CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System

The development of the CRISPR–Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR–Cas9 system, which may lead to the modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. We introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the efficiency to edit the induced targets is maximized. We suggest several approaches for designing the optimal individual sgRNA and an approach to compute the optimal set of sgRNAs for cases when the experimental platform allows for more than one. The latter may optionally account for the homologous relationships among gene-family members. We further show that CRISPys outperforms simpler alignment-based techniques by in silico examination over all gene families in the Solanum lycopersicum genome.     

Natural killer cells in perinatally HIV-1-infected children exhibit less degranulation compared to HIV-1-exposed uninfected children and their expression of KIR2DL3, NKG2C, and NKp46 correlates with disease severity

NK cells play an integral role in the innate immune response by targeting virally infected and transformed cells with direct killing and providing help to adaptive responses through cytokine secretion. Whereas recent studies have focused on NK cells in HIV-1-infected adults, the role of NK cells in perinatally HIV-1-infected children is less studied. Using multiparametric flow cytometric analysis, we assessed the number, phenotype, and function of NK cell subsets in the peripheral blood of perinatally HIV-1-infected children on highly active antiretroviral therapy and compared them to perinatally exposed but uninfected children. We observed an increased frequency of NK cells expressing inhibitory killer Ig-like receptors in infected children. This difference existed despite comparable levels of total NK cells and NK cell subpopulations between the two groups. Additionally, NK cell subsets from infected children expressed, with and without stimulation, significantly lower levels of the degranulation marker CD107, which correlates with NK cell cytotoxicity. Lastly, increased expression of KIR2DL3, NKG2C, and NKp46 on NK cells correlated with decreased CD4+ T-lymphocyte percentage, an indicator of disease severity in HIV-1- infected children. Taken together, these results show that HIV-1-infected children retain a large population of cytotoxically dysfunctional NK cells relative to perinatally exposed uninfected children. This reduced function appears concurrently with distinct NK cell surface receptor expression and is associated with a loss of CD4+ T cells. This finding suggests that NK cells may have an important role in HIV-1 disease pathogenesis in HIV-1-infected children.     

South temperate birds have higher apparent adult survival than tropical birds in Africa

Life history theory predicts an inverse relationship between annual adult survival and fecundity. Globally, clutch size shows a latitudinal gradient among birds, with south temperate species laying smaller clutches than north temperate species, but larger clutches than tropical species. Tropical birds often have higher adult survival than north temperate birds associated with their smaller clutches. However, the prediction that tropical birds should also have higher adult survival than south temperate birds because of smaller clutch sizes remains largely untested. We measured clutch size and apparent annual breeding adult survival for 17 south temperate African species to test two main predictions. First, we found strong support for a predicted inverse relationship between adult survival and clutch size among the south temperate species, consistent with life‐history theory. Second, we compared our clutch size and survival estimates with published estimates for congeneric tropical African species to test the prediction of larger clutch size and lower adult survival among south temperate than related tropical species. We found that south‐temperate species laid larger clutches, as predicted, but had higher, rather than lower, apparent adult survival than related tropical species. The latter result may be an artefact of different approaches to measuring survival, but the results suggest that adult survival is generally high in the south temperate region and raises questions about the importance of the cost of reproduction to adult survival.     

Age‐specific survival and movement among major African Penguin Spheniscus demersus colonies

Reliable estimates of survival and dispersal are crucial to understanding population dynamics, but for seabirds, in which some individuals spend years away from land, mortality and emigration are often confounded. Multistate mark–recapture methods reduce bias by incorporating movement into the process of estimating survival. We used a multistate model to provide unbiased age‐specific survival and movement probabilities for the Endangered African Penguin Spheniscus demersus based on 5281 nestlings and 31 049 adults flipper‐banded and resighted in the Western Cape, South Africa, between 1994 and 2012. Adult survival was initially high (≥ 0.74) but declined after 2003–2004 coincident with a reduction in the availability of Sardine Sardinops sagax and Anchovy Engraulis encrasicolus on the west coast of South Africa. Juvenile survival was poorly estimated, but was lower and more variable than adult survival. Fidelity to the locality of origin varied over time, but was high in adults at Robben and Dassen islands (≥ 0.88) and above 0.55 for juvenile and immature Penguins at all localities. Movement occurred predominantly during 1994–2003 and was indicative of immigration to Robben and Dassen islands. Our results confirm that a prolonged period of adult mortality contributed to the observed decline in the African Penguin population and suggest a need for approaches operating over large spatial scales to ensure food security for marine top predators.     

