The mainstay of hemodynamic treatment of septic shock is fluid resuscitation followed by vasopressors where fluids alone are insufficient to achieve target blood pressure. Norepinephrine, a catecholamine, is the first-line vasopressor used worldwide but given that all routinely used catecholamines target the same adrenergic receptors, many clinicians may add a non-catecholamine vasopressor where refractory hypotension due to septic shock is present. However, the timing of this additional intervention is variable. This decision is based on three key factors: availability, familiarity, and safety profile. In our opinion, further consideration should be potential vasopressor response because following appropriate volume resuscitation, the response to different vasopressor classes is neither uniform nor predictable. Critically ill patients who are non-responders to high-dose catecholamines have a dismal outcome. Similarly, patients have a variable response to non-catecholamine agents including vasopressin and angiotensin II: but where patients exhibit a blood pressure response the outcomes are improved over non-responders. This variable responsiveness to vasopressors is similar to the clinical approach of anti-microbial sensitivity. In this commentary, the authors propose the concept of “broad spectrum vasopressors” wherein patients with septic shock are started on multiple vasopressors with a different mechanism of action simultaneously while the vasopressor sensitivity is assessed. Once the vasopressor sensitivities are assessed, then the vasopressors are ‘de-escalated’ accordingly. We believe that this concept may offer a new approach to the treatment of septic shock. Sepsis remains the most common cause of vasodilatory shock worldwide. International consensus guidelines describe specific recommendations regarding treatment. These include the timing of important interventions comprising blood culture collection, initiation of broad-spectrum antibiotics, blood glucose targets, use of steroids, and restoration of optimal hemodynamic status . The maiGiven that all routinely used catecholamines target the same adrenergic receptors, most clinicians are inclined to add a non-catecholamine vasopressor to treat patients with refractory hypotension due to septic shock, a decision based on three key factors: availability (a relatively rigid constraint), familiarity (often governed by previous practice), and safety profile. In our opinion, further consideration should be potential vasopressor response. Following appropriate volume resuscitation, the response to different vasopressor classes is neither uniform nor predictable. Furthermore, the responsiveness to a vasopressor may impact the outcome. Indeed, norepinephrine, recognized as the first-line vasopressor, often demonstrates a variable response which may be due to various factors which include pre-existing therapy/medications, genetics, the underlying pathophysiology of septic shock, and/or receptor responsiveness –10. BothNon-responders to high-dose catecholamines have a dismal outcome . In termWe propose the notion of “broad spectrum vasopressors” wherein patients with septic shock are started on multiple vasopressors with a different mechanism of action simultaneously while the vasopressor sensitivity is assessed. Vasopressor sensitivity could be assessed by sequential removal of vasopressors or developing a vasopressor sensitivity panel. Once the vasopressor sensitivities are assessed, then the vasopressors are de-escalated accordingly. However, this concept is hampered by several issues. Firstly, there is currently no bedside test that predicts the blood pressure response to catecholamines, vasopressin, or angiotensin II. Secondly, not all of these vasopressors are currently available worldwide due to either a lack of regulatory approval or cost considerations. Thirdly, there are no prospective data supporting this approach. Despite these hurdles, we feel that this is a testable hypothesis: Does time to sensitive vasopressor response improve outcomes in septic shock? We suggest this is a question worth answering and may prove an essential approach in managing these critically ill individuals.


Porites astreoides coral larvae using in situ chambers on reefs differing in habitat quality . Mean larval settlement was twice as high in an acoustic environment with high levels of low-frequency sounds, typical of a high-quality, healthy reef; this result was observed in both natural light and dark treatments. Overall, the enhancement of coral settlement by soundscapes typical of healthy reefs suggests a positive feedback where soundscape properties of reefs with elevated coral and fish abundance may facilitate coral recruitment.The settlement of reef-building corals is critical to the survival and recovery of reefs. Recent evidence indicates that coral larvae orient towards reef sound, yet the components of the acoustic environment that may attract coral larvae and induce settlement are unknown. Here we investigated the effects of ambient soundscapes on settlement of  Most marine invertebrates with sessile juvenile and adult life stages, including reef-building corals, produce planktonic larvae whose settlement into a favourable habitat is critical to the maintenance and growth of adult populations. For these organisms, larval habitat selection and settlement are key life-history processes . SettlemCoral planulae respond to physical variables such as light and pressure, possibly facilitating their encounter with the substrate and the selection of favourable depths and light regimes in which to settle –7. Reef Orbicella faveolata moved toward speakers playing reef sounds [Porites astreoides coral larvae at well-characterized reef sites while testing for the light dependence of the acoustic cues. The aim was to expand upon the previous study of soundscape effects on coral settlement, using larvae of a common coral species in the Caribbean [Biological and physical acoustic cues emanating from potential settlement sites can provide larvae with relevant sensory information complementary to the local substrate and water properties. Soundscape variation has been found to influence reef-fish and crustacean settlement –12, but f sounds . A fieldf sounds , but ligaribbean  and reef2.2.1.Porites astreoides were collected on reefs off the island of St John, in the US Virgin Islands  on 22 June 2017 from 10 m depth. These broodstock colonies were not collected at the experimental test sites but rather an adjacent reef outside the Virgin Islands National Park where we were permitted for coral collection. Prior to the field tests, colonies were maintained in a shaded outdoor ambient seawater-supplied aquarium. Six coral colonies spawned overnight during the July new moon (22–24 July) and larvae were collected each morning and maintained in 0.2 µm filtered seawater. Larval production ranged from 53 to 388 larvae over three nights. On 25 July, larvae from all colonies and spawning nights were pooled, and 18 groups of 55 actively swimming larvae were selected . Groups were randomly assigned to one of nine light or nine dark 140 ml polypropylene chambers (preconditioned with reef water for one month) filled with 0.7 µm filtered seawater to remove zooplankton grazers but retain seawater bacteria and smaller phytoplankton.Eight colonies of the brooding coral Each chamber contained two preconditioned (one month) settlement surfaces: a clay stilt (3.8 cm diameter) and a red cable tie . Sites differed in biophysical habitat characteristics (c). Light sensors were positioned facing upward and sampled every 10 min. The six chambers on each pole were positioned in random vertical order, alternating the side of the pole to which they were affixed so that no chamber was directly above another, to limit shading effects. For the reef sites, the experimental set-up was placed in the approximate centre of the reef structure.Following larval addition, three light and three dark settlement chambers were each affixed to a 0.75 m vertical pole deployed at three sites: Tektite Reef , Cocoloba Reef  and an off-reef sand site with no reef structure within 100 m  c. Sites eristics  known toeristics c. Light −1 [Larvae were completely isolated within settlement chambers, allowing exposure to ambient sounds (polypropylene plastics have high acoustic transparency with attenuation between 0.24 and 0.5 dB mm−1 ) while p−1 ,14. In o2.3.Acoustic recordings were analysed to compare the experimental soundscapes, initially by examining acoustic spectra (sound power as a function of frequency). Digital recording samples were analysed using MATLAB code written to compare the acoustic characteristics of the three sites for the duration of the experiment. Frequency composition of the ambient soundscapes was compared using acoustic spectra (sound power as a function of frequency) across measured frequencies (50–20 000 Hz). Mean power spectral densities were estimated  within 1-min samples across the total experiment length (62 h). Root-mean-square (RMS) sound pressure levels  were calculated for each site in the 1-min samples, within two frequency bands of interest. The lower analysis band (50–1000 Hz) contains the majority of fish-produced acoustic signals, as well as noise generated by wind and waves, while the higher analysis band (1000–20 000 Hz) primarily represents the acoustic energy derived from invertebrate sounds (e.g. snapping shrimp) . AdditioBenthic cover and fish diversity and abundance at the sites were characterized during July 2017 using SCUBA-based visual surveys as previously described [3.3.1.