Search

SUMMARYChitin is a homopolymer of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) and a major structural component of fungal cell walls. In plants, chitin acts as a microbe-associated molecular pattern (MAMP) that is recognized by lysin motif (LysM)-containing plant cell surface-localized pattern recognition receptors (PRRs) that activate a plethora of downstream immune responses. In order to deregulate chitin-induced plant immunity and successfully establish infection, many fungal pathogens secrete LysM domain-containing effector proteins during host colonization. It was previously shown that the LysM effector Ecp6 from the tomato leaf mould fungus Cladosporium fulvum can outcompete plant PRRs for chitin binding because two of its three LysM domains cooperate to form a composite groove with ultra-high (pM) chitin-binding affinity. However, most functionally characterized LysM effectors contain only two LysMs, including Magnaporthe oryzae MoSlp1, Verticillium dahliae Vd2LysM, and Colletotrichum higginsianum ChElp1 and ChElp2. Here, we performed modelling, structural and functional analyses to investigate whether such dual-domain LysM effectors can also form ultra-high chitin-binding affinity grooves through intramolecular LysM dimerization. However, our study suggests that intramolecular LysM dimerization does not occur. Rather, our data support the occurrence of intermolecular LysM dimerization for these effectors, associated with a significantly lower chitin binding affinity than monitored for Ecp6. Interestingly, the intermolecular LysM dimerization allows for the formation of polymeric complexes in the presence of chitin. Possibly, such polymers may precipitate at infection sites in order to eliminate chitin oligomers, and thus suppress the activation of chitin-induced plant immunity.

Control of the arboviral disease vector Aedes aegypti has shown variable levels of efficacy around the globe. We evaluated an Autocidal Gravid Ovitrap (AGO) intervention as a stand‐alone control tool for population suppression of A. aegypti in US communities bordering Mexico.We conducted a cluster randomized crossover trial with weekly mosquito surveillance of sentinel households from July 2017 to December 2018. The intervention took place from August to December of both years. Multilevel models (generalized linear and additive mixed models) were used to analyse the changes in population abundance of female A. aegypti.We observed that female populations were being suppressed 77% (2018) and four times lower outdoor female abundance when AGO coverage (number of intervention AGO traps that surrounded a sentinel home) was high (2.7 AGOs/house). However, we also observed that areas with low intervention AGO coverage resulted in no difference (2017) or slightly higher abundance compared to the control. These results suggest that coverage rate might play a critical role on how populations of female A. aegypti are being modulated in the field. The lack of larval source habitat reduction and the short duration of the intervention period might have limited the A. aegypti population suppression observed in this study. Synthesis and applications. The mosquito, A. aegypti, is a public health concern in most tropical and subtropical regions. With the rise of insecticide resistance, the evaluation of non‐chemical tools has become pivotal in the fight against arboviral disease transmission. Our study shows that the AGO intervention, as a stand‐alone control tool, is limited by its coverage in human settlements. Vector control programmes should consider, that if the target coverage rate is not achieved, measures will be ineffective unless coupled with other control approaches. Although our multilevel modelling was focused on A. aegypti and the AGO, the approach can be applied to other mosquito vector species., The mosquito, A. aegypti, is a public health concern in most tropical and subtropical regions. With the rise of insecticide resistance, the evaluation of non‐chemical tools has become pivotal in the fight against arboviral disease transmission. Our study shows that the AGO intervention, as a stand‐alone control tool, is limited by its coverage in human settlements. Vector control programmes should consider, that if the target coverage rate is not achieved, measures will be ineffective unless coupled with other control approaches. Although our multilevel modelling was focused on A. aegypti and the AGO, the approach can be applied to other mosquito vector species.

Knowledge of host associations of blood-feeding vectors may afford insights into managing disease systems and protecting public health. However, the ability of methods to distinguish bloodmeal sources varies widely. We used two methods—Sanger sequencing and amplicon deep sequencing—to target a 228 bp region of the vertebrate Cytochrome b gene and determine hosts fed upon by triatomines (n = 115) collected primarily in Texas, USA. Direct Sanger sequencing of PCR amplicons was successful for 36 samples (31%). Sanger sequencing revealed 15 distinct host species, which included humans, domestic animals (Canis lupus familiaris, Ovis aries, Gallus gallus, Bos taurus, Felis catus, and Capra hircus), wildlife (Rattus rattus, Incilius nebulifer, Sciurus carolinensis, Sciurus niger, and Odocoileus virginianus), and captive animals (Panthera tigris, Colobus spp., and Chelonoidis carbonaria). Samples sequenced by the Sanger method were also subjected to Illumina MiSeq amplicon deep sequencing. The amplicon deep sequencing results (average of 302,080 usable reads per sample) replicated the host community revealed using Sanger sequencing, and detected additional hosts in five triatomines (13.9%), including two additional blood sources (Procyon lotor and Bassariscus astutus). Up to four bloodmeal sources were detected in a single triatomine (I. nebulifer, Homo sapiens, C. lupus familiaris, and S. carolinensis). Enhanced understanding of vector-host-parasite networks may allow for integrated vector management programs focusing on highly-utilized and highly-infected host species.

Planar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesized that this glycosaminoglycan–PCP interaction may regulate CE, but, surprisingly, DiO labelling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube., Summary: A gene–environment interaction involving the Vangl2 gene and sulfated glycosaminoglycans prevents mouse neural tube closure. Convergent extension shaping of the embryo is unaffected, implicating other mechanism(s) in this process.

The aim of this work was to report the detection of a putative novel Ehrlichia strain associated with the tick Amblyomma triste. Free-living adult ticks determined as A. triste were collected by drag-sampling in Argentina and Uruguay. Molecular detection of Ehrlichia agents was performed targeting three different loci: 16S rRNA gene, dsb gene and a fragment of groESL heat shock operon. In total, 164 adults of A. triste (38 from INTA E.E.A Delta del Parana in Argentina and 126 from Toledo Chico in Uruguay) were analyzed. One tick (0.6%) collected in INTA E.E.A. Delta del Parana (Argentina) was positive. The phylogenetic analyses show that the Ehrlichia strain found in this study (named Ehrlichia sp. strain Delta) represents an independent lineage within the genus Ehrlichia, close to E. chaffeensis and E. muris. This is also the first report of an Ehrlichia agent infecting the tick A. triste. The medical and veterinary significance of Ehrlichia sp. strain Delta remains to be demonstrated. However, it is important to mention that adults of A. triste are aggressive to humans and domestic mammals. Therefore, the potential role of A. triste in the transmission of Ehrlichia agents to humans or domestic animals across its distributional range should be highlighted, even more considering that Ehrlichia sp. strain Delta is phylogenetically related to the zoonotic E. chaffeensis, which is recognized as pathogenic to both humans and animals.