Photon Statistics of a Hybrid Quantum Dot-Metal Nanoparticle Cluster

A theoretical study of photon statistics of an optically driven quantum dot located near a metal nanoparticle cluster (composed of one or two nanoparticles) is presented. Considering the system in the weak Rabi frequency regime, an analytical formula for anti-bunching time is derived. Using a photon Green’s function method based on the exact quantization of electromagnetic field in a dissipative medium, the dependence of the anti-bunching time on the geometrical parameters (quantum dot radius and quantum dot distance from the metal nanoparticle) is studied. The results show that these geometrical parameters have pronounced impacts on the photon statistics. Furthermore, our findings reveal that the quantum dot dipole orientation possesses an important role in the quantum dot photon emission.     

Nest survival and productivity of translocated and resident burrowing owls in Arizona

Development activities associated with urbanization can directly displace animals, causing high mortality and dispersal rates. Wildlife managers have attempted to mitigate the impacts of development on burrowing owls (Athene cunicularia), a species susceptible to ground-disturbing activities, by translocating them away from areas slated for development. In this study, we evaluated the effects of translocation on burrowing owl reproduction by comparing nest survival and productivity of owls involved in an ongoing translocation program in Arizona, USA, with that of resident owls. We used nest survival models to evaluate differences in nest survival and generalized linear models with Poisson error to assess differences in productivity. In 2017, cumulative nest survival (CNS) was lower among current-year translocated owls (i.e., owls translocated within the last year; CNS = 0.34, 95% CI = 0.18, 0.51) compared to non-translocated resident owls (0.83, 95% CI = 0.71, 0.94), but CNS was similar between previously translocated owls (i.e., those translocated >1 yr ago, CNS = 0.81, 95% CI = 0.64, 0.98) and residents. Likewise, in 2018, CNS was lower for current-year translocated owls (CNS = 0.12, 95% CI = 0.00, 0.24) compared to residents (CNS = 0.68, 95% CI = 0.50, 0.87) and previously translocated owls (CNS = 0.65, 95% CI = 0.40, 0.90). Productivity was significantly lower for current-year translocated owls ($\bar{x}$ = 0.5 fledglings/nest) compared to residents ($\bar{x}$ = 2.4 fledglings/nest) and previously translocated owls ($\bar{x}$ = 1.5 fledglings/nest) across sites and years. With the current methods of translocation, owls had poor reproductive success in the first year after release, but if they survived 2 years after release, they had similar nest survival and productivity compared to residents. Our results demonstrate that the current practice of releasing translocated burrowing owls in the Phoenix, Arizona area during the breeding season depresses reproduction and should be substantially changed. Nest survival was low and this was mainly attributed to nest establishment of translocated owls within release tents (acclimation structures used in translocations that involve soft release) where 85% of nests failed following tent removal. Translocation approaches that reflect a closer fit to the timing and behavior of the species should be tested to determine whether they result in better success. In particular, future studies should examine the translocation results of releasing owls as male-female pairs or single birds during the nonbreeding period.     