−1) with minimal settling on red zip ties or chamber walls (0–4 chamber−1). Larval settlement differed significantly by site . Notably, the standard deviations were generally smaller in the dark than light treatments for all sites.Coral larvae settled in all chambers, primarily on clay stilts . Tektite Reef was highest in lower-frequency acoustic amplitudes. Low frequencies on coral reefs tend to reflect higher fish abundance and diversity and are also the frequencies sensitive to many marine invertebrates [figure 2d). The Tektite Reef soundscape differed from the other sites predominantly with a notable peak in acoustic power between 300 and 800 Hz (figure 2a). Cocoloba Reef and the off-reef site showed a similar frequency composition (figure 2a), with broad peaks in spectra from 100 to 300 Hz and 2 to 10 kHz, but Cocoloba Reef was approximately 10 dB (ca 3×) louder.Sound levels differed significantly between experimental sites across frequencies . In the lower-frequency, fish-dominated band, Tektite Reef demonstrated crepuscular chorusing, with increased activity throughout the daytime (figure 2b(i)); this fish chorusing was less evident at Cocoloba Reef and absent off-reef ). In the high-frequency snapping-shrimp band, Cocoloba Reef and the off-reef site showed strong crepuscular peaks and night-time dominance while Tektite Reef exhibited asymmetrical peaks (figure 2b). Overall, reef soundscapes were higher in sound levels at all frequencies (figure 2c) compared with the off-reef site, and Tektite Reef showed the highest sound levels in the fish-dominant frequency band.Temporal trends reveal marked acoustic differences among sites b. In the4.Porites astreoides. Our data establish that soundscape variation can influence coral settlement in this species, regardless of light environment, and demonstrate highest larval settlement under exposure to the soundscape of a healthier reef environment, with abundant hard corals, sponges and fishes. Previously, larvae of the free-spawning coral Orbicella faveolata were found to orient and move towards reef sound [a). This implies that the acoustic driver of larval response to the Tektite Reef soundscape was more specific than simply higher sound levels, and could have resulted from the sounds of specific fish (e.g. the 300–800 Hz peak) and/or the strong crepuscular chorusing that were both absent from the Cocoloba Reef and off-reef sites. An alternative possibility is that coral larvae may only respond to low-frequency (50–1000 Hz) acoustic cues above a certain intensity threshold. This would also lead to the results we observed if the threshold intensity fell between that of Tektite Reef and Cocoloba Reef. Distinguishing between the two possible drivers of the observed results  warrants further study.This field experiment was designed to test the effect of habitat-related soundscape variability on the larval settlement of a common Caribbean brooding coral ef sound  and to sef sound . Similaref sound . Indeed,Together, the habitat surveys, acoustic monitoring and settlement results indicate a possible link between habitat quality, soundscape and coral settlement whereby diverse and abundant soniferous communities may provide cues for settling corals. However, precisely which acoustic cues are important and whether variation in their levels or diel patterns affect settlement remain open questions. Moreover, while this study expands knowledge of coral settlement processes by establishing that soundscape cues can influence settlement for a coral species with a brooding reproductive strategy, the roles of genetic variation, parental effects and local adaptation in this larval response are unknown. Further work is needed to test how widely applicable acoustic settlement cues are across coral species and geographies.Coral larval settlement was enhanced at Tektite Reef both in the dark and ambient light treatments, suggesting that the soundscape is a cue used both in the natural light and dark environments. While settlement responses to soundscape variation were found irrespective of light environment, the influence of soundscape was most pronounced when coral larvae were required to settle in the dark. Sound may be a particularly important cue in light-limited environments such as at night or on mesophotic reefs and is a compelling avenue for future settlement cue investigation. Owing to the nature of the light scattering environment, our study sites did differ in light intensity, but this variation did not appear to relate to observed settlement patterns. There also tended to be greater standard deviations under natural light compared with dark conditions. Perhaps this was related to the naturally varying light cue, or perhaps there is an interaction of light and sound conditions. Further study would be needed to disentangle the individual and synergistic effects of these variables. To our knowledge, no previous studies have examined coral settlement in darkness, and here we demonstrate that the settlement of algal symbiont-bearing corals can indeed occur in darkness. Coral larvae are known to have settlement preference for particular light intensities  and specOverall, we demonstrate that increased coral settlement occurs in the presence of a low-frequency, fish-chorus dominated soundscape. Such a soundscape is often representative of a healthier, more diverse reef. Further, the influence of the soundscape is important to coral larvae independent of light environment. The coral larvae in this study appear to respond to the soundscape of a favourable settlement site, shown here to be a reef with higher sound levels in fish calling acoustic frequencies. This suggests a positive feedback loop where reefs of higher coral cover and fish abundance generate soundscapes best suited to attract coral larvae, which in turn sustain the healthy coral community and habitat for fish (similar to a mechanism proposed for fish ). It fol


ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci  also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the  Alternative splicing of pre-messenger RNA (mRNA) generates multiple transcript isoforms from a single gene that can code for proteins with distinct functions. Examples include distinct ligand recognition of the b- versus c-isoforms of fibroblast growth factor (FGF) receptors , the proBone marrow continuously replenishes differentiated blood cells from a small pool of stem cells. Studies of this process have provided many of the concepts of tissue regeneration from resident stem cells ,13,14. HHealthy Bone Marrow Donors—The study was reviewed and considered as “exempt” by the Institutional Review Board of Georgetown University (IRB # 2002-022). All methods were carried out in accordance with relevant guidelines and regulations. Freshly harvested bone marrow tissues were collected from discarded healthy human bone marrow collection filters that had been de-identified. cDNA libraries for the short-read RNA-seq and the full-length RNA-seq were isolated from two separate healthy donors. From one donor, three cDNA library preparations were generated for total bone marrow, lineage-negative, and lineage-positive cells. Full-length RNA-seq of the samples was done at Pacific Biosciences. From an additional donor, a cDNA library was generated from lineage-negative cells. This was sequenced at the Mt. Sinai Sequencing Facility. Independent sequencing results from the same donor and from the additional donor were used for validation of the full-length RNA-seq results . Lineage-positive cells bound to the antibodies were removed by magnetic beads and lineage-negative cells obtained from the flow-through. To increase purity, lineage-negative cells were enriched two times.Short-read RNA-seq: Sequencing of fragmented cDNAs—Total RNA was submitted to Otogenetics Corporation  for RNA-seq. Briefly, the integrity and purity of total RNA were assessed using Agilent Bioanalyzer by OD260/280 ratio. Altogether, 5 μg of total RNA was subjected to rRNA depletion using the RiboZero Human/Mouse/Rat