The purpose of this study was to evaluate the inter-device reliability of three VERT devices (Mayfonk Athletic, Florida, USA) when worn on the waist (W), left-hip (LH), and right-hip (RH) during single- and double-leg counter movement jumps (CMJ) in collegiate athletes. Thirty-two female and twenty-eight male NCAA Division II athletes (n = 60) participated in the present study. Jump height (JH) values for double-leg CMJs were analyzed by each device using a one-way repeated measures ANOVA whereas a 2 (jump leg) x 3 (wear location) repeated measures ANOVA was employed to evaluate single-leg CMJs. Reliability of the VERT devices were based upon intraclass correlation coefficients (ICC). Double-leg CMJs revealed an excellent ICC between all three VERT devices (ICC = 0.969). However, JH for RH and LH (45.69 ± 9.84 and 45.82 ± 10.45 cm, respectively) were on average lower than W (50.44 ± 12.37cm; both p < 0.001). The ICCs were excellent for right- and left-leg CMJs (ICC = 0.939 and 0.941, respectively). However, an interaction was observed (p < 0.001). No differences existed for left- or right-leg when VERT was worn on the waist. However, JH was higher when VERT devices were worn on the opposite hip of the jump leg (i.e., LH>RH for right-leg CMJs; RH>LH for leftleg CMJs; all p < 0.001). Results suggest that LH and RH are interchangeable for double-leg CMJs, but not with waist despite excellent reliability. In addition, all wear locations provided excellent ICCs for single-leg CMJs. However, waist provides more consistent JH values for right- and left-leg CMJs while RH and LH show more variability.

Antimicrobial resistance (AMR) in pathogenic strains of bacteria, such as Escherichia coli (E. coli), adversely impacts personal and public health. In this study, we examine competing hypotheses for the evolution of AMR including (i) 'genetic capitalism' in which genotypes that confer antibiotic resistance are gained and not often lost in lineages, and (ii) 'stabilizing selection' in which genotypes that confer antibiotic resistance are gained and lost often. To test these hypotheses, we assembled a dataset that includes annotations for 409 AMR genotypes and a phylogenetic tree based on genome-wide single nucleotide polymorphisms from 29 255 isolates of E. coli collected over the past 134 years. We used phylogenetic methods to count the times each AMR genotype was gained and lost across the tree and used model-based clustering of the genotypes with respect to their gain and loss rates. We demonstrate that many genotypes cluster to support the hypothesis for genetic capitalism while a few genotypes cluster to support the hypothesis for stabilizing selection. Comparing the sets of genotypes that fall under each of the hypotheses, we found a statistically significant difference in the breakdown of resistance mechanisms through which the AMR genotypes function. The result that many AMR genotypes cluster under genetic capitalism reflects that strong positive selective forces, primarily induced by human industrialization of antibiotics, outweigh the potential fitness costs to the bacterial lineages for carrying the AMR genotypes. We expect genetic capitalism to further drive bacterial lineages to resist antibiotics. We find that antibiotics that function via replacement and efflux tend to behave under stabilizing selection and thus may be valuable in an antibiotic cycling strategy.

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 102 to 5.50 x 106 RNA copies and in unfed females from 5.42 x 103 to 8.70 x 106 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females.

Aedes aegypti is the main vector of arboviral diseases such as dengue, chikungunya and Zika. A key feature for disease transmission modeling and vector control planning is adult mosquito dispersal. We studied Ae aegypti adult dispersal by conducting a mark-capture study of naturally occurring Ae. aegypti from discarded containers found along a canal that divided two residential communities in Donna, Texas, USA. Stable isotopes were used to enrich containers with either 13C or 15N. Adult mosquitoes were collected outdoors in the yards of households throughout the communities with BG Sentinel 2 traps during a 12-week period. Marked mosquito pools with stable isotopes were used to estimate the mean distance travelled using three different approaches (Net, Strip or Circular) and the probability of detecting an isotopically marked adult at different distances from the larval habitat of origin. We consistently observed, using the three approaches that male (Net: 220 m, Strip: 255 m, Circular: 250 m) Ae. aegypti dispersed further in comparison to gravid (Net: 135 m, Strip: 176 m, Circular: 189 m) and unfed females (Net: 192 m, Strip: 213 m, Circular: 198 m). We also observed that marked male capture probability slightly increased with distance, while, for both unfed and gravid females, such probability decreased with distance. Using a unique study design documenting adult dispersal from natural larval habitat, our results suggest that Ae. aegypti adults disperse longer distances than previously reported. These results may help guide local vector control authorities in their fight against Ae. aegypti and the diseases it transmits, suggesting coverage of 200 m for the use of insecticides and innovative vector control tools.

Effective mosquito surveillance and management depend on a thorough understanding of the biology and feeding patterns unique to species and sex. Given that a propensity to sugar feed is necessary for some mosquito surveillance and newer control strategies, we sought to document the amount of total sugar in wild Aedes aegypti (L.) and Culex quinquefasciatus (Say) captured from five different locations in the Lower Rio Grande Valley (LRGV) of South Texas over 2 yr. We used Biogents Sentinel 2 (BGS2) traps in year 1 and aspirators, BGS2, and CDC resting traps in years 2 and 3 to collect adult mosquitoes. The hot anthrone test was used to quantify total sugar content in each mosquito. Additionally, the cold and hot anthrone tests were used to distinguish fructose content from total sugars for mosquitoes captured in 2019. Overall, Ae. aegypti females had significantly lower total sugar content than Ae. aegypti males as well as both sexes of Cx. quinquefasciatus. However, the percentage of Ae. aegypti positive for fructose consumption was four to eightfold higher than Ae. aegypti previously reported in other regions. The difference between locations was significant for males of both species, but not for females. Seasonality and trapping method also revealed significant differences in sugar content of captured mosquitoes. Our results reinforce that sugar feeding in female Ae. aegypti is less than Cx. quinquefasciatus, although not absent. This study provides necessary data to evaluate the potential effectiveness of sugar baits in surveillance and control of both Ae. aegypti and Cx. quinquefasciatus mosquitoes.

CreERT2‐mediated gene recombination is widely applied in developmental biology research. Activation of CreERT2 is typically achieved by injection of tamoxifen in an oily vehicle into the peritoneal cavity of mid‐gestation pregnant mice. This can be technically challenging and adversely impacts welfare. Here we characterize three refinements to this technique: Pipette feeding (not gavage) of tamoxifen, ex vivo CreERT2 activation in whole embryo culture and injection of cell‐permeable TAT‐Cre into Cre‐negative cultured embryos. We demonstrate that pipette feeding of tamoxifen solution to the mother on various days of gestation reliably activates embryonic CreERT2, illustrated here using β‐Actin CreERT2, Sox2 CreERT2, T CreERT2, and Nkx1.2 CreERT2. Pipette feeding of tamoxifen induces dose‐dependent recombination of Rosa26 mTmG reporters when administered at E8.5. Activation of two neuromesodermal progenitor‐targeting Cre drivers, T CreERT2, and Nkx1.2 CreERT2, produces comparable neuroepithelial lineage tracing. Dose‐dependent CreERT2 activation can also be achieved by brief exposure to 4OH‐tamoxifen in whole embryo culture, allowing temporal control of gene deletion and eliminating the need to treat pregnant mice. Rosa26 mTmG reporter recombination can also be achieved regionally by injecting TAT‐Cre into embryonic tissues at the start of culture. This allows greater spatial control over Cre activation than can typically be achieved with endogenous CreERT2, for example by injecting TAT‐Cre on one side of the midline. We hope that our description and application of these techniques will stimulate refinement of experimental methods involving CreERT2 activation for gene deletion and lineage tracing studies. Improved temporal (ex vivo treatment) and spatial (TAT‐Cre injection) control of recombination will also allow previously intractable questions to be addressed.

