Integrating high‐speed videos in capture‐mark‐recapture studies of insects

Capture–mark–recapture (CMR) studies have been used extensively in ecology and evolution. While it is feasible to apply CMR in some animals, it is considerably more challenging in small fast‐moving species such as insects. In these groups, low recapture rates can bias estimates of demographic parameters, thereby handicapping effective analysis and management of wild populations. Here, we use high‐speed videos (HSV) to capture two large dragonfly species, Anax junius and Rhionaeschna multicolor, that rarely land and, thus, are particularly challenging for CMR studies. We test whether HSV, compared to conventional “eye” observations, increases the “resighting” rates and, consequently, improves estimates of both survival rates and the effects of demographic covariates on survival. We show that the use of HSV increases the number of resights by 64% in A. junius and 48% in R. multicolor. HSV improved our estimates of resighting and survival probability which were either under‐ or overestimated with the conventional observations. Including HSV improved credible intervals for resighting rate and survival probability by 190% and 130% in A. junius and R. multicolor, respectively. Hence, it has the potential to open the door to a wide range of research possibilities on species that are traditionally difficult to monitor with distance sampling, including within insects and birds.     

Multiple coping strategies maintain stability of a small mammal population in a resource‐restricted environment

In semi‐arid environments, aperiodic rainfall pulses determine plant production and resource availability for higher trophic levels, creating strong bottom‐up regulation. The influence of climatic factors on population vital rates often shapes the dynamics of small mammal populations in such resource‐restricted environments. Using a 21‐year biannual capture–recapture dataset (1993 to 2014), we examined the impacts of climatic factors on the population dynamics of the brush mouse (Peromyscus boylii) in semi‐arid oak woodland of coastal‐central California. We applied Pradel''s temporal symmetry model to estimate capture probability (p), apparent survival (φ), recruitment (f), and realized population growth rate (λ) of the brush mouse and examined the effects of temperature, rainfall, and El Niño on these demographic parameters. The population was stable during the study period with a monthly realized population growth rate of 0.993 ± SE 0.032, but growth varied over time from 0.680 ± 0.054 to 1.450 ± 0.083. Monthly survival estimates averaged 0.789 ± 0.005 and monthly recruitment estimates averaged 0.175 ± 0.038. Survival probability and realized population growth rate were positively correlated with rainfall and negatively correlated with temperature. In contrast, recruitment was negatively correlated with rainfall and positively correlated with temperature. Brush mice maintained their population through multiple coping strategies, with high recruitment during warmer and drier periods and higher survival during cooler and wetter conditions. Although climatic change in coastal‐central California will likely favor recruitment over survival, varying strategies may serve as a mechanism by which brush mice maintain resilience in the face of climate change. Our results indicate that rainfall and temperature are both important drivers of brush mouse population dynamics and will play a significant role in predicting the future viability of brush mice under a changing climate.     

Population dynamics of little brown bats (Myotis lucifugus) at summer roosts: Apparent survival,fidelity, abundance,and the influence of winter conditions

1. White‐nose syndrome (WNS) has caused the death of millions of bats, but the impacts have been more difficult to identify in western North America. Understanding how WNS, or other threats, impacts western bats may require monitoring other roosts, such as maternity roosts and night roosts, where bats aggregate in large numbers.
2. Little brown bats (Myotis lucifugus) are experiencing some of the greatest declines from WNS. Estimating survival and understanding population dynamics can provide valuable data for assessing population declines and informing conservation efforts.
3. We conducted a 5‐year mark–recapture study of two M. lucifugus roosts in Colorado. We used the robust design model to estimate apparent survival, fidelity, and abundance to understand population dynamics, and environmental covariates to understand how summer and winter weather conditions impact adult female survival. We compared the fidelity and capture probability of M. lucifugus between colonies to understand how bats use such roosts.
4. Overwinter survival increased with the number of days with temperatures below freezing (β > 0.100, SE = 0.003) and decreased with the number of days with snow cover (β < −0.40, SE < 0.13). Adult female fidelity was higher at one maternity roost than the other. Overwinter and oversummer adult female survival was high (>0.90), and based on survival estimates and fungal‐swabbing results, we believe these populations have yet to experience WNS.
5. Recapture of M. lucifugus using antennas that continuously read passive integrated transponder tags allows rigorous estimation of bat population parameters that can elucidate trends in abundance and changes in survival. Monitoring populations at summer roosts can provide unique population ecology data that monitoring hibernacula alone may not. Because few adult males are captured at maternity colonies, and juvenile males have low fidelity, additional effort should focus on understanding male M. lucifugus population dynamics.