kit . cDNA was generated from the depleted RNA using random hexamers or custom primers and Superscript III . The resulting cDNA was purified and fragmented using a Covaris fragmentation kit , profiled using an Agilent Bioanalyzer, and Illumina libraries were prepared using NEBNext reagents . The quality, quantity, and the size distribution of the Illumina libraries were determined using an Agilent Bioanalyzer 2100. The libraries were then submitted for Illumina HiSeq2000 sequencing. Paired-end 90 or 100 nucleotide reads were generated and checked for data quality using FASTQC , and DNAnexus  was used on the platform provided by the Center for Biotechnology and Computational Biology  . ORFs accepted were the first ORFs and not necessarily the largest. In some cases, both the first and the largest ORF were included and designated with letters a, b, etc., appended to the end of the name assigned.Multiple Sequence Alignment—multiple sequence alignment was done using Clustal Omega available at EBI .Sequence Alignment Editing—the sequence alignments were edited using BioEdit version 7.2.5. This Sequence Alignment Editor written for the windows environment was run on OSX Yosemite on a Mac Book Pro through the use of the wine version 1.6.2, a windows emulator available for download, and installation through Home Brew version 0.9.5.Naming Convention—the names generated by the cDNA Primer software were used to create the nomenclature that relates to the deposited transcript structures as well as the uniprot deposited protein isoforms.Quantitation of Abundance—all figures were generated through custom R scripts permitting alignment of abundance with isoforms. The full-length sequence reads were aligned to the hg19 reference genome using gsnap. The fragmented sequence reads were aligned to the hg19 reference using Tophat 2 and the aligned and paired reads were assembled using Cufflinks 2 and genes.gtf also from hg19 annotation as a guide [ a guide . QuantitCode availability—custom code is available upon request.total human bone marrow cell population (T) dominated by differentiated cells with the small (<1%) subpopulation of lineage-negative progenitor cells (N) using single-molecule real-time (SMRT) sequencing of unfragmented cDNAs . Utilizing complete transcriptomes generated either by full-length or short-read RNA-seq, we then describe the composite results and provide a comparison with protein fragments identified by mass spectrometry. The overall experimental flow is shown in We compared the Distinct novel transcript isoforms of EEF1A1 detected in bone marrow cell subpopulations by full-length RNA-seq—eukaryotic translation elongation factor 1 α 1 (EEF1A1) is a highly abundant, conserved protein that delivers aminoacyl-tRNAs to the ribosome during protein synthesis but has also been found to contribute to additional cellular functions [EEF1A1 gene spans 5.2 kb on chromosome 6q13 and results in a 3.5 kb transcript that contains six protein coding exons  of approximately 70 amino acids, each with a motif for calcium binding. These sequences are highlighted in yellow and the alignment of the predicted ORFs from transcript isoforms in total and lineage-negative bone marrow cells shows the overlaps with the canonical protein and with each other  code for the canonical  UTR see c. ORF 2  UTR see . FinallyHigh complexity of the transcriptome revealed by full-length RNA-seq—as described above for ANXA1 and EEF1A1, full-length RNA-seq of bone marrow cell populations revealed an up to 10-fold higher number of transcript isoforms than found by short-read RNA-seq. To investigate the universality of isoform complexity, we evaluated genes having <6 coding exons  and found that RNA-seq detected 1 to 4 isoforms while full-length RNA-seq identified 12 to 36 isoforms. In a repeat sample, we identified as many as 15 isoforms with full-length RNA-seq, and in a sample from an additional donor we identified a maximum number of 48 isoforms for these genomic loci. We then considered loci with >6 coding exons. Short-read RNA-seq yielded a similar number of isoforms as it had with <6 exons, i.e., 1 to 3 isoforms compared with 31 to 43 transcript isoforms identified by full-length RNA-seq ( RNA-seq a. Similap = 0.0014). It is worth noting that an increase in sequencing depth for short-read RNA-seq from 20 to 100 million reads did not impact this maximum significantly ; 26% to 58% of aligned reads showed the top matchAnnot score of >5 relative to known transcript isoforms deposited in the data base  4- to 6-fold gain in information. Given that over half of the detected transcripts are from multi-exon genes, the ability to span two exons with short reads may be inadequate to resolve a full-length transcript successfully without the addition of longer reads [ANXA1 and EEF1A1 above and an overview of genomic loci interrogated, full-length RNA-seq reveals complex posttranscriptional processing . From a combination of these two approaches we confirmed an average of 83% of the transcript isoforms  approach established earlier [Our transcriptome analysis of the human bone marrow progenitor and differentiated cell populations revealed a previously unknown complexity of the transcripts when analyzed by single-molecule real-time (SMRT) sequencing of unfragmented cDNAs, i.e., full-length RNA-seq. With a direct reading of full-length RNA sequences—rather than computational assembly from read fragments—open reading frames are predicted more reliably and a proteomics analysis for the respective predicted proteins showed that one third of them were present at high enough abundance to be detected by mass spectrometry. The full-length RNA-seq analysis described here is the most recent approach that relies on the long reads possible with the PacBio technology  and the  earlier  and favoD. melanogaster and C. elegans but not in H. sapiens. This is likely due to the lower number of transcript isoforms per genomic locus found in the invertebrates—fewer than 25% of genes in C. elegans and D. melanogaster give rise to more than two transcript isoforms, whereas human genes are currently annotated with an average of five transcript isoforms. Beyond this, short-read RNA-seq may identify too few isoforms due to ambiguous assignment of a given read to multiple loci. Altogether, this will impede the ability to detect novel isoforms [Numerous computational experiments were conducted exploring alternative methods, including using Trinity and Oasis to perform de novo assembly of the short reads alone, then combined with the full-length reads. These were experiments with varied success. Parts of transcripts would often be confirmed, but the process was both time consuming and computationally costly with unsatisfactory outcome. It is remarkable that the complexities of the transcriptome and hence distinction between subpopulations of bone marrow cells described above were confirmed but not identifiable de novo by the conventional, short-read RNA-seq of fragmented cDNAs. Indeed, short-read RNA-seq uses computational methods to reconstruct transcripts from the sequence fragments and can uncover novel exon-exon junctions. However, for acceptable performance, reconstruction requires a transcript isoform database to resolve the origin of isoforms. This will bias assignments of sequence fragments and limits discovery of isoforms of higher complexity . Thus, wisoforms ,31,32.HLA-A and -C, respectively (The most striking examples that reveal the limitations of the short-read RNA-seq are paralogous gene loci where only full-length RNA-seq detected gene expression c. Mass sectively . These fAn analysis of splicing complexity by gene size showed that the combination potential for exon splicing can increase exponentially . The autWe conclude that single-molecule real-time full-length RNA-seq provides a map of the complexity of transcript isoform structures that eludes the transcriptome analysis by conventional short-read RNA-seq. In addition, open reading frames of proteins derived from these full-length transcript isoforms enables the comparison of the presence and absence of specific binding sites, domains, and regulatory regions in proteins that can be related to the cell population and physiologic or pathologic status . In partFinally, as shown above, protein isoforms contain isoform-specific amino acid sequence junctions that represent epitopes specific for cell subpopulations in complex tissues. It is conceivable that pathologic regulation of splicing in diseases such as neurodegenerative disorders, metabolic syndrome, or cancer can generate neo-epitopes and elicit an immune response irrespective of genomic mutagenesis. Unraveling the complexity of the transcriptome complemented by analysis of resulting proteins thus may provide new diagnostic options and unique therapeutic targets.