The use of stable isotope enrichment to mark mosquitoes has provided a tool to study the biology of vector species. In this study, we evaluated isotopic marking of Aedes aegypti (L.) (Diptera: Culicidae) in a laboratory setting. We determined the optimal dosage for marking adult Ae. aegypti mosquitoes with 13C and 15N. Additionally, Ae. aegypti mosquitoes were single and dually marked with 13C and 15N for up to 60 d postemergence without changes to adult body size or transgenerational marking. This report adds to the growing literature that explores the use of alternative marking methods for ecological and vector biology studies.

Canine monocytic ehrlichiosis (CME) is an infectious disease caused by the bacterium Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato, a tick with worldwide distribution. When not diagnosed and treated early, disease can be severe. Currently, the disease is confirmed by serological or molecular assays. The objective of this study was to compare a serological assay based on immunochromatography (SPEED® EHRLI immunochromatographic test; BVT, France) and a molecular assay (a screening PCR followed by a nested PCR specific for E. canis) for the diagnosis of E. canis in suspected dogs from Buenos Aires city and southern Greater Buenos Aires, Argentina. Blood samples from 20 clinically healthy dogs (Control Group) and from 80 sick dogs suspected of having CME (Groups 1 to 4) were tested in parallel. Neither the immunochromatographic test nor the PCR assay was able to detect the presence of E. canis in the Control Group. In the group which had been previously tested by serology, the agreement between the tests was low (kappa: 0.200), whereas in the group which had been previously tested by PCR, the concordance between the tests was adequate (kappa: 0.650). The concordance between the tests evaluated in the total population studied was moderate (kappa: 0.496). The results of our study suggest that the use of rapid serological tests as a first approach, together with subsequent confirmation by PCR, will improve the diagnosis of CME., A ehrlichiose monocítica canina (CME) é uma doença infecciosa transmitida pelo carrapato Rhipicephalus sanguineus sensu lato com distribuição mundial causada por Ehrlichia canis, que pode produzir uma doença grave se não foi diagnosticada e tratada precocemente. A confirmação da doença é feita diretamente pela detecção do DNA fazendo a reação em cadeia da polimerase (PCR) ou indiretamente por métodos sorológicos. O objetivo deste estudo foi comparar o método sorológico baseado na imunocromatografia e a técnica de PCR para o diagnóstico de E. canis em cães suspeitos da Cidade de Buenos Aires e da região sul da Grande Buenos Aires. As amostras de sangue de 20 cães clinicamente saudáveis (Grupo Controle) e de 80 cães com suspeita clínica de CME (Grupo 1-4) foram avaliadas em paralelo. O diagnóstico serológico foi feito pelo teste imunocromatográfico SPEED® EHRLI (BVT, França). Para a detecção molecular, foi utilizada uma PCR de triagem para amplificar um fragmento de 345 pb do gene que codifica a subunidade 16S do rRNA da família Anaplasmataceae. As amostras positivas depois foram processadas pela PCR aninhada específica para E. canis. No Grupo Controle, a presença de E. canis não foi detectada por PCR ou anticorpos específicos com o teste imunocromatográfico. No grupo em que a sorologia foi solicitada inicialmente (1 e 2), a concordância entre os testes foi baixo (kappa: 0,200) enquanto que no grupo onde o teste inicialmente solicitado foi a PCR, a concordância entre os testes era adequado (kappa: 0,650). A concordância entre os testes avaliados na população total estudada foi moderada (kappa: 0,496). Em conclusão, os resultados do nosso estudo sugerem que o uso de testes serológicos rápidos inicialmente, juntamente com a confirmação subsequente por PCR, permitirá melhorar o diagnóstico de CME., Facultad de Ciencias Veterinarias

Blood sources used for insect colonies and their effects on fecundity and fertility have been studied in multiple mosquito species, but the effect of anticoagulants that prevent clotting of blood has received minimal attention. Here, we identify the effect two anticoagulants have on the mortality, fecundity, and fertility of Culex quinquefasciatus (Sebring and BCS strains) and Aedes aegypti Liverpool. Each mosquito species was provided with one of three treatments: direct feeding on live chicken (LC), blood from freshly exsanguinated chicken treated with heparin (EXS) or commercially purchased chicken blood treated with Alsever's solution (ART). No significant effect of treatment on mortality was observed. Both Cx. quinquefasciatus Sebring and BCS strains demonstrated a significant effect of treatment type on fecundity with the number of eggs laid for LC being 1.40-fold higher than EXS and 2.14-fold higher than ART for Sebring. For BCS strain mosquitoes, LC was 1.55-fold higher than ART, and EXS was 1.57-fold higher than ART, but there was no significant difference between LC and EXS. For Ae. aegypti mosquitoes, only a significant difference in mean egg counts was observed between LC and ART treatments, with LC laying 1.46-fold more eggs. No significant effect on fertility was observed among any mosquitoes for any treatment. These results demonstrate the negative effect of anticoagulants on the fecundity for multiple mosquito taxa. This may affect the ability of labs to produce large numbers of mosquitoes or colonize wild mosquito populations and should be taken into account when considering colony maintenance or vector biology research.