Survival and fidelity of translocated and resident burrowing owls in Arizona

Given the rapid pace of urbanization, mitigation translocations are increasingly being used to reduce human-wildlife conflicts, especially to move animals out of the path of land development. Burrowing owls (Athene cunicularia) are commonly subjected to translocations because of their association with flat, open lands that are attractive to development. But outcomes of burrowing owl translocations and the effects they have on their demographic parameters are seldom documented. From 2017–2019, we tracked the fates of 42 resident (non-translocated) burrowing owls and 43 translocated owls using very high frequency radio-telemetry across 4 release sites in southcentral Arizona, USA. Translocations were conducted by Wild At Heart (WAH), a non-profit raptor rehabilitation program, and owls were moved from areas with planned development activities to release sites that contained artificial burrows. Each year, WAH held translocated owls in groups of 6–10 owls with varying male to female ratios in aviaries prior to taking the owls to release sites where the owls stayed in soft-release tents for an acclimation period of 30 days before release. Resident owls were located at or near the 4 release sites. We used the joint live encounter and dead recovery model to evaluate differences in survival and fidelity probabilities between resident (non-translocated) and translocated burrowing owls and to assess factors that influenced these probabilities including sex, year, site, captivity duration, and the number of males in release cohorts for translocated owls. Annual survival was consistently lower for translocated owls (0.35, 95% CI = 0.10, 0.61 and 0.01, 95% CI = 0.00, 0.03 for 2017 and 2018, respectively) compared to resident owls (0.68, 95% CI = 0.45, 0.92 and 0.69, 95% CI = 0.47, 0.90 for 2017 and 2018, respectively). Annual fidelity was lower for translocated owls (0.05, 95% CI = 0.00, 0.15) compared to resident owls (0.79, 95% CI = 0.57, 1.00) in 2018, but fidelity did not differ significantly between translocated (0.54, 95% CI = 0.19, 0.89) and resident owls (0.62, 95% CI = 0.37, 0.87) in 2017. For translocated owls, the number of males in release cohorts negatively affected survival ($\hat{\mathrm{\beta }}$ = −0.74, 95% CI = −1.07, −0.40) and fidelity ($\hat{\mathrm{\beta }}$ = −0.73, 95% CI = −1.17, −0.29). Our results indicate the need for substantial changes to the current translocation methodology for burrowing owls in Arizona. Breeding season releases that included owl groups and multiple males within cohorts resulted in agonistic interactions, high mortality, and low fidelity. Translocations of this territorial species should be restricted to male-female pairs or single individuals. Earlier releases may allow owls time to become established on release sites prior to the breeding season, which could promote fidelity. We recommend these changes be implemented and evaluated along with an examination of other release methods.     

Habitat utilisation and nesting behaviour of two sympatric weavers in Mbalmayo District,Cameroon

Avian ecology and behaviour data provides crucial information for bird conservation and management, but are still missing for many species, particularly in developing countries. This study examines habitat utilisation, nesting behaviour, anthropogenic disturbances and impacts on agricultural crops of Village Weaver Ploceus cucullatus and Vieillot’s Black Weaver Ploceus nigerrimus in Mbalmayo District, Cameroon. Abundance was estimated by nest counts and point counts of birds at 40 previously located colonies, where also tree metrics and anthropogenic disturbances were measured. In total, more than 1 700 birds of the two species were estimated within a 9 km2 study area, divided into two blocks and five different habitat types. Village Weavers were generally more abundant, especially in the urban habitat, whereas Vieillot’s Black Weavers were most numerous in the rural habitat. Among the tree species used for nesting, mango Mangifera indica and African border tree Newbouldia laevis had the highest nest counts, which also was positively related to canopy cover diameter. Traffic noise disturbance was associated with lower bird and nest counts, whereas the opposite was observed on farming disturbance. The relative impacts on agricultural crops were estimated for African oil palm Elaeis guineensis (38%), maize Zea mays (36%), Musa spp. (18%) and Mangifera indica (8%). The implications for long-term coexistence between farmland birds and humans are discussed, and the challenge is to minimise both kinds of bird–human interactions, i.e. anthropogenic disturbances and agricultural crop damage.     

Notes on Hysterochelifer afghanicus Beier (Pseudoscorpiones: Cheliferidae), with a key to the species of Hysterochelifer Chamberlin

The pseudoscorpion Hysterochelifer afghanicus Beier, 1966 is redescribed and illustrated based on 21 specimens (13♂, 8♀) collected from different sites in northern, western, and southern Iran. A number of morphological characters and new provincial records are given. In addition, a key for 13 species of the pseudoscorpion genus Hysterochelifer Chamberlin, 1932 is proposed.