Bacillus thuringiensis (Bt) toxin perforation. The complete ABC transporter is composed of two hydrophobic transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). Conformational changes that are needed for their action are mediated by ATP hydrolysis. According to the similarity among their sequences and organization of conserved ATP-binding cassette domains, insect ABC transporters have been divided into eight subfamilies (ABCA–ABCH). This review describes the functions and mechanisms of ABC transporters in insecticide detoxification, plant toxic secondary metabolites transport and insecticidal activity of Bt toxin. With improved understanding of the role and mechanisms of ABC transporter in resistance to insecticides and Bt toxins, we can identify valuable target sites for developing new strategies to control pests and manage resistance and achieve green pest control.ATP-binding cassette (ABC) transporters, a large class of transmembrane proteins, are widely found in organisms and play an important role in the transport of xenobiotics. Insect ABC transporters are involved in insecticide detoxification and  Escherichia coli that was directly energized by hydrolysis of ATP [Salmonella typhimurium (coded by the hisP gene) and maltose-maltodextrin transport system of E. coli  were cloned [ATP-binding cassette (ABC) proteins comprise an extensive and variable transporter superfamily within P-loop motif and are found in all living organisms ,2,3. Stus of ATP ,5. In 19e cloned ,7. Concue cloned ,9. Evente cloned . On the e cloned .The primary function of most ABC proteins is ATP-dependent active transport of a broad spectrum of substrates including amino acids, sugars, heavy metal ions and conjugates, peptides, lipids, polysaccharides, xenobiotics and chemotherapeutic drugs across cellular membranes ,12,13,14white gene in Drosophila melanogaster was the first identified insect ABC transporter gene and is involved in the transport of eye pigment precursors [white has also been confirmed in Bombyx mori and Tribolium castaneum [ABCC genes are associated with the resistance to Cry toxins from Bacillus thuringiensis (Bt) by reducing the binding affinity of Cry toxins to the brush border membrane vesicles in different lepidopteran species [Although the ABC transporters have been recognized to be associated with multidrug resistance in humans, bacteria and nematodes, their functional role in arthropods has not been fully studied . Althougecursors . The rolastaneum ,20. The astaneum ,22,23. I species ,25,26. OExcellent reviews on the role of insect ABC transporters in the transport and resistance to Bt toxin and insecticide have been published previously ,27,28, sE. coli and exporters such as the multidrug exporter Sav1866 from Staphylococcus aureus or related flippases such as MsbA lipid flippase from E. coli [Structural models of ABC transporters are based on the crystal structure of different bacterial proteins that act as importers such as vitamin B12 transporter BtuCDF from  E. coli ,30,31. O E. coli ,33. The  E. coli ,35,36,37 E. coli ,15; therd-loop, which contains an aspartate residue and is responsible for forming a salt bridge; Q-loop, which contains a glutamate residue and acts as the attacking nucleophile in ATP hydrolysis; H motif, which has an invariant histidine active site that may be involved in maintaining the stability of the pre-hydrolytic state; and an α-helical region that has the ABC signature sequence (LSGGQ motif) [The structure of ABC transporters is highly conserved among most eukaryotic organisms, including insects. A functional ABC transporter is characterized by the presence of a P-type traffic ATPase, which comprises two cytosolic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) ,12,38 FA. The foQ motif) ,39. The ABC transporters have a common mechanism for exporting substrates across the membrane by hydrolyzing ATP as a pump, but other models have been proposed for the ABC transporter mechanism based on structural and biochemical evidence, including the ATP-switch , alternaD. melanogaster, B. mori, Helicoverpa armigera and Plutella xylostella, have been reported . ABCA genes that encode only FTs are present in T. castaneum, Anopheles gambiae, D. melanogaster, Apis mellifera, Daphnia pulex, Tetranychus urticae and Bemisia tabaci [B. mori genome [TcABCA-9A or TcABCA-9B in T. castaneum by injection of dsRNA leads to developmental defects in the wings and elytra and to about 30% mortality at the adult eclosion stage [According to the distinctive conserved traits, ABCA subfamily proteins can be divided into full transporters (FTs) and half transporters (HTs); the number of insect a tabaci ,58. Two i genome , whereasi genome . In mammi genome , but liton stage . This sion stage ,67. As ion stage .T. castaneum, An. gambiae, D. melanogaster, B. mori and A. mellifera, the number of FTs are 2, 2, 4, 5 and 1, and the number of HTs are 4, 3, 4, 4 and 4, respectively. A. mellifera also contains two incomplete ABCB sequences. Similarly, two genes encoding FTs, and five and two genes encoding HTs were found in the D. pulex and T. urticae genomes, respectively. The function of the ortholog of the human half transporter HsABCB7 in D. melanogaster was studied by RNA interference and found to play a key role in cellular iron homeostasis [CG4225, homologous to human ABCB6 in D. melanogaster, is also known as DmHMT-1, which is responsible for resistance against cadmium [CG7955 in D. melanogaster, is correlated with chill coma stress resistance [+/K+-ATPase [ABCB subfamily transporters also contain FTs and HTs. In the genome of eostasis . CG4225, cadmium . Anothersistance . The per+-ATPase . ABCC genes, with the most found to date in T. urticae, followed by T. castaneum and Chrysomela populi. There are 35 genes encoding ABCC transporters in T. castaneum, and expansion of TcABCC gene mainly occurs on chromosome 5 and affects genes of the “short” MRP group [B. mori can be divided into two groups, one of which has very high similarity with the ABCB subfamily [B. mori ABCC subfamily consists of five FTs, seven HTs and three incomplete ABC sequences. The ABCC transporters of most insects with known genomes are also composed of FTs and HTs, except for D. melanogaster; its 14 ABCC transporters are FTs. Some ABCC transporters also contain an additional N-terminal TMD (TMD0) [D. pulex, An. gambiae, D. melanogaster and T. castaneum [The functions of ABCC subfamily in mammals mainly include ion transport, signal transduction, and toxin secretion . The ABCRP group . The ABCubfamily . The B. D (TMD0)  and haveastaneum ,47,48. ABCD genes, but a few insects, such as Danaus plexippus and P. xylostella, contain three [The ABCD subfamily is composed of highly conserved HTs, located on the peroxisome membrane, that form heterodimers and mediate the transport of acyl coenzyme A esters in organisms ,74. Mostin three ,60. Becain three .ABCE gene, which is highly conserved [ABCE is annotated as RNase L (Rli1) inhibitor in eukaryotes and may have a general function in innate immunity [T. castaneum with either TcABCE-3A- or TcABCF-2A-specific dsRNA leads to a lethal phenotype [Compared with other ABC transporters, both ABCE and ABCF proteins have no TMDs, and each member contains a pair of adjacent NBDs . Due to onserved . The ABConserved . ABCE anonserved ,77,78. Aimmunity . Injectihenotype .ABCG genes in an insect species ranges from 2 to 24. The white proteins of D. melanogaster can form dimers with one of the other two HTs named Brown and Scarlet and play a crucial role in the cellular uptake of pigment precursors in the eye [white gene of D. melanogaster is present in all known insect genomes, but the brown and scarlet genes are only present in some insects [white ortholog of T. castaneum found that the transport precursor of a pigment across the membrane of Malpighian tubule cells is ABCG-dependent [B. mori. The ABCG HTs homologous to brown in aphids, lepidopteran and hymenopteran species can form dimers with white to participate in the transport of urate [B. mori genome, white and scarlet are juxtaposed in a head-to-tail orientation, which indicates that the origin of white and scarlet resulted from the tandem duplication of an ancestral gene. The same arrangement was also found in other insect species that lack a functional ortholog of the brown gene [ABCG transporters are typical HTs, which must form homo- or heterodimers to fulfill their transport function. Compared with other half-transporter proteins, ABCG transporters have a reverse domain structure; its TMD is connected to the C-terminal region of NBD . Some AB the eye . An orth insects . A studyependent . White iof urate . In the own gene . D. melanogaster, then in other insects; they have not been found in mammals, plants, fungi and C. elegans [ABCH genes were identified in the Ptychadena nana genome [ABCH1 gene of P. xylostella with a high dose of dsRNA results in lethal larval and pupal phenotypes [ABCH1 may be an excellent target for pest control. Injection of dsRNA of TcABCH-9C causes dehydration and death of T. castaneum larvae [Locusta migratoria [D. melanogaster [ABCH transporters were first found in  elegans ,82,83. H elegans , but rep elegans ,86. Foura genome . Similara genome . Three Aenotypes ; thus ABm larvae . ABCH-9Cgratoria ,89 and DGenerally, insect resistance to xenobiotics is due to point mutations at target sites, or the effective metabolism or sequestration of toxic substances ,91. The T. castaneum), the Cap’n’collar C isoform (CncC) is involved in all three phases of insecticide detoxification and regulates the expression of ABCA-UB, ABCA-1A and ABCA-1AL. RNAi and an insecticide bioassay showed that these ABCA genes contributed to the susceptibility of T. castaneum to pyrethroid [Xenobiotic