Background:The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a pandemic of coronavirus disease (COVID-19), which continues to cause infections and mortality worldwide. SARS-CoV-2 is transmitted primarily via the respiratory route and has experimentally been found to be stable on surfaces for multiple days. Flies (Diptera) and other arthropods mechanically transmit several pathogens, including turkey coronavirus. A previous experimental study demonstrated house flies, Musca domestica, can mechanically transmit SARS-CoV-2, but the ability of flies in general to acquire and deposit this virus in natural settings has not been explored. The purpose of this study was to explore the possibility of mechanical transmission of SARS-CoV-2 by peridomestic insects and their potential as a xenosurveillance tool for detection of the virus.Methods:In order to optimize chances of viral detection, flies were trapped in homes where at least one confirmed human COVID-19 case(s) resided. Sticky and liquid baited fly traps were deployed inside and outside of the homes of SARS-CoV-2 human cases in Brazos, Bell, and Montgomery Counties, from June to September 2020. Flies from sticky traps were identified, pooled by taxa, homogenized, and tested for the presence of SARS-CoV-2 RNA using qRT-PCR. Liquid traps were drained, and the collected fluid similarly tested after RNA concentration. Experimental viral detection pipeline and viral inactivation were confirmed in a Biosafety Level 3 lab. As part of a separate ongoing study, companion animals in the home were sampled and tested for SARS-CoV-2 on the same day of insect trap deployment.Results:We processed the contents of 133 insect traps from 44 homes, which contained over 1,345 individual insects of 11 different Diptera families and Blattodea.These individuals were grouped into 243 pools, and all tested negative for SARS-CoV-2 RNA. Dead flies exposed to SARS-CoV-2 in a BSL3 lab were processed using the same methods and viral RNA was detected by RT-PCR. Fourteen traps in seven homes were deployed on the day that cat or dog samples tested positive for SARS-CoV-2 RNA by nasal, oral, body, or rectal samples.Conclusions:This study presents evidence that biting and non-biting flies are not likely to contribute to mechanical transmission of SARS-CoV-2 or be useful in xenosurveillance for SARS-CoV-2.

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII–XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms., M.L.-P. was supported by a FPU contract from Ministerio de Ciencia, Innovación y Universidades (ref. FPU15/01775). This work was supported by projects ERA CoBioTech H2020 MeMBrane (Grant agreement: 722361) to A.Q. and A.G. and PCI2018-093190 (AEI/FEDER, U.E.) and PROMETEO/2020/014 Project from the Generalitat Valenciana to A.Q. and BBSRC (BB/R02152X/1) to A.G. We thank the Genomics Unit of the Central Service for Experimental Research (SCSIE), University of Valencia, for their genome sequencing support.

To preserve genome integrity when faced with DNA lesions, cells activate and coordinate a multitude of DNA repair pathways to ensure timely error correction or tolerance, collectively called the DNA damage response (DDR). These interconnecting damage response pathways are molecular signal relays, with protein kinases (PKs) at the pinnacle. Focused efforts in model eukaryotes have revealed intricate aspects of DNA repair PK function, including how they direct DDR pathways and how repair reactions connect to wider cellular processes, including DNA replication and transcription. The Kinetoplastidae, including many parasites like Trypanosoma spp. and Leishmania spp. (causative agents of debilitating, neglected tropical infections), exhibit peculiarities in several core biological processes, including the predominance of multigenic transcription and the streamlining or repurposing of DNA repair pathways, such as the loss of non-homologous end joining and novel operation of nucleotide excision repair (NER). Very recent studies have implicated ATR and ATM kinases in the DDR of kinetoplastid parasites, whereas DNA-dependent protein kinase (DNA-PKcs) displays uncertain conservation, questioning what functions it fulfills. The wide range of genetic manipulation approaches in these organisms presents an opportunity to investigate DNA repair kinase roles in kinetoplastids and to ask if further kinases are involved. Furthermore, the availability of kinase inhibitory compounds, targeting numerous eukaryotic PKs, could allow us to test the suitability of DNA repair PKs as novel chemotherapeutic targets. Here, we will review recent advances in the study of trypanosomatid DNA repair kinases.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility., Whether the adult testis harbours a somatic progenitor population is unknown. Here, the authors provide evidence that the testis interstitial cells expressing the transcription factor Tcf21 maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury.

Background Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches’ broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. Results Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host–pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. Conclusions Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

Understanding the ecological and epidemiological roles of pets in the transmission of SARS-CoV-2 is critical for animal and human health, identifying household reservoirs, and predicting the potential enzootic maintenance of the virus. We conducted a longitudinal household transmission study of 76 dogs and cats living with at least one SARS-CoV-2-infected human in Texas and found that 17 pets from 25.6% of 39 households met the national case definition for SARS-CoV-2 infections in animals. This includes three out of seventeen (17.6%) cats and one out of fifty-nine (1.7%) dogs that were positive by RT-PCR and sequencing, with the virus successfully isolated from the respiratory swabs of one cat and one dog. Whole-genome sequences of SARS-CoV-2 obtained from all four PCR-positive animals were unique variants grouping with genomes circulating among people with COVID-19 in Texas. Re-sampling showed persistence of viral RNA for at least 25 d-post initial test. Additionally, seven out of sixteen (43.8%) cats and seven out of fifty-nine (11.9%) dogs harbored SARS-CoV-2 neutralizing antibodies upon initial sampling, with relatively stable or increasing titers over the 2–3 months of follow-up and no evidence of seroreversion. The majority (82.4%) of infected pets were asymptomatic. ‘Reverse zoonotic’ transmission of SARS-CoV-2 from infected people to animals may occur more frequently than recognized.

The world’s most important mosquito vector of viruses, Aedes aegypti, is found around the world in tropical, subtropical and even some temperate locations. While climate change may limit populations of Ae. aegypti in some regions, increasing temperatures will likely expand its territory thus increasing risk of human exposure to arboviruses in places like Europe, Northern Australia and North America, among many others. Most studies of Ae. aegypti biology and virus transmission focus on locations with high endemicity or severe outbreaks of human amplified urban arboviruses, such as dengue, Zika, and chikungunya viruses, but rarely on areas at the margins of endemicity. The objective in this study is to explore previously published global patterns in the environmental suitability for Ae. aegypti and dengue virus to reveal deviations in the probability of the vector and human disease occurring. We developed a map showing one end of the gradient being higher suitability of Ae. aegypti with low suitability of dengue and the other end of the spectrum being equal and higher environmental suitability for both Ae. aegypti and dengue. The regions of the world with Ae. aegypti environmental suitability and no endemic dengue transmission exhibits a phenomenon we term ‘aegyptism without arbovirus’. We then tested what environmental and socioeconomic variables influence this deviation map revealing a significant association with human population density, suggesting that locations with lower human population density were more likely to have a higher probability of aegyptism without arbovirus. Characterizing regions of the world with established populations of Ae. aegypti but little to no autochthonous transmission of human-amplified arboviruses is an important step in understanding and achieving aegyptism without arbovirus., Author summary The preeminent vector of arboviruses, Aedes aegypti, is distributed globally and capable of transmitting deadly pathogens to over half the world’s population. While most studies focus on areas where Ae. aegypti and human-amplified urban arboviruses such as dengue and Zika viruses are locally established, our study explores the margins of endemicity where Aedes aegypti can be found, but arboviral illness is rare. These areas where we find environmental suitability for the vector but an absence of established arboviral transmission we term ‘aegyptism without arbovirus’. This builds on the long-held observation of ‘anophelism without malaria’ in which some regions having Plasmodium-competent Anopheles spp. mosquitoes but not the associated human malaria. This study uses previously published maps to reveal locations with higher suitability of Ae. aegypti and low suitability of dengue. Additionally, we analyzed the resulting map of deviations between Ae. aegypti and dengue and found significant associations with human population density, infant mortality rate, temperature, and precipitation. To the best of our knowledge, this is the first study to characterize places around the world that exhibit ‘aegyptism without arbovirus’ which is an important first step in our ongoing battle with human-amplified urban arboviruses.