     

Sources of variation in breeding-ground fidelity of mallards (Anas platyrhynchos)

Generalizations used to support hypotheses about the evolutionof fidelity to breeding areas in birds include the tendencyfor fidelity to be greater in adult birds than in yearlings.In ducks, in contrast to most bird species, fidelity is thoughtto be greater among females than males. Researchers have suggestedthat fidelity in ducks is positively correlated with pond availability.However, most estimates of fidelity on which these inferences have been based represent functions of survival and recapture—resighting probabilities in addition to fidelity. We applied the modelingapproach developed by Burnham to recapture and band recoverydata of mallard ducks to test the above hypotheses about fidelity.We found little evidence of sex differences in adult philopatry,with females being slightly more philopatric than males inone study area, but not in a second study area. However, yearlingfemales were more philopatric than yearling males in both study areas. We found that adults were generally more philopatricthan yearlings. We could find no relationship between fidelityand pond availability. Our results, while partially supportingcurrent theory concerning sex and age differences in philopatry,suggest that adult male mallards are more philopatric thanonce thought, and we recommend that other generalizations aboutphilopatry be revisited with proper estimation techniques.     

Integrating growth and survival models for flexible estimation of size‐dependent survival in a cryptic,endangered snake

Estimates of demographic rates for animal populations and individuals have many applications for ecological and conservation research. In many animals, survival is size‐dependent, but estimating the form of the size–survival relationship presents challenges. For elusive species with low recapture rates, individuals’ size will be unknown at many points in time. Integrating growth and capture–mark–recapture models in a Bayesian framework empowers researchers to impute missing size data, with uncertainty, and include size as a covariate of survival, capture probability, and presence on‐site. If there is no theoretical expectation for the shape of the size–survival relationship, spline functions can allow for fitting flexible, data‐driven estimates. We use long‐term capture–mark–recapture data from the endangered San Francisco gartersnake (Thamnophis sirtalis tetrataenia) to fit an integrated growth–survival model. Growth models showed that females reach longer asymptotic lengths than males and that the magnitude of sexual size dimorphism differed among populations. The capture probability and availability of San Francisco gartersnakes for capture increased with snout–vent length. The survival rate of female snakes exhibits a nonlinear relationship with snout–vent length (SVL), with survival flat between 300 mm and 550 mm SVL before decreasing for females between 550 mm and 700 mm SVL. For male snakes, survival decreased for adult males >550 mm SVL. The survival rates of the smallest and largest San Francisco gartersnakes were highly uncertain because recapture rates were very low for these sizes. By integrating growth and survival models and using penalized splines, we found support for size‐dependent survival in San Francisco gartersnakes. Our results have applications for devising management activities for this endangered subspecies, and our methods could be applied broadly to the study of size‐dependent demography among animals.     

Female and male Eurasian lynx have distinct spatial tactics at different life‐history stages in a high‐density population

Knowledge regarding the spatial behavior of the Eurasian lynx is mainly inferred from populations in Europe. We used GPS telemetry to record the spatial behavior of nine individuals in northwestern Anatolia obtaining eleven home ranges (HRs). Analyses revealed the smallest mean HR sizes (n_{HR ♀}  = 4) at 57 km² (95% kernel utilization distribution, KUD) and 56 km² (95% minimum convex polygon, MCP), ever reported for adult female Eurasian lynx. Adult males either occupied small permanent territories (n_HR♂._T = 2), with a mean of 176 km² (95% KUD) and 150 km² (95% MCP), or were residents without territories (floaters, n_HR♂. _F  = 2) roaming across large, stable HRs with a mean size of 2,419 km² (95% KUD) and 1,888 km² (95% MCP), comparable to HR sizes of Scandinavian lynx populations. Three disperser subadult males did not hold stable HRs (mean 95% KUD = 203 km², mean 95% MCP = 272 km²). At 4.9 individuals per 100 km², population density was one of the highest recorded, suggesting that the presence of adult male floaters was a consequence of a landscape fully occupied by territorials and revealing a flexibility of spatial behavior of Eurasian lynx not previously recognized. Such a high population density, small HRs, and behavioral flexibility may have been aided by the legal protection from and apparent low levels of poaching of this population. The observed spatial tactics are unlikely to be seen in most of the previously studied Eurasian lynx populations, as they either suffer medium to high levels of human‐caused mortality or were unlikely to be at carrying capacity. For effective and appropriate conservation planning, data from felid populations in a reasonably natural state such as ours, where space, density, prey, and pathogens are likely to be the key drivers of spatial dynamics, are therefore essential.     