transcription factors (XTFs) are also important in regulating the expression of genes that encode proteins involved in detoxification. Cap’n’collar (Cnc) transcription factors are members of the XTF superfamily ,96. In trethroid . This stAedes aegypti, temephos toxicity significantly increased (57%) in the P-gp-silenced mutant [H. armigera to abamectin and indoxacarb [H. armigera to insecticides [D. melanogaster is directly related to abamectin resistance, and the level of P-gp in blood-brain barrier of resistant D. melanogaster is significantly higher than in the susceptible control [D. melanogaster [ABCB4 through gene amplification has been detected in pyrethroid-resistant Ae. aegypti [D. melanogaster. Among them, the resistance conferred by Mdr49 was related to its splice-form variant and amino acid residue changes [D. melanogaster with a Mdr65-gene knockout via CRISPR/cas9 had increased susceptibility to all neuroactive insecticides tested. Deficiency crosses, synergism with the ABC inhibitor verapamil and accumulation of pesticides in the Mdr65-knockout individuals further confirmed that Mdr65 plays an important role in pesticide transport [Mdr65 also confirmed the role of the gene in reduced toxicity of multiple insecticides in D. melanogaster [Among ABC transporters family, the ABCB subfamily mostly represents the P-gps and multiple drug resistance (Mdr) proteins. P-gps are ATP-dependent efflux pumps, which are closely related to the transmembrane transport of substances. Some of these proteins are associated with multidrug resistance . When RNd mutant . Jin et doxacarb . Previoucticides . An incr control ,102. In-nogaster . Over-ex aegypti . ABCB tr changes ,104. D. ransport . Tissue-nogaster . TrnMRP1 and TrnMRP4 are highly expressed in the Malpighian tubules in the lepidopteran Trichoplusia ni and may be involved in excreting metabolic wastes or ingested xenobiotics [B. tabaci showed that an ABC transporter of subfamily G is upregulated when exposed to thiamethoxam [P. xylostella (diamondback moth), suggesting that these ABC transporters may be related to the transport or detoxification of different pesticides [HaOG200303 from subfamily C, HaOG200310, HaOG200353 and HaOG200354 from subfamily G and HaOG200341 from subfamily H were significantly upregulated in H. armigera when treated with both lambda-cyhalothrin and indoxacarb [PhABCC4 of Pediculus humanus humanus expressed in Xenopus oocytes functioned in ivermectin efflux, suggesting that PhABCC4 might be involved in ivermectin transport [Many ABCC transporters can specifically transport a variety of drugs, so they are also called multidrug resistance proteins (MRPs). obiotics . Microarmethoxam ,109. Comsticides ,111. Thedoxacarb . Howeverransport .D. melanogaster indicates that Mdr is involved in the detoxification of plant secondary metabolite cardenolides [CpMRP (ABC subfamily C) of C. populi revealed that CpMRP acts as a pacemaker, transporting specific metabolites from the hemolymph to defensive secretions, and eventually excreting them from the body. Silencing of CpMRP makes larvae defenseless, indicating CpMRP plays a key role in secretion [CpMRP were also identified in the defensive glands of two related leaf beetle species, suggesting that this ABC transporter is the key component of insect resistance to noxious phytochemicals. Plants can release a variety of toxic secondary metabolites to deter the feeding of herbivorous insects, and insects have evolved corresponding countermeasures via natural selection, specialization, sequestration during the long-term interactions between plants and insects . As partenolides . A studyecretion . HomologABC genes was studied in tissues of 5th instar larvae of H. armigera after they were fed an artificial diet supplemented with various plant secondary metabolites. Taxol induced the expression of ABCE1 in Malpighian tubules, upregulated the expression of ABCB1 in the gut and expression of ABCB1, ABCC2 and ABCC6 in the rest of the body [HaABCG11 was upregulated after larvae were fed a diet supplemented with nicotine compared to the control samples, and HaABCB3 expression was very high in the gut of H. armigera larvae after they fed on a diet containing nicotine or tomatine [ABCB3 was upregulated in Manduca sexta larvae that fed on Datura and Solanum. This plant-specific expression pattern may be related to the adaptability of insect to these plants [The expression level of the body . ABCB1 hthe body . The exptomatine . Similare plants . The funH. armigera. The resistance of two H. armigera strains to Cry2Ab was an independent evolutionary event involving different deletion mutations, which were located in different exons of the same ABCA2 gene [HaABCA2 was also detected in resistant lines of H. punctigera Cry2Ab. A similar situation was also found in resistant lines of Pectinophora gossypiella; a loss of PgABCA2 exon 6 was caused by alternative splicing in resistant larvae of laboratory-selected strains in Arizona and field-selected strains in India [H. armigera with CRISPR/cas9 knockout of HaABCA2 showed that HaABCA2 plays an important role in mediating resistance to Cry2Aa and Cry2Ab [The ABC transporters that act as Bt-toxin receptor belong to subfamilies A, B, C and D . With thCA2 gene . A homolin India . In addiin India ,119. A bd Cry2Ab .Chrysomela tremuela strain survived and reproduced on transgenic poplar trees that expressed a high level of Cry3Aa Bt toxin, and its Cry3Aa resistance was an autosomal recessive trait. Candidate resistance genes were analyzed using midgut transcriptome of larvae, demonstrating that a mutation of the ABCB homolog of P-gp was closely linked to the resistance of Cry3Aa, and named CtABCB1. A 4-bp deletion in CtABCB1 introduced a frame shift with a premature stop codon, resulting in the loss of transporter signature motifs 1 (TpM1) and transmembrane domain 2 (TMD2) [CtABCB1 homologous gene in western corn rootworm (Diabrotica virgifera virgifera) was also shown to be genetically linked to Cry3Bb1 resistance [A 2 (TMD2) . A CtABCsistance . PerhapsHeliothis virescens and cytotoxicity of three Cry1A toxins  in Spodoptera frugiperda Sf9 cells and found that ABCC2 is the central target of Cry1A toxin action [D. melanogaster has been used to validate the involvement of ABCC2 in Cry1Ac toxin binding [ABCC2 or a mutation causing a single amino acid change, could reduce binding of Cry1Ac toxin, conferring high levels of Bt resistance in the target pest [P. xylostella with a 30-bp deletion in exon 20, was expected to cause the removal of the 12th transmembrane (TM) domain and carboxyl terminal of TM12 that is located outside the cell. The mutation may result in the lack of function of a core ATP-binding loop [Some ABCC transporters are a functional receptor for more than one Cry1A toxin. The binding of Cry1Ac toxin to ABCC2 on membrane vesicles can lead to membrane perforation. Bretschneider et al. studied the relationship between the ABCC2 of n action . Heterol binding . An inacget pest ,124,134.ing loop . In addiing loop ,129. A cing loop . ABCC gene expression. The study of Bt resistance in hybrid larvae of B. mori revealed a trans-regulatory mechanism involved in the allele-specific expression of ABCC2 in response to Cry1Ab toxin, which may play an important role in insect Bt resistance [MAP4K4) in the MAPK signaling pathway [ABCC2 and ABCC3 genes in Sf9 cells [Bt resistance mediated by ABCC transporters involves not only different forms of gene mutations, but also the regulation of sistance . The bin pathway . In addif9 cells . ABCC2 aABCG1 (white) gene is one of the most widely studied members of the ABCG protein subfamily. The white protein in P. xylostella is located on the cell membrane, and the expression of Pxwhite gene in the midgut of a Bt-resistant population is significantly lower than in a susceptible population. After RNAi silencing of the midgut Pxwhite gene, the sensitivity of the larvae to Bt-Cry1Ac toxin significantly decreased [Ostrinia furnacalis is also related to its resistance to Cry1Ab and Cry1Ac toxins [ecreased . Geneticc toxins .Functional study of insect ABC transporters and their role in resistance to chemical insecticides and Bt toxins has shown that several ABC transporters are involved in toxin resistance. However, more progress is needed to fully understand the functions and detailed mechanisms of action. Different ABC transporters may recognize different chemicals or Bt toxins, and their TMDs have distinct binding sites that recognize substrates with highly diverse chemical properties . ABCA2 iABC transporters are the main receptors for most Bt toxins, so it is not surprising that Bt resistance in pests is commonly related to a mutation in an ABC transporter. Upstream regulatory factors such as MAP4K4 and FOXA can also change gene expression for ABC transporters, thereby altering susceptibility of pests to Bt toxins ,128. Theβ-actin gene in Colorado potato beetle was transferred to potato chloroplasts, and the transgenic plants were lethal to Colorado potato beetle larvae [acetylcholinesterase (AChE) gene involved in neuronal transmission and the ecdysone receptor (EcR) gene involved in transcriptional activation of development in transgenic plants can effectively control whitefly populations [Plant-mediated RNA interference provides a new method and model for pest control in the field. The dsRNA fragment targeting the e larvae . The useulations . BecauseWith the deepening of our knowledge on the structure and physiological function of insect ABC transporters, the mechanism of action of these transporters in pesticide resistance, Bt insecticidal activity and other physiological processes will become clearer, and new technologies will be developed for the effective control of insect pests.