An integral part to integrated mosquito management is to ensure chemical products used for area-wide control are effective against a susceptible population of mosquitoes. Prior to conducting an intervention trial using an insect growth regulator, pyriproxyfen, in South Texas to control Aedes aegypti, we conducted a larval bioassay to evaluate baseline levels of susceptibility. We used seven serially-diluted doses ranging from 2.5 ppb to 6.3 × 10−4 ppb. We observed 100% inhibition emergence (IE) at even the lowest dose of 6.3 × 10−4 ppb in our susceptible reference colony of Ae. aegypti Liverpool. In our field strain of Ae. aegypti (F5 colonized from South Texas) we observed 79.8% IE at 6.3 × 10−4 ppb, 17.7% IE at 1.25 × 10−3 ppb, 98.7% IE at 1.25 × 10−2 ppb, and 100% emergence inhibition for the remainder of the doses. Given that commercial pyriproxyfen products are labeled for doses ranging to 50 ppb, we conclude that the field population sampled by this study are susceptible to this insect growth regulator.

South Texas is recognized as a potential area for the emergence and re-emergence of mosquito-borne diseases due to recent circulation of Zika, chikungunya, and dengue viruses. During 2017, high Aedes aegypti abundance found in the city of Brownsville, TX, in combination with the previous year's local transmission of Zika virus, triggered the activation of the Texas Department of State Health Services Emergency Mosquito Control Contingency Contract. A contract with the Clarke Environmental and Mosquito Control was a response to control Ae. aegypti, using a ground-based wide-area larvicide spray (WALS™) containing Bacillus thuringiensis israelensis. The WALS application was evaluated through a field-based bioassay and by comparing surveillance data pre- and post-WALS application. The WALS application bioassay demonstrated that the larvicide was effective up to 60 m into the target properties. Additionally, the number of Ae. aegypti captured in traps decreased in the WALS intervention areas compared with the untreated control areas, with an estimated 29% control.

African swine fever virus (ASFV) continues to threaten global animal health and agricultural biosecurity. Mitigating the establishment of ASFV in the United States (U.S.) is contingent on (1) the identification of arthropod vectors and vertebrate hosts that are capable of viral maintenance and transmission in the U.S. and (2) knowledge of vector-host associations that may permit transmission. We aggregated data on vector competence, host competence and tick–host associations by systematic review of published articles and collection records to identify species that may support the invasion of ASFV in the U.S. Three species of competent soft ticks occur in the U.S., Ornithodoros coriaceus, Ornithodoros turicata, and Ornithodoros puertoricensis, however, vector competence for the majority of soft ticks in the U.S. remains unknown. Three species of competent vertebrate hosts currently occur in the U.S.: domestic pigs (Sus scrofa domesticus), feral hogs (Sus scrofa), and common warthogs (Phacochoerus africanus). Hierarchical hazard categories based on vector competence, tick–host contact rates, and vector abundance were used to semiquantitatively rank U.S. soft tick species by their relative risk for contributing to ASFV transmission to identify which soft tick species are a priority for future studies. High-risk vector and host species identified in this study can be used to focus ASFV risk assessments in the U.S., guide targeted surveillance and control strategies, and proactively prepare for an ASFV incursion event. Results indicate O. coriaceus, O. turicata, and O. puertoricensis demonstrate the highest relative risk for contributing to ASFV transmission in the U.S., however, many gaps in knowledge exist preventing the full evaluation of at least 30 soft tick species in the U.S. Further study is required to identify soft tick vectors that interact with feral swine populations, elucidate vector competence, and further understand the biology of soft tick species.

African swine fever virus (ASFV) causes a high-consequence foreign animal disease that has emerged along international trade routes. Owing to high lethality and resulting trade sanctions, establishment of this disease in the United States would have devastating economic consequences. ASFV can be transmitted by soft ticks in the genus Ornithodoros or directly between swine, including domestic, feral, and wild swine. Consequently, the spatial risk of ASFV establishment depends on where susceptible animals, with or without competent vectors, co-occur. We synthesized county-level historical records of soft tick occurrence, current maps of feral swine distribution, and domestic swine inventory to evaluate the risk of ASFV establishment and spillover in the United States. Areas of California, Florida, and much of the southwestern United States were classified as high risk for ASFV establishment and spillover should an introduction event occur. Our analyses indicate that California, Texas, Georgia, and Florida are high-priority candidates for proactive risk reduction strategies. Domestic swine are often produced in high-biosecurity environments, mitigating health risks associated with contacting infected hosts and vectors. However, small-scale and organic pig producers in much of the southern United States remain more vulnerable to disease emergence.

The yellow fever mosquito, Aedes aegypti, has facilitated the re-emergence of dengue virus (DENV) and emergence of chikungunya virus (CHIKV) and Zika virus (ZIKV) in the Americas and the Caribbean. The recent transmission of these arboviruses in the continental United States has been limited, to date, to South Florida and South Texas despite Ae. aegypti occurring over a much larger geographical region within the country. The main goal of our study was to provide the first long term longitudinal study of Ae. aegypti and enhance the knowledge about the indoor and outdoor relative abundance of Ae. aegypti as a proxy for mosquito-human contact in South Texas, a region of the United States that is at high risk for mosquito-borne virus transmission. Here, the relative abundance of indoors and outdoors mosquitoes of households in eight different communities was described. Surveillance was done weekly from September 2016 to April 2018 using the CDC Autocidal Gravid Ovitraps in low- and middle-income communities. A total of 69 houses were included in this survey among which 36 were in the low-income communities (n = 11 for Donna, n = 15 for Progresso, n = 5 for Mesquite, n = 5 for Chapa) and 33 in middle-income communities (n = 9 for La Feria, n = 8 for Weslaco, n = 11 for McAllen, and n = 5 for Rio Rico). Overall, Ae. aegypti was the dominant species (59.2% of collections, n = 7255) followed by Culex spp. mosquitoes (27.3% of collections, n = 3350). Furthermore, we demonstrated for Ae. aegypti that 1) outdoor relative abundance was higher compared to indoor relative abundance, 2) low-income communities were associated with an increase in mosquito relative abundance indoors when compared to middle-income communities, 3) no difference was observed in the number of mosquitoes collected outdoors between low-income and middle-income communities, and 4) warmer months were positively correlated with outdoor relative abundance whereas no seasonality was observed in the relative abundance of mosquitoes indoors. Additionally, Ae. aegypti mosquitoes collected in South Texas were tested using a specific ZIKV/CHIKV multiplex real-time PCR assay, however, none of the mosquitoes tested positive. Our data highlights the occurrence of mosquitoes indoors in the continental United States and that adults are collected nearly every week of the calendar year. These mosquito data, obtained concurrently with local ZIKV transmission of 10 locally acquired cases in nearby communities, represent a baseline for future studies in the Lower Rio Grande Valley (LRGV) including vector control interventions relying on the oviposition behavior to reduce mosquito populations and pathogen transmission.