Extracting spatial networks from capture–recapture data reveals individual site fidelity patterns within a marine mammal’s spatial range

1. Estimating the impacts of anthropogenic disturbances requires an understanding of the habitat‐use patterns of individuals within a population. This is especially the case when disturbances are localized within a population''s spatial range, as variation in habitat use within a population can drastically alter the distribution of impacts.
2. Here, we illustrate the potential for multilevel binomial models to generate spatial networks from capture–recapture data, a common data source used in wildlife studies to monitor population dynamics and habitat use. These spatial networks capture which regions of a population''s spatial distribution share similar/dissimilar individual usage patterns, and can be especially useful for detecting structured habitat use within the population''s spatial range.
3. Using simulations and 18 years of capture–recapture data from St. Lawrence Estuary (SLE) beluga, we show that this approach can successfully estimate the magnitude of similarities/dissimilarities in individual usage patterns across sectors, and identify sectors that share similar individual usage patterns that differ from other sectors, that is, structured habitat use. In the case of SLE beluga, this method identified multiple clusters of individuals, each preferentially using restricted areas within their summer range of the SLE.
4. Multilevel binomial models can be effective at estimating spatial structure in habitat use within wildlife populations sampled by capture–recapture of individuals, and can be especially useful when sampling effort is not evenly distributed. Our finding of a structured habitat use within the SLE beluga summer range has direct implications for estimating individual exposures to localized stressors, such as underwater noise from shipping or other activities.

     

Testing the precision and sensitivity of density estimates obtained with a camera‐trap method revealed limitations and opportunities

The use of camera traps in ecology helps affordably address questions about the distribution and density of cryptic and mobile species. The random encounter model (REM) is a camera‐trap method that has been developed to estimate population densities using unmarked individuals. However, few studies have evaluated its reliability in the field, especially considering that this method relies on parameters obtained from collared animals (i.e., average speed, in km/h), which can be difficult to acquire at low cost and effort. Our objectives were to (1) assess the reliability of this camera‐trap method and (2) evaluate the influence of parameters coming from different populations on density estimates. We estimated a reference density of black bears (Ursus americanus) in Forillon National Park (Québec, Canada) using a spatial capture–recapture estimator based on hair‐snag stations. We calculated average speed using telemetry data acquired from four different bear populations located outside our study area and estimated densities using the REM. The reference density, determined with a Bayesian spatial capture–recapture model, was 2.87 individuals/10km² [95% CI: 2.41–3.45], which was slightly lower (although not significatively different) than the different densities estimated using REM (ranging from 4.06–5.38 bears/10km² depending on the average speed value used). Average speed values obtained from different populations had minor impacts on REM estimates when the difference in average speed between populations was low. Bias in speed values for slow‐moving species had more influence on REM density estimates than for fast‐moving species. We pointed out that a potential overestimation of density occurs when average speed is underestimated, that is, using GPS telemetry locations with large fix‐rate intervals. Our study suggests that REM could be an affordable alternative to conventional spatial capture–recapture, but highlights the need for further research to control for potential bias associated with speed values determined using GPS telemetry data.     

Unique multiple paternity in the endangered big‐headed turtle (Platysternon megacephalum) in an ex situ population in South China

Understanding the mating system and reproductive strategies of an endangered species is critical to the success of captive breeding. The big‐headed turtle (Platysternon megacephalum) is one of the most threatened turtle species in the world. Captive breeding and reintroduction are necessary to re‐establish wild populations of P. megacephalum in some of its historical ranges in China, where the original populations have been extirpated. However, the captive breeding of P. megacephalum is very difficult and this may be due to its mysterious reproductive strategies and special behavior (e.g., aggressive temperament and territoriality). In this study, we achieved successful captive breeding of P. megacephalum by creating a habitat that mimics natural conditions and then investigated its mating system using microsatellite makers. A total of 16 clutches containing 79 eggs of P. megacephalum were collected, and 52 were hatched successfully over two breeding seasons. Of the 15 effective clutches, 6 clutches (40%) exhibited multiple paternity. There was no significant correlation between clutch size and multiple paternity, and no significant difference in hatching success between multiple‐sired and single‐sired clutches. However, there was significant correlation between male body size and the number of offspring, with higher‐ranked males contributing to more clutches. Our results provide the first evidence of multiple paternity and male hierarchy in P. megacephalum. These findings suggest that multiple paternity and male hierarchy should be considered in captive breeding programs for P. megacephalum, and creating a habitat that mimics natural conditions is an effctive way to achieve successful captive breeding and investigate the mating systems of this species.     