Sexually transmitted infections (STIs) are predicted to play an important role in the evolution of host mating strategies, and vice versa, yet our understanding of host‐STI coevolution is limited. Previous theoretical work has shown mate choice can evolve to prevent runaway STI virulence evolution in chronic, sterilizing infections. Here, I generalize this theory to examine how a broader range of life‐history traits influence coevolution; specifically, how host preferences for healthy mates and STI virulence coevolve when infections are acute and can cause mortality or sterility, and hosts do not form long‐term sexual partnerships. I show that mate choice reduces both mortality and sterility virulence, with qualitatively different outcomes depending on the mode of virulence, costs associated with mate choice, recovery rates, and host lifespan. For example, fluctuating selection—a key finding in previous work—is most likely when hosts have moderate lifespans, STIs cause sterility and long infections, and costs of mate choice are low. The results reveal new insights into the coevolution of mate choice and STI virulence as different life‐history traits vary, providing increased support for parasite‐mediated sexual selection as a potential driver of host mate choice, and mate choice as a constraint on the evolution of virulence. Columba livia) prefer males without lice , suggesting support for PMSS—whereas in other cases females appear unable to distinguish between infected males—for instance, female milkweed leaf beetles  is predicted to lead to the evolution of reproductive strategies that limit the risk of infection from mating , in the evolution of host mating strategies, and the role of host mating behavior in the evolution of STIs  and disease causes variable mortality or sterility virulence, and for different recovery rates. Using evolutionary invasion analysis, I first show how mate choice leads to lower optimal levels of mortality and sterility virulence. I then show how and when mate choice is likely to evolve under different disease characteristics. Finally, I consider host‐STI coevolution, showing that coevolutionary cycling is typically more common under sterility virulence, whereas mortality virulence tends to lead to more stable outcomes. I also identify conditions when polymorphism in host mate choice can evolve through evolutionary branching, but this outcome only occurs under a narrow set of conditions. Finally, I examine how costs associated with mate choice, the rate of recovery from infection, and the lifespan of the host impact host‐STI coevolution. Combined with previous studies, these results show that PMSS can occur for a broad range of host and STI life‐history traits.S and I are the densities of susceptible and infected individuals, respectively; b is the host birth rate, which depends on the fecundity of infected hosts relative to uninfected hosts, f, with 0≤f≤1, the strength of mate choice , g≥0, and v, which is used to relate the mode of virulence to mate choice (defined below); d is the natural mortality rate; α is the disease‐associated mortality rate; β is the transmission probability per sexual contact; γ is the recovery rate; and [XY] is the mating rate between individuals in classes X∈{S,I} and Y∈{S,I} (see Table α=α(β), f=f(β)) as parasites may need to damage their hosts or use host resources to produce transmission stages, and the more transmission stages produced the greater the damage is likely to be to the host =1−ηβ for ηβ<1 and 0 otherwise for sterility virulence, and α(β)=κβ for mortality virulence, with η and κ parameters that define the strength of these relationships. Such functions correspond to a situation where the damage caused to the host is proportional to the number of transmission stages produced by the STI. I restrict my analysis to how one mode of virulence at most varies and affects mate choice by setting η=0 and/or κ=0 and by using v=v(β) to relate the current mode of virulence to mate choice, with v(β)=1−f(β) in the case of sterility virulence and v(β)=α(β)/κ in the case of mortality virulence.I model the dynamics of an STI in a well‐mixed host population, which for simplicity I treat as a single hermaphroditic sex as there is assumed to be no sex‐specific variation in disease characteristics. The epidemiological and mating dynamics of a one‐host‐one‐STI system are described bydSdt=b. If the prospective partner is currently uninfected, the probability that the focal host accepts the mate is mS(g), with dmSdg≤0. The function mS(g) allows for the fact that hosts may imperfectly assess the condition of other individuals and those who are choosier ) may be generally more cautious in their approach to mating, potentially declining healthy prospective partners. If the prospective partner is currently infected, the probability of accepting them as a mate is mI) with ∂mI∂g,∂mI∂v≤0. By causing more damage to their hosts, more virulent STIs may be easier to detect by prospective mates . Preferential mating with uninfected hosts is somewhat comparable to the notion of disease causing lower contact rates  in classical evolution of virulence theory  and mI) are the actual probabilities of accepting an uninfected or infected prospective partner as a mate, respectively. I use a dummy variable for the strength of mate choice so that the host responses to susceptible and infected individuals are correlated. Throughout, it is assumed that the probability of accepting an uninfected individual as a mating partner is at least as large as the probability of accepting an infected individual: mS(g)≥mI). Without this assumption, there would never be any advantages to mate choice, as choosier individuals would mate with infected members of the population at a higher rate than less choosy individuals. I therefore set mI)=mS(g)m∼I) with m∼I)≤1 the mate choice response specific to prospective partners who are infected. In the analysis that follows, I set mS(g)=1−ζg for ζg<1 and 0 otherwise, where ζ is the cost of mate choice, and either m∼I)=1−gv(β) (linear response) or m∼I)=1−gv(β)2 (nonlinear response) with both functions restricted to m∼I)≥0. I only examine a linear function for mS(g) because g is a dummy variable and therefore one only needs to consider linear and nonlinear forms of one of the two correlated mate choice functions. Biologically, the linear and nonlinear functions for m∼I) mean that the effects of mate choice either increase proportionately or accelerate with virulence. In other words, the linear function implies that the probability of accepting an infected mate is proportional to the damage caused by the STI, and the nonlinear function implies that hosts are disproportionately choosier when STIs are more virulent or that the STI is increasingly easier to detect. Although empirical evidence for how mate choice varies with virulence is currently lacking, it is plausible that either linear or nonlinear relationships could exist and therefore varying the shape of these functions is important for a better understanding of potential host‐STI coevolutionary dynamics.Note that S and I classes are given byThe mating rates for each combination of the [SI] appears when there is mating between individuals in different classes and is