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the TnSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5-to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

Four species of bivalve molluscs (Anomalocardia brasiliana, Iphigenia brasiliana, Lucina pectinata and Trachycardium muricatum) were collected in the Todos os Santos Bay (TSB), Bahia, Brazil, in order to evaluate As, Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, V and Zn levels and, consequently, the risk of bivalve mollusc consumption in humans. The samples were analyzed by inductively coupled plasma optical emission spectrometry (ICP OES) and inductively coupled plasma mass spectrometry (ICP-MS) after closed-vessel microwave digestion. The accuracy was confirmed using the certified reference materials of oyster tissue (NIST 1566b) and mussel tissue (NIST 2977), and the results were statistically equivalent to the certified values. Application of principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed a tendency to form two groups between samples of Lucina pectinata and Trachycardium muricatum. All species showed As and Cr concentrations higher than the maximum tolerable limit specified in Brazilian legislation.

Moniliophthora perniciosaandMoniliophthora roreriare hemibiotrophic fungi that harbor a large number of Pathogenesis-Related 1 genes, many of which are induced in the biotrophic interaction withTheobroma cacao.Here, we provide evidence that the evolution of PR-1 inMoniliophthorawas adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Phylogenetic analysis revealed conserved PR-1 genes, shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity inMoniliophthora, as well as in recent specialization cases within both species. PR-1 families inMoniliophthorawith higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arm race. Furthermore, we show that the highly diversifiedMpPR-1genes are not induced by two phytoalexins, suggesting detoxification might not be their main function as proposed before.

Aedes aegypti control requires dedicated resources that are usually scarce, limiting the reach and sustainability of vector control programs. This generates a need to focus on areas at risk of disease transmission and also understand the factors that might modulate local mosquito abundance. We evaluated the eco-bio-social factors that modulate indoor and outdoor relative abundance of female Ae. aegypti in communities of South Texas. We conducted housing quality and Knowledge Attitudes and Practices surveys in households that were part of a weekly mosquito surveillance program in November of 2017 and 2018. Our results showed widespread knowledge of mosquitoes and Zika virus by our participants. However, less than 35% considered them as serious problems in this region. The presence of window-mounted air conditioning units increased the risk of female mosquito relative abundance indoors. An increase in outdoor relative abundance was associated with larger properties and a higher number of children between 6 to 17 years of age. Interestingly, we observed that an increasing number of children &lt, 5 years of age modulated both indoor and outdoor relative abundance, with a 52% increase indoors and 30% decrease outdoors. The low perception of mosquito and disease risk highlights engagement needs for vector-borne disease prevention in this region. The identified risk factors can help guide public health officials in their efforts to reduce human and vector contact.

Arthropod vectors are frequently exposed to a diverse assemblage of parasites, but the consequence of these infections on their biology and behavior are poorly understood. We experimentally evaluated whether the ingestion of a common protozoan parasite of avian hosts (Haemoproteus spp.; Haemosporida: Haemoproteidae) impacted the survivorship of Culex quinquefasciatus (Say) (Diptera: Culicidae). Blood was collected from wild northern cardinals (Cardinalis cardinalis) in College Station, Texas, and screened for the presence of Haemoproteus spp. parasites using microscopic and molecular methods. Experimental groups of Cx. quinquefasciatus mosquitoes were offered Haemoproteus-positive cardinal blood through an artificial feeding apparatus, while control groups received Haemoproteus-negative cardinal blood or domestic canary (Serinus canaria domestica) blood. Culex quinquefasciatus mosquitoes exposed to Haemoproteus infected cardinal blood survived significantly fewer days than mosquitoes that ingested Haemoproteus-negative cardinal blood. The survival of mosquitoes fed on positive cardinal blood had a median survival time of 18 days post-exposure and the survival of mosquitoes fed on negative cardinal blood exceeded 50% across the 30 day observation period. Additionally, mosquitoes that fed on canary controls survived significantly fewer days than cardinal negative controls, with canary control mosquitoes having a median survival time of 17 days. This study further supports prior observations that Haemoproteus parasites can be pathogenic to bird-biting mosquitoes, and suggests that Haemoproteus parasites may indirectly suppress the transmission of co-circulating vector-borne pathogens by modulating vector survivorship. Our results also suggest that even in the absence of parasite infection, bloodmeals from different bird species can influence mosquito survivorship.

The ecology and host feeding patterns of many soft ticks (Ixodida: Argasidae) remain poorly understood. To address soft tick–host feeding associations, we fed Ornithodoros turicata Dugès on multiple host species and evaluated quantitative PCR (qPCR) and stable isotope analyses to identify the vertebrate species used for the bloodmeal. The results showed that a qPCR with host-specific probes for the cytochrome b gene successfully identified bloodmeals from chicken (Gallus gallus L.), goat (Capra aegagrus hircus L), and swine (Sus scrofa domesticus) beyond 330 days post-feeding and through multiple molting. Also, qPCR-based bloodmeal analyses could detect multiple host species within individual ticks that fed upon more than one species. The stable isotope bloodmeal analyses were based on variation in the natural abundance of carbon (13C/12C) and nitrogen (15N/14N) isotopes in ticks fed on different hosts. When compared to reference isotope signatures, this method discerned unique δ13C and δ15N signatures in the ticks fed on each host taxa yet could not discern multiple host species from O. turicata that fed on more than one host species. Given the significance of soft tick-borne zoonoses and animal diseases, elucidating host feeding patterns from field-collected ticks using these methods may provide insight for an ecological basis to disease management.

Tick-host bloodmeal associations are important factors when characterizing risks of associated pathogen transmission and applying appropriate management strategies. Despite their biological importance, comparatively little is known about soft tick (Argasidae) host associations in the United States compared to hard ticks (Ixodidae). In this study, we evaluated a PCR and direct Sanger sequencing method for identifying the bloodmeal hosts of soft ticks. We collected 381 cave-associated Ornithodoros turicata near San Antonio, Texas, USA, and also utilized eight colony-reared specimens fed artificially on known host blood sources over 1.5 years ago. We correctly identified the vertebrate host bloodmeals of two colony-reared ticks (chicken and pig) up to 1,105 days post-feeding, and identified bloodmeal hosts from 19 out of 168 field-collected soft ticks, including raccoon (78.9%), black vulture (10.5%), Texas black rattlesnake (5.3%), and human (5.3%). Our results confirm the retention of vertebrate blood DNA in soft ticks and advance the knowledge of argasid host associations in cave-dwelling O. turicata.

Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. Here we report that neural fold elevation during mouse spinal neurulation is vulnerable to deletion of the VANGL planar cell polarity protein 2 (Vangl2) gene in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves diminished myosin-II localisation on neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted neuroepithelial cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap localisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects., Mutations that cause tissue mosaicism have been identified in individuals with severe congenital defects. Here, the authors show that mosaic deletion of Vangl2 in the murine neuroepithlium causes spina bifida by preventing apical constriction via reduced myosin II and tubulin organisation.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We used single cell RNA-sequencing (scRNA-seq) to capture enhancer activity at single cell resolution and delineate specification of cells labeled by enhancers in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We combine enhancer-based reporter labeling with single-cell transcriptional readout to characterize enhancer activity and define cell populations in vivo. Seven enhancers had diverse activities in specific progenitor and neuronal populations within the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization (ISH) data to distinguish subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. This work demonstrates how the power of scRNA-seq can be extended by enhancer-based labelling and leveraging ISH data and reveals novel lineage specification paths underlying patterning of developing mouse brain.

Ocular coloboma is a congenital eye malformation, resulting from a failure in optic fissure closure (OFC) and causing visual impairment. There has been little study of the epithelial fusion process underlying closure in the human embryo and coloboma aetiology remains poorly understood. We performed RNAseq of cell populations isolated using laser capture microdissection to identify novel human OFC signature genes and probe the expression profile of known coloboma genes, along with a comparative murine analysis. Gene set enrichment patterns showed conservation between species. Expression of genes involved in epithelial-to-mesenchymal transition was transiently enriched in the human fissure margins during OFC at days 41-44. Electron microscopy and histological analyses showed that cells transiently delaminate at the point of closure, and produce cytoplasmic protrusions, before rearranging to form two continuous epithelial layers. Apoptosis was not observed in the human fissure margins. These analyses support a model of human OFC in which epithelial cells at the fissure margins undergo a transient epithelial-to-mesenchymal-like transition, facilitating cell rearrangement to form a complete optic cup.

The natural infections and epidemiological roles of household pets in SARS-CoV-2 transmission are not understood. We conducted a longitudinal study of dogs and cats living with at least one SARS-CoV-2 infected human in Texas and found 47.1% of 17 cats and 15.3% of 59 dogs from 25.6% of 39 households were positive for SARS-CoV-2 via RT-PCR and genome sequencing or neutralizing antibodies. Virus was isolated from one cat. The majority (82.4%) of infected pets were asymptomatic. Re-sampling of one infected cat showed persistence of viral RNA at least 32 d-post human diagnosis (25 d-post initial test). Across 15 antibody-positive animals, titers increased (33.3%), decreased (33.3%) or were stable (33.3%) over time. A One Health approach is informative for prevention and control of SARS-CoV-2 transmission.

Background Co-infection with malaria and intestinal parasites is common in children in Africa and may affect their immune response to a malaria parasite infection. Prior studies suggest that co-infections may lead to increased susceptibility to malaria infection and disease severity; however, other studies have shown the reverse. Knowledge on how co-morbidities specifically affect the immune response to malaria antigens is limited. Therefore, this study sought to determine the prevalence of co-infection of malaria and intestinal parasites and its association with antibody levels to malaria merozoite antigens. Methods A cross sectional study was carried out in two villages with high transmission of malaria in Cameroon (Ngali II and Mfou) where mass drug administration (MDA) had been administered at ~6-month intervals (generally with albendazole or mebendazole). Children aged 1–15 years were enrolled after obtaining parental consent. A malaria rapid diagnostic test was used on site. Four (4) ml of peripheral blood was collected from each participant to determine Plasmodium falciparum infections by microscopy, haemoglobin levels and serology. Fresh stool samples were collected and examined by wet mount, Kato-Katz method and modified Ritchie concentration techniques. A Multiplex Analyte Platform assay was used to measure antibody levels. Results A total of 320 children were enrolled. The prevalence of malaria by blood smear was 76.3% (244/320) and prevalence of malaria and intestinal parasites was 16.9% (54/320). Malaria prevalence was highest in young children; whereas, intestinal parasites (IP+) were not present until after 3 years of age. All children positive for malaria had antibodies to MSP142, MSP2, MSP3 and EBA175. No difference in antibody levels in children with malaria-co infections compared to malaria alone were found, except for antibody levels to EBA-175 were higher in children co-infected with intestinal protozoa (p = 0.018), especially those with Entamoeba histolytica infections (p = 0.0026). Conclusion Antibody levels to EBA175 were significantly higher in children co-infected with malaria and E. histolytica compared to children infected with malaria alone. It is important to further investigate why and how the presence of these protozoans might modulate the immune response to malaria antigens.

Quantification of cortical bone mass and architecture using μCT is commonplace in osteoporosis and osteoarthritis research. Different groups often report substantially divergent mouse cortical bone responses to nominally comparable interventions. In the case of studies assessing bones' responses to externally applied loading, these differences are commonly associated with methodological differences in the loading regime. This chapter describes a widely published, standardized method of in vivo mouse tibia axial loading to produce lamellar bone formation. Despite uniform application of axial loading, changes in bone mass are highly site-specific within individual bones. For example, the mouse proximal tibia rapidly accrues new bone following axial loading, but this osteogenic response tapers to produce undetectable differences distally. Consequently, the bone sites selected for comparisons substantially influence the magnitude of differences observed. Application of the freely available Site Specificity software allows site-specific responses to be identified by rapidly quantifying cortical bone mass at each 1% site along the bone's length. This high-content screening tool has been informatively applied to study the local effects of changes in loading as well as systemic interventions including hormonal treatment and aging. Automated multisite analyses of cortical mass is increasingly identifying site-specific effects of "systemic" interventions such as global gene deletions. Biological mechanisms underlying this apparent regionalization of cortical responses are largely unknown but may start to be elucidated by increasingly widespread application of Site Specificity methods.

The genus Verticillium contains 10 species of plant-associated fungi, some of which are notorious pathogens. Verticillium species evolved by frequent chromosomal rearrangements that contribute to genome plasticity. Centromeres are instrumental for separation of chromosomes during mitosis and meiosis, and failed centromere functionality can lead to chromosomal anomalies. Here, we used a combination of experimental techniques to identify and characterize centromeres in each of the Verticillium species. Intriguingly, we could strongly associate a single repetitive element to the centromeres of some of the Verticillium species. The presence of this element in the centromeres coincides with increased centromere sizes and genome-wide repeat expansions. Collectively, our findings signify a role of repetitive elements in the function, organization, and rapid evolution of centromeres in a set of closely related fungal species., Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the 10 species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (∼150-kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen Verticillium dahliae. Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single degenerated long terminal repeat (LTR) retrotransposon is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, of which some could be linked to centromeres, suggesting that centromeres contributed to chromosomal evolution. The size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus Verticillium.