Survival and abundance of polar bears in Alaska’s Beaufort Sea, 2001–2016

The Arctic Ocean is undergoing rapid transformation toward a seasonally ice‐free ecosystem. As ice‐adapted apex predators, polar bears (Ursus maritimus) are challenged to cope with ongoing habitat degradation and changes in their prey base driven by food‐web response to climate warming. Knowledge of polar bear response to environmental change is necessary to understand ecosystem dynamics and inform conservation decisions. In the southern Beaufort Sea (SBS) of Alaska and western Canada, sea ice extent has declined since satellite observations began in 1979 and available evidence suggests that the carrying capacity of the SBS for polar bears has trended lower for nearly two decades. In this study, we investigated the population dynamics of polar bears in Alaska''s SBS from 2001 to 2016 using a multistate Cormack–Jolly–Seber mark–recapture model. States were defined as geographic regions, and we used location data from mark–recapture observations and satellite‐telemetered bears to model transitions between states and thereby explain heterogeneity in recapture probabilities. Our results corroborate prior findings that the SBS subpopulation experienced low survival from 2003 to 2006. Survival improved modestly from 2006 to 2008 and afterward rebounded to comparatively high levels for the remainder of the study, except in 2012. Abundance moved in concert with survival throughout the study period, declining substantially from 2003 and 2006 and afterward fluctuating with lower variation around an average of 565 bears (95% Bayesian credible interval [340, 920]) through 2015. Even though abundance was comparatively stable and without sustained trend from 2006 to 2015, polar bears in the Alaska SBS were less abundant over that period than at any time since passage of the U.S. Marine Mammal Protection Act. The potential for recovery is likely limited by the degree of habitat degradation the subpopulation has experienced, and future reductions in carrying capacity are expected given current projections for continued climate warming.     

An open spatial capture–recapture model for estimating density,movement, and population dynamics from line‐transect surveys

The purpose of many wildlife population studies is to estimate density, movement, or demographic parameters. Linking these parameters to covariates, such as habitat features, provides additional ecological insight and can be used to make predictions for management purposes. Line‐transect surveys, combined with distance sampling methods, are often used to estimate density at discrete points in time, whereas capture–recapture methods are used to estimate movement and other demographic parameters. Recently, open population spatial capture–recapture models have been developed, which simultaneously estimate density and demographic parameters, but have been made available only for data collected from a fixed array of detectors and have not incorporated the effects of habitat covariates. We developed a spatial capture–recapture model that can be applied to line‐transect survey data by modeling detection probability in a manner analogous to distance sampling. We extend this model to a) estimate demographic parameters using an open population framework and b) model variation in density and space use as a function of habitat covariates. The model is illustrated using simulated data and aerial line‐transect survey data for North Atlantic right whales in the southeastern United States, which also demonstrates the ability to integrate data from multiple survey platforms and accommodate differences between strata or demographic groups. When individuals detected from line‐transect surveys can be uniquely identified, our model can be used to simultaneously make inference on factors that influence spatial and temporal variation in density, movement, and population dynamics.     

Considering sampling bias in close‐kin mark–recapture abundance estimates of Atlantic salmon

