippers of pups tagged in early lactation (n= 20) and at weaning (n= 19) on the Isle of May, Scotland (56°11′N, 02°33′W) from October to December 2008. Pups tagged in early lactation were sampled again at late lactation, at weaning and then every 3 d for an average of 29 d post‐tagging while pups tagged at weaning were sampled every 3 d for an average of 17 d post‐tagging. Tag sites were also scored for signs of infection or swelling at each sampling. Results showed that (1) small temperature increases associated with wound healing processes around the tag site returned to pre‐tagging levels before animals leave the island and (2) there was little evidence of tagging‐related infections or tag loss irrespective of age at tagging.     

Visible implant elastomer (VIE) tagging and simulated predation risk elicit similar physiological stress responses in three‐spined stickleback Gasterosteus aculeatus

The effect of visible implant elastomer (VIE) tagging on the immediate physiological stress response was tested in female three‐spined stickleback Gasterosteus aculeatus, using non‐invasive waterborne cortisol analysis. Post‐tagging cortisol levels were significantly higher compared with pretreatment baseline concentrations; however, when comparing post‐tagging cortisol levels with cortisol levels after exposure to a simulated aerial predator, no significant differences were found. This study indicates that VIE tagging elicits a physiological stress response similar to those occurring in the everyday lives of this important biological model organism.     

Contrasting responses of heterotrophic and autotrophic respiration to experimental warming in a winter annual‐dominated prairie

Understanding how soil respiration (Rs) and its source components respond to climate warming is crucial to improve model prediction of climate‐carbon (C) feedback. We conducted a manipulation experiment by warming and clipping in a prairie dominated by invasive winter annual Bromus japonicas in Southern Great Plains, USA. Infrared radiators were used to simulate climate warming by 3 °C and clipping was used to mimic yearly hay mowing. Heterotrophic respiration (Rh) was measured inside deep collars (70 cm deep) that excluded root growth, while total soil respiration (Rs) was measured inside surface collars (2–3 cm deep). Autotrophic respiration (Ra) was calculated by subtracting Rh from Rs. During 3 years of experiment from January 2010 to December 2012, warming had no significant effect on Rs. The neutral response of Rs to warming was due to compensatory effects of warming on Rh and Ra. Warming significantly (P < 0.05) stimulated Rh but decreased Ra. Clipping only marginally (P < 0.1) increased Ra in 2010 but had no effect on Rh. There were no significant interactive effects of warming and clipping on Rs or its components. Warming stimulated annual Rh by 22.0%, but decreased annual Ra by 29.0% across the 3 years. The decreased Ra was primarily associated with the warming‐induced decline of the winter annual productivity. Across the 3 years, warming increased Rh/Rs by 29.1% but clipping did not affect Rh/Rs. Our study highlights that climate warming may have contrasting effects on Rh and Ra in association with responses of plant productivity to warming.     

In situ tagging technique for fishes provides insight into growth and movement of invasive lionfish

Information on fish movement and growth is primarily obtained through the marking and tracking of individuals with external tags, which are usually affixed to anesthetized individuals at the surface. However, the quantity and quality of data obtained by this method is often limited by small sample sizes owing to the time associated with the tagging process, high rates of tagging‐related mortality, and displacement of tagged individuals from the initial capture location. To address these issues, we describe a technique for applying external streamer and dart tags in situ, which uses SCUBA divers to capture and tag individual fish on the sea floor without the use of anesthetic. We demonstrate this method for Indo‐Pacific lionfish (Pterois volitans/P. miles), species which are particularly vulnerable to barotrauma when transported to and handled at the surface. To test our method, we tagged 161 individuals inhabiting 26 coral reef locations in the Bahamas over a period of 3 years. Our method resulted in no instances of barotrauma, reduced handling and recovery time, and minimal post‐tagging release displacement compared with conventional ex situ tag application. Opportunistic resighting and recapture of tagged individuals reveals that lionfish exhibit highly variable site fidelity, movement patterns, and growth rates on invaded coral reef habitats. In total, 24% of lionfish were resighted between 29 and 188 days after tagging. Of these, 90% were located at the site of capture, while the remaining individuals were resighted between 200 m and 1.1 km from initial site of capture over 29 days later. In situ growth rates ranged between 0.1 and 0.6 mm/day. While individuals tagged with streamer tags posted slower growth rates with increasing size, as expected, there was no relationship between growth rate and fish size for individuals marked with dart tags, potentially because of large effects of tag presence on the activities of small bodied lionfish (i.e., <150 mm), where the tag was up to 7.6% of the lionfish's mass. Our study offers a novel in situ tagging technique that can be used to provide critical information on fish site fidelity, movement patterns, and growth in cases where ex situ tagging is not feasible.     