Skeletal elements have a diverse range of shapes and sizes specialized to their various roles including protecting internal organs, locomotion, feeding, hearing, and vocalization. The precise positioning, size, and shape of skeletal elements is therefore critical for their function. During embryonic development, bone forms by endochondral or intramembranous ossification and can arise from the paraxial and lateral plate mesoderm or neural crest. This review describes inductive mechanisms to position and pattern bones within the developing embryo, compares and contrasts the intrinsic vs extrinsic mechanisms of endochondral and intramembranous skeletal development, and details known cellular processes that precisely determine skeletal shape and size. Key cellular mechanisms are employed at distinct stages of ossification, many of which occur in response to mechanical cues (eg, joint formation) or preempting future load‐bearing requirements. Rapid shape changes occur during cellular condensation and template establishment. Specialized cellular behaviors, such as chondrocyte hypertrophy in endochondral bone and secondary cartilage on intramembranous bones, also dramatically change template shape. Once ossification is complete, bone shape undergoes functional adaptation through (re)modeling. We also highlight how alterations in these cellular processes contribute to evolutionary change and how differences in the embryonic origin of bones can influence postnatal bone repair., Key Findings Compares and contrasts Endochondral and intramembranous bone developmentReviews embryonic origins of different bonesDescribes the cellular and molecular mechanisms of positioning skeletal elements.Describes mechanisms of skeletal growth with a focus on the generation of skeletal shape

The world’s most important mosquito vector of viruses, Aedes aegypti, is found around the world in tropical, subtropical and even some temperate locations. While climate change may limit populations of Ae. aegypti in some regions, increasing temperatures will likely expand its territory thus increasing risk of human exposure to arboviruses in places like Europe, Northern Australia and North America, among many others. Most studies of Ae. aegypti biology and virus transmission focus on locations with high endemicity or severe outbreaks of human amplified urban arboviruses, such as dengue, Zika, and chikungunya viruses, but rarely on areas at the margins of endemicity. Our objective in this study is to explore global patterns in the environmental suitability for Ae. aegypti and dengue virus to reveal deviations in the probability of the vector and human disease occurring. We developed a map showing one end of the gradient being higher suitability of Ae. aegypti with low suitability of dengue and the other end of the spectrum being equal and higher environmental suitability for both Ae. aegypti and dengue. The regions of the world with Ae. aegypti environmental suitability and no endemic dengue transmission exhibits a phenomenon we term ‘aegyptism without arbovirus’. We then tested what environmental and socioeconomic variables influence this deviation map revealing a significant association with population density, suggesting that locations with lower population density were more likely to have a higher probability of aegyptism without arbovirus. Characterizing regions of the world with established populations of Ae. aegypti but little to no autochthonous transmission of human-amplified arboviruses is an important step in understanding and achieving aegyptism without arbovirus.

Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration., Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules.

Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the ten species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (∼150 kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen V. dahliae. Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single repetitive element is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, yet only a minority could be linked to centromeres, suggesting that centromeres played a minor role in chromosomal evolution. Nevertheless, the size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus Verticillium.IMPORTANCEThe genus Verticillium contains ten species of plant-associated fungi, some of which are notorious pathogens. Verticillium species evolved by frequent chromosomal rearrangements that contribute to genome plasticity. Centromeres are instrumental for separation of chromosomes during mitosis and meiosis, and failed centromere functionality can lead to chromosomal anomalies. Here, we used a combination of experimental techniques to identify and characterize centromeres in each of the Verticillium species. Intriguingly, we could strongly associate a single repetitive element to the centromeres of some of the Verticillium species. The presence of this element in the centromeres coincides with increased centromere sizes and genome-wide repeat expansions. Collectively, our findings signify a role of repetitive elements in the function, organization and rapid evolution of centromeres in a set of closely related fungal species.

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuroinflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

SummaryTesticular development and function relies on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity decline in aging and disease. Whether the adult testis maintains a reserve progenitor population with repair or regenerative capacity remains uncertain. Here, we characterized a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that Tcf21+cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as reserve somatic progenitors following injury.In vitro, Tcf21+cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, Tcf21+cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be leveraged in development of future therapies for hypoandrogenism and/or infertility.HighlightsMultipotent Tcf21+MPs can differentiate into somatic testis cell typesTcf21+cells contribute to testis and ovary somatic cells during gonadal developmentTcf21+cells replenish somatic cells of the aging testis and in response to tissue injuryTestis Tcf21 cells resemble resident fibroblast populations in multiple organs

Root systems are dynamic and adaptable organs that play critical roles in plant development. However, how roots grow and accumulate biomass during plant life cycle and in relation to shoot growth phenology remains understudied. A comprehensive time-dependent root morphological analysis integrated with molecular signatures is then required to advance our understanding of root growth and development. Here we studied Brachypodium distachyon rooting process by monitoring root morphology, biomass production, and C/N ratios during developmental stages. To provide insight into gene regulation that accompanies root growth, we generated comprehensive transcript profiles of Brachypodium whole-root system at four developmental stages. Our data analysis revealed that multiple biological processes including trehalose metabolism and various families of transcription factors (TFs) were differentially expressed in root system during plant development. In particular, the AUX/IAA, ERFs, WRKY, NAC, and MADS TF family members were upregulated as plant entered the booting/heading stage, while ARFs and GRFs were downregulated suggesting these TF families as important factors involved in specific phases of rooting, and possibly in regulation of transition to plant reproductive stages. We identified several Brachypodium candidate root biomass-promoting genes and cis-regulatory elements for further functional validations and root growth improvements in grasses.

Mosquito-borne viruses are emerging or re-emerging globally, afflicting millions of people around the world. Aedes aegypti, the yellow fever mosquito, is the principal vector of dengue, Zika, and chikungunya viruses, and has well-established populations across tropical and subtropical urban areas of the Americas, including the southern United States. While intense arboviral epidemics have occurred in Mexico and further south in the Americas, local transmission in the United States has been minimal. Here, we study Ae. aegypti and Culex quinquefasciatus host feeding patterns and vertebrate host communities in residential environments of South Texas to identify host-utilization relative to availability. Only 31% of Ae. aegypti blood meals were derived from humans, while 50% were from dogs and 19% from other wild and domestic animals. In Cx. quinquefasciatus, 67% of blood meals were derived from chicken, 22% came from dogs, 9% from various wild avian species, and 2% from other mammals including one human, one cat, and one pig. We developed a model for the reproductive number, R0, for Zika virus (ZIKV) in South Texas relative to northern Mexico using human disease data from Tamaulipas, Mexico. We show that ZIKV R0 in South Texas communities could be greater than one if the risk of human exposure to Ae. aegypti bites in these communities is at least 60% that of Northern Mexico communities. The high utilization of non-human vertebrates and low risk of human exposure in South Texas diminishes the outbreak potential for human-amplified urban arboviruses transmitted by Ae. aegypti.