Genetic methods for the estimation of population size can be powerful alternatives to conventional methods. Close‐kin mark–recapture (CKMR) is based on the principles of conventional mark–recapture, but instead of being physically marked, individuals are marked through their close kin. The aim of this study was to evaluate the potential of CKMR for the estimation of spawner abundance in Atlantic salmon and how age, sex, spatial, and temporal sampling bias may affect CKMR estimates. Spawner abundance in a wild population was estimated from genetic samples of adults returning in 2018 and of their potential offspring collected in 2019. Adult samples were obtained in two ways. First, adults were sampled and released alive in the breeding habitat during spawning surveys. Second, genetic samples were collected from out‐migrating smolts PIT‐tagged in 2017 and registered when returning as adults in 2018. CKMR estimates based on adult samples collected during spawning surveys were somewhat higher than conventional counts. Uncertainty was small (CV < 0.15), due to the detection of a high number of parent–offspring pairs. Sampling of adults was age‐ and size‐biased and correction for those biases resulted in moderate changes in the CKMR estimate. Juvenile dispersal was limited, but spatially balanced sampling of adults rendered CKMR estimates robust to spatially biased sampling of juveniles. CKMR estimates based on returning PIT‐tagged adults were approximately twice as high as estimates based on samples collected during spawning surveys. We suggest that estimates based on PIT‐tagged fish reflect the total abundance of adults entering the river, while estimates based on samples collected during spawning surveys reflect the abundance of adults present in the breeding habitat at the time of spawning. Our study showed that CKMR can be used to estimate spawner abundance in Atlantic salmon, with a moderate sampling effort, but a carefully designed sampling regime is required.     

TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

A global synthesis of survival estimates for microbats

Accurate survival estimates are needed to construct robust population models, which are a powerful tool for understanding and predicting the fates of species under scenarios of environmental change. Microbats make up 17% of the global mammalian fauna, yet the processes that drive differences in demographics between species are poorly understood. We collected survival estimates for 44 microbat species from the literature and constructed a model to determine the effects of reproductive, feeding and demographic traits on survival. Our trait-based model indicated that bat species which produce more young per year exhibit lower apparent annual survival, as do males and juveniles compared with females and adults, respectively. Using 8 years of monitoring data for two Australian species, we demonstrate how knowledge about the effect of traits on survival can be incorporated into Bayesian survival analyses. This approach can be applied to any group and is not restricted to bats or even mammals. The incorporation of informative priors based on traits can allow for more timely construction of population models to support management decisions and actions.     

Selection analysis on the rapid evolution of a secondary sexual trait

Evolutionary analyses of population translocations (experimental or accidental) have been important in demonstrating speed of evolution because they subject organisms to abrupt environmental changes that create an episode of selection. However, the strength of selection in such studies is rarely measured, limiting our understanding of the evolutionary process. This contrasts with long-term, mark–recapture studies of unmanipulated populations that measure selection directly, yet rarely reveal evolutionary change. Here, we present a study of experimental evolution of male colour in Trinidadian guppies where we tracked both evolutionary change and individual-based measures of selection. Guppies were translocated from a predator-rich to a low-predation environment within the same stream system. We used a combination of common garden experiments and monthly sampling of individuals to measure the phenotypic and genetic divergence of male coloration between ancestral and derived fish. Results show rapid evolutionary increases in orange coloration in both populations (1 year or three generations), replicating the results of previous studies. Unlike previous studies, we linked this evolution to an individual-based analysis of selection. By quantifying individual reproductive success and survival, we show, for the first time, that males with more orange and black pigment have higher reproductive success, but males with more black pigment also have higher risk of mortality. The net effect of selection is thus an advantage of orange but not black coloration, as reflected in the evolutionary response. This highlights the importance of considering all components of fitness when understanding the evolution of sexually selected traits in the wild.     

Site fidelity and behavioral plasticity regulate an ungulate’s response to extreme disturbance

With rapid global change, the frequency and severity of extreme disturbance events are increasing worldwide. The ability of animal populations to survive these stochastic events depends on how individual animals respond to their altered environments, yet our understanding of the immediate and short‐term behavioral responses of animals to acute disturbances remains poor. We focused on animal behavioral responses to the environmental disturbance created by megafire. Specifically, we explored the effects of the 2018 Mendocino Complex Fire in northern California, USA, on the behavior and body condition of black‐tailed deer (Odocoileus hemionus columbianus). We predicted that deer would be displaced by the disturbance or experience high mortality post‐fire if they stayed in the burn area. We used data from GPS collars on 18 individual deer to quantify patterns of home range use, movement, and habitat selection before and after the fire. We assessed changes in body condition using images from a camera trap grid. The fire burned through half of the study area, facilitating a comparison between deer in burned and unburned areas. Despite a dramatic reduction in vegetation in burned areas, deer showed high site fidelity to pre‐fire home ranges, returning within hours of the fire. However, mean home range size doubled after the fire and corresponded to increased daily activity in a severely resource‐depleted environment. Within their home ranges, deer also selected strongly for patches of surviving vegetation and woodland habitat, as these areas provided forage and cover in an otherwise desolate landscape. Deer body condition significantly decreased after the fire, likely as a result of a reduction in forage within their home ranges, but all collared deer survived for the duration of the study. Understanding the ways in which large mammals respond to disturbances such as wildfire is increasingly important as the extent and severity of such events increases across the world. While many animals are adapted to disturbance regimes, species that exhibit high site fidelity or otherwise fixed behavioral strategies may struggle to cope with increased climate instability and associated extreme disturbance events.     