Con‐specific neighbours may enhance compensation capacity in an invasive plant

Facilitation, both by inter‐ and intra‐specific neighbours, is known to be an important process in structuring plant communities. However, only a small number of experiments have been reported on facilitation in plant invasions, especially between invasive con‐specific individuals. Here, we focus on how con‐specific neighbours of the invasive alien plant alligator weed affect the tolerance of alligator weed to herbivory by the introduced biological control agent, Agasicles hygrophila. We conducted greenhouse and garden experiments in which invasive plant density and herbivory intensity (artificial clipping and real herbivory) were manipulated. In the greenhouse experiment, artificial clipping significantly reduced plant biomass when plants were grown individually, but when con‐specific neighbours were present in the same pot, biomass was not significantly different from control plants. Similarly, when compared to control plants, plants that were subjected to herbivory by A. hygrophila produced more biomass when grown with two con‐specific neighbours than when grown alone. Real herbivory also resulted in an increased number of vegetative buds, and again when two con‐specific neighbours were present this effect was increased (a 55.3% increase in buds when there was no neighbour, but a 111.6% increase in buds when two con‐specific neighbours were present). In the garden experiment, in which plants were grown at high density (6 plants per pot), alligator weed fully recovered from defoliation caused by insects at levels from 20–30% to 100%. Our results indicate that the con‐specific association may increase the compensatory ability to cope with intense damage in this invasive plant.     

Effects of repeated damage and fertilization on palatability of Vaccinium myrtillus to grey sided voles, Clethrionomys rufocanus

In this study we examined the responses of the dominant understorey plant species, Vaccinium myrtillus, in a Swedish boreal forest to nitrogen applications and repeated damage by clipping. Four years of clipping V. myrtillus reduced its abundance, regardless of whether the clipping was combined with fertilization or not. The treatments also induced changes in growth form and concentration of phenolic compounds in the shoots. Repeated damage to the shrub caused reductions in both the length and diameter of the shoots, while fertilization alone increased their diameter. Fertilization also decreased the concentration of condensed tannins in shoots of V. myrtillus, while clipping had no significant effect in this respect. Condensed tannin concentrations were higher in shoots given the combined fertilization and clipping treatment than in shoots that were fertilized but not clipped. The effect on tannins is in accordance with the predictions of the CNB‐hypothesis. Among the seven individual phenolic compounds analysed only one, a cinnamic acid derivate, showed a significant effect of the treatments. Repeated damage resulted in decreased concentration of this phenolic acid. In addition there was a tendency towards treatment effects on both nitrogen and carbon concentration of the V. myrtillus shoots, but none of these effects were statistically significant. The treatment‐induced changes in V. myrtillus also affected the food preferences of grey‐sided voles (Clethrionomys rufocanus), resulting in the following order of preference among the treatments: 1) fertilization and clipping, 2) fertilization, 3) control and 4) clipping. Not only biochemical changes, but also changes in growth form were found to influence the preferences, as the voles avoided the smallest shoots. This size‐dependent feeding may partly explain the observed differences in their preferences. Thus, induced changes in growth form need to be considered when conclusions about changes in herbivores’ preference are made.     

Metabolic rates and the energetic cost of external tag attachment in juvenile blacktip sharks Carcharhinus limbatus

This study reports on the metabolic rate of the blacktip shark Carcharhinus limbatus and the energetic costs of external tag attachment. Metabolic rates, swimming speed and tail‐beat (B_T) frequency were measured in a static respirometer with untagged animals and animals equipped with a small data logger. Tagged sharks showed significantly higher routine oxygen consumption and lower swimming speeds than untagged animals, indicating that tagging significantly affected the swimming efficiency and energetic requirements in these small sharks, and that these effects must be accounted for when interpreting telemetry data from free‐ranging individuals.