required to balance the total mating rate, M:The factor of 2 in the equation for mS(g)=mI)=1 , the total mating rate reduces to M=pN. For the general case, hosts that have mated produce offspring at a total rate ofr is the maximum reproduction rate per pair and the birth rate is subject to density‐dependent competition given by the parameter h.Note that for the specific case when = of this system occurs at(mS(g))2pr>d . A newly introduced STI will spread in a susceptible population when the basic reproductive ratio, R0, is greater than 1, whereThe disease‐free equilibrium nh hosts each with strength of mate choice gi, where i∈{1,…,nh} and np STIs each with transmissibility βj and virulence vj where j∈{1,…,np},are fully described by the following system of ordinary differential equations:[SiI∘j]=2pmISiN∑kmS(gk)Ikj, which is the total mating rate between susceptible hosts with trait gi and all hosts infected by STIs with traits βj and vj, and the birth rate for each host type isS∘ is the sum over all uninfected hosts, Ii∘ is the sum over all hosts of type i that are infected, I∘j is the sum over all hosts that are infected with parasite type j, and I∘∘ is the sum over all infected hosts), so thatThe above model describes the dynamics when there is only one host type and one STI type in the population. To account for situations where hosts vary in their strength of mate choice and STIs in the transmissibility/virulence, I adapt the above monomorphic model for populations that are polymorphic in these traits. The dynamics for mS(g) and mI) for easier interpretation and so that different functions governing mate choice can be readily explored.This model is related to the pair formation framework proposed in Ashby and Boots , but theI use evolutionary invasion analysis to determine the long‐term trait dynamics of the host and STI  in a population at equilibrium N∗=S∗+I∗ isThe invasion fitness of a rare mutant strain of the STI >0, which requiresREFF is the effective reproductive ratio of the STI. Because STI fitness can be written in this form, we know that parasite evolution maximizes its basic reproductive ratio R0  does not feature in this equation, we do not need to consider the effects of costs of mate choice on STI evolution. In general, mI) and f(β) will be decreasing (or constant) functions of β, and α(β) will be an increasing (or constant) function. In the absence of mate choice (mS(g)=mI)=1), a continuously stable strategy (CSS)—analogous to an evolutionary stable strategy or ESS—can only exist when α(β) is concave up . In the presence of mate choice, however, a CSS can exist under a broader set of conditions, such as concave down mortality‐transmission trade‐offs and with sterility‐transmission trade‐offs. This is clear from the equation for R0 ) and β  eq. , which f(dfdβ=0). When there is no mate choice and mortality virulence is a linear function of β with α(β)=κβ, we have ∂R0∂β>0, so the STI will evolve to maximize transmission at β=1 and virulence at α(1)=κ. If, however, mate choosiness is a linear function of mortality virulence  such that v(β)=α(β)/κ and m∼I)=1−gα(β)κ for gα(β)<κ and 0 otherwise, then a singular strategy exists atTo illustrate the above, suppose first that sterility virulence is constant (dαdβ=0) and sterility virulence is a function of the transmission probability such that f(β)=1−ηβ, with v(β)=1−f(β). If host mate choosiness is a linear function of sterility virulence such that m∼I)=1−g(1−f(β)), then the singular strategy occurs at β∗=12gη  in the denominator  in a resident population at equilibrium are given byThe initial dynamics of a rare mutant host (subscript Γβ=d+α(β)+γ, fβ=f(β), mSg=mS(g), and mIgm,vβ=mI) for the sake of brevity. The host will evolve in the direction of∂wH∂g until g is minimized at 0, one or both populations are driven extinct, or a singular strategy, g∗, is reached at ∂wH∂g|g=g∗=0.Using the next‐generation method =1) and that mate choice is a linear function of virulence such that m∼I)=1−gv(β). In this scenario, there may be one or two singular strategies. The singular strategy at g1∗=(2pβ−d−α(β)−γ)/2pβv(β) always exists, and corresponds to the point where the host drives the STI extinct. The second singular strategy (g2∗), if it exists, is an evolutionary repeller  with 0<g2∗<g1∗, in which case the outcome depends on the initial conditions, with g<g2∗ causing selection against mate choice, and g>g2∗ leading to STI extinction due to mate choice , mate choice is likely to evolve for intermediate transmission probabilities  and if the probability of transmission is close to 1, there may be selection against weak mate choice caused by the evolutionary repeller. This is because disease prevalence is high and so most attempted matings are with infected individuals, meaning that even weak mate choice dramatically reduces the mating rate for invading host mutants compared to the resident population. If, however, there is already a sufficient level of mate choice in the resident population , disease prevalence is sufficiently low to allow runaway selection for mate choice, eventually driving the disease extinct. This pattern is similar regardless of whether virulence has fixed effects on mortality or sterility <1 for g>0), and so one must find the evolutionary dynamics using numerical analysis. Although many of the results are qualitatively similar to the no‐cost scenario, there are some notable exceptions. In particular, if the host evolves mate choice, then it no longer drives the STI extinct, and is instead likely to reach a CSS with the STI endemic in the population. When virulence is correlated with transmission, the host only evolves mate choice de novo at sufficiently high values of β  interact with the mode of virulence  and the shape of the mate choice response to infected individuals. The model exhibits the same range of qualitative outcomes under both sterility and mortality virulence: (1) co‐CSS where STI virulence and host mate choice are at a stable equilibrium , the recovery rate (γ), and the natural mortality rate of the host ium Fig. C, D; andium Fig. E, F. Notd) and long (low d) host lifespans  and partner fertility . Another potentially important extension to the current model would be to split the host population into males and females with different disease outcomes and transmission rates between the sexes. Here, the hosts were treated as a single hermaphroditic sex for the sake of simplicity, but such an extension would add greater realism and expand predictions for an even broader set of STIs with sex‐specific characteristics.Neisseria gonorrhoeae infections (Gonnorrhea) in humans  must be weighed against the potential benefits of avoiding infection. Alternatively, hosts may have more effective forms of defense against STIs, such as post‐copulatory grooming or urination to remove parasites (Hart et al. Given the general lack of predictions and data on host‐STI coevolution, there are clearly a number of important avenues for future theoretical and empirical research in this area. Still, the present study, combined with previous theoretical work (Ashby and Boots The author declares there is no conflict of interest.Associate Editor: K. KoelleHandling Editor: D. W. Hall   Click here for additional data file.


Currently, the assembly of helical plant viruses is poorly understood. Theviral assembly and infection may be affected by the charge distribution on thevirion surface. However, only the total virion charge (isoelectric point) hasbeen determined for most plant viruses. Here, we report on the firstapplication of positively charged magnetic nanoparticles for mapping thesurface charge distribution of helical plant viruses. The charge wasdemonstrated to be unevenly distributed on the surface of viruses belonging todifferent taxonomic groups, with the negative charge being predominantlylocated at one end of the virions. This charge distribution is mainlycontrolled by viral RNA. Investigation of the physico-chemical characteristics of plant virus virions,including the charge distribution on their surface, may lead to a betterunderstanding of the molecular mechanisms of infection development in theprimary infected cell or during the movement of a transport form of RNA viruses(virions/vRNPs) to neighboring uninfected cells. To date, studies exist thatdescribe the features of surface charge formation in icosahedral viruses -3;isoelTobamovirus genus andpotato virus X (PVX) and alternanthera mosaic virus (AltMV) of thePotexvirus genus), as well as in virus-like particles (VLPs)and viral ribonucleoproteins (vRNPs) derived from virion components.In this work, we propose a method for surface charge mapping in helical virionsusing fluidMAG-DEAE magnetic nanoparticles (MNPs). We studied the surfacecharge distribution in helical plant viruses from different taxonomic groups(tobacco mosaic virus (TMV) of the A suggestion of a relationship between either the surface charge distributionin plant viruses or the surface charge heterogeneity throughout the entirevirion and the accessibility of encapsidated RNA for interaction with ribosomesand RNA packaging in the coat protein (CP) has not yet been discussed.Isolation of viruses, viral RNA, and coat proteinsPVX and TMV samples were isolated according to  and 7],,7], respPreparation of TMV and AltMV CP (VLP) repolymers and PVX vRNPsTMV and AltMV CP repolymers were prepared using the techniques described in, 12, resTreatment of virions and virus-like particles with RNase A andmicrococcal nuclease2. The reactionwas stopped by adding 250 mM EGTA. tRNA was used as a co-precipitator duringRNA isolation from nuclease-treated virions.Virions and vRNPs with a final concentration of 0.05 mg/mL were treated withRNase A at the ratio 1 μg of enzyme per 4 μg of the virus. Incubationwas carried out for 30 min; the reaction was stopped by placing the samples inice. Micrococcal nuclease (MN) (50 units of active enzyme per 1 μg of RNA)was added to the samples pre-treated with 100 mM CaClUltrasonic treatment of TMV virionsTMV particles were sonicated using an Ultrasonic Processor homogenizer. Thetreatment was performed at a TMV concentration of 0.05 mg/mL, in ice for 60 s.Preparation of virion/virus-like particle (VLP)–magneticnanoparticle complexesVirions/VLPs were incubated with fluidMAG-DEAE magnetic particles  in an aqueous solution at a final virion/VLP concentration of 0.05mg/mL for 20 min.Transmission electron microscopy and nanoparticle tracking analysisSamples were adsorbed on copper grids and contrasted according to the proceduredescribed in . The anaSamples in liquid were studied by nanoparticle tracking analysis using aNanoSight NS500 instrument and NanoSight NTA 2.3 software . Theparticles’ Brownian motion was recorded and processed using the followingsettings: 10 video recordings 60 seconds long each at a camera level of 14 anda detection threshold of 5. The mean hydrodynamic diameter and particleconcentration are presented as a 95% confidence interval.Virion surface charges were mapped using fluidMAG-DEAE magnetic nanoparticles(MNPs)  with a specified hydrodynamic diameter of 50 nm.These MNPs consist of an iron oxide magnetic core and a shell composed ofstarch functionalized with diethylaminoethyl groups. Due to the positive chargeof these groups, the MNPs can be used to map a negative charge on the surfaceof biological structures. The magnetite core enables detection of the MNPposition in a complex with viral particles by transmission electron microscopy(TEM).The mean MNP hydrodynamic diameter measured by nanoparticle tracking analysiswas 72 ± 3 nm. According to the TEM data, individual MNPs were assembledinto aggregates of 2 to 20 nanoparticles.14 and (1.6± 0.2) × 1014 particles/ml, respectively, which indicatedcomplex formation.The assembly of TMV virion–MNP complexes was performed at a totalnegative charge of viral particles. The interaction between MNPs and TMV inliquid was studied by nanoparticle tracking analysis. The mean equivalenthydrodynamic diameter of TMV was 115 ± 3 nm. Addition of MNPs to the virusled to an increase of the mean diameter to 134 ± 8 nm at a constantparticle concentration of (1.6 ± 0.1) × 10.The so-called "spider" complexes were alsodetected, which are a group of virions simultaneously interacting by one end with magneticnanoparticles .No complexes of MNPs interacting simultaneously with two opposite TMV ends were detected.Analysis of TMV–MNP complexes by TEM showed that the magnetic particleseffectively formed complexes with TMV bounding to only one end of the virionUS). The mean length of theseparticles was 149 ± 83 nm.This fact (MNPs interacting with only one end of TMV particles) attracted ourattention. In further studies, we used a TMV preparation of 300 nm nativeparticles "broken" by ultrasound . Nocomplexes between MNPs and TMV VLP ends were observed. Because the binding ofrepolymers to MNPs occurred at pH 5.6 (the condition for TMV repolymerassembly), the TMV–MNP complexes obtained under the same conditions wereused as controls. At pH 5.6, MNPs were found to interact also with only one endof the TMV virions.To elucidate the role of individual virion components in the formation of anincreased negative charge area at one end of the TMV virus particle, weprepared TMV CP repolymers  with a helicalstructure similar to that of the virion, but lacking RNA. An analTherefore, the negative charge is uniformly distributed on the surface of VLPsobtained by polymerization of the TMV CP in the absence of RNA and is notlocalized at one of the ends, in contrast to the virion. RNA is likely tocontribute significantly to the formation of an increased negative chargedensity area at one end of the native TMV.To assess the contribution of viral RNA to the surface charge formation, TMVvirions were treated with two nucleases: RNase A and micrococcal nuclease.The ability of RNases to affect the TMV virion RNA was analyzed in preliminaryexperiments. Virions were treated with the nucleases. Then, RNA was isolatedand analyzed by electrophoresis in 1% agarose gel. RNase A hydrolyzed thenucleic acid to fragments with an electrophoretic mobility comparable to thatof the tRNA used as a co-precipitator or less (data not shown).Micrococcal nuclease, as shown previously , does no.The surface charge of TMVvirions containing degraded RNA was evenly distributed, as in TMV VLPs.However, an unusual pattern was observed upon theincubation of MNPs with MN-treated virions. Magnetic nanoparticles were notonly located at the virion ends but also interacted with the entire surface ofvirions .One may assume MN to be able to partiallyhydrolyze an RNA fragment that forms an increased negative charge density area.This is consistent with a model of the cotranslational TMV disassemblymechanism [There was no predominant affinity of magnetic nanoparticles to the ends of thevirions treated with RNase A; the interaction occurred over the entire surfaceof the viral particlesechanism . Most liPotexvirus genus: potato virus X (PVX) and alternantheramosaic virus (AltMV). We did not find any published data on the chargedistribution on the AltMV or PVX surface; only the PVX isoelectric point isknown (pI of 4.4).Further investigation of the virion surface charge distribution was carried outon viruses with a flexible filamentous virion, which belong to thein vitro stable extended particles similar in length andmorphology to AltMV virions – AltMV virus-like particles (AltMV VLPs)[Like the TMV CP, the AltMV CP is able (in the absence of RNA) to formMV VLPs).Figure 5,MNPs, as in the case of TMV, formcomplexes with AltMV, binding to one end of the native virion.As seen from Fig. 5B).Therefore, as in the case of TMV, thecharge is unevenly distributed over the AltMV surface and the increasednegative charge density area is also located at one end of the virion and isdue to the presence of RNA. Similar results were obtained for PVX virions.An analysis of MNP–AltMV VLP complexes by electron microscopy showed thatMNPs were distributed over the VLP surface. However, MNPs had no affinity tothe VLP ends .Therefore, the surface charge of the PVX virionscontaining degraded RNA is unevenly distributed, like in native virions.It may be assumed that an RNA fragment located atthe viral particle end remains bound to the CP and forms an increased negativecharge density area. This result is consistent with the differences in thetranslational properties of encapsulated RNA in PVX and TMV virions.The unexpected result was obtained for RNase A-treated PVX virions. Previously,treatment of PVX with RNases (A and T1) was shown to lead to RNA degradationinto short segments (5 to 6 nucleotides) .In thisin vitro, the PVX CP is able to form RNA-containing vRNPs andPVX CPs that have a helical "head," whose structure is identical to that of theprotein helix of PVX virions, and a "tail"–CP-free RNA (one-tailedparticles) [Unlike TMV, the PVX CP is incapable of polymerization in the absence of RNA. Howeverrticles) .Fig. 7A).The lack of interaction between MNPs and the vRNP head surfacemay be explained by the competitive binding of all available MNPs to free RNA.To prevent this, vRNPs were treated with MN. The PVX CP is known to cap the5’-end of