Genotyping validates the efficacy of photographic identification in a capture‐mark‐recapture study based on the head scale patterns of the prairie lizard (Sceloporus consobrinus)

Population studies often incorporate capture‐mark‐recapture (CMR) techniques to gather information on long‐term biological and demographic characteristics. A fundamental requirement for CMR studies is that an individual must be uniquely and permanently marked to ensure reliable reidentification throughout its lifespan. Photographic identification involving automated photographic identification software has become a popular and efficient noninvasive method for identifying individuals based on natural markings. However, few studies have (a) robustly assessed the performance of automated programs by using a double‐marking system or (b) determined their efficacy for long‐term studies by incorporating multi‐year data. Here, we evaluated the performance of the program Interactive Individual Identification System (I³S) by cross‐validating photographic identifications based on the head scale pattern of the prairie lizard (Sceloporus consobrinus) with individual microsatellite genotyping (N = 863). Further, we assessed the efficacy of the program to identify individuals over time by comparing error rates between within‐year and between‐year recaptures. Recaptured lizards were correctly identified by I³S in 94.1% of cases. We estimated a false rejection rate (FRR) of 5.9% and a false acceptance rate (FAR) of 0%. By using I³S, we correctly identified 97.8% of within‐year recaptures (FRR = 2.2%; FAR = 0%) and 91.1% of between‐year recaptures (FRR = 8.9%; FAR = 0%). Misidentifications were primarily due to poor photograph quality (N = 4). However, two misidentifications were caused by indistinct scale configuration due to scale damage (N = 1) and ontogenetic changes in head scalation between capture events (N = 1). We conclude that automated photographic identification based on head scale patterns is a reliable and accurate method for identifying individuals over time. Because many lizard or reptilian species possess variable head squamation, this method has potential for successful application in many species.     

FastD: Fast detection of insecticide target‐site mutations and overexpressed detoxification genes in insect populations from RNA‐Seq data

Target‐site mutations and detoxification gene overexpression are two major mechanisms conferring insecticide resistance. Molecular assays applied to detect these resistance genetic markers are time‐consuming and with high false‐positive rates. RNA‐Seq data contains information on the variations within expressed genomic regions and expression of detoxification genes. However, there is no corresponding method to detect resistance markers at present. Here, we collected 66 reported resistance mutations of four insecticide targets (AChE, VGSC, RyR, and nAChR) from 82 insect species. Next, we obtained 403 sequences of the four target genes and 12,665 sequences of three kinds of detoxification genes including P450s, GSTs, and CCEs. Then, we developed a Perl program, FastD, to detect target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a web server for FastD (http://www.insect-genome.com/fastd). The estimation of FastD on simulated RNA‐Seq data showed high sensitivity and specificity. We applied FastD to detect resistant markers in 15 populations of six insects, Plutella xylostella, Aphis gossypii, Anopheles arabiensis, Musca domestica, Leptinotarsa decemlineata and Apis mellifera. Results showed that 11 RyR mutations in P. xylostella, one nAChR mutation in A. gossypii, one VGSC mutation in A. arabiensis and five VGSC mutations in M. domestica were found to be with frequency difference >40% between resistant and susceptible populations including previously confirmed mutations G4946E in RyR, R81T in nAChR and L1014F in VGSC. And 49 detoxification genes were found to be overexpressed in resistant populations compared with susceptible populations including previously confirmed detoxification genes CYP6BG1, CYP6CY22, CYP6CY13, CYP6P3, CYP6M2, CYP6P4 and CYP4G16. The candidate target‐site mutations and detoxification genes were worth further validation. Resistance estimates according to confirmed markers were consistent with population phenotypes, confirming the reliability of this program in predicting population resistance at omics‐level.     

Site‐directed mutagenesis of Serine‐72 reveals the location of the fructose 6‐phosphate regulatory site of the Agrobacterium tumefaciens ADP‐glucose pyrophosphorylase

The allosteric regulation of ADP–glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP‐glucose pyrophosphorylase. By X‐ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate‐binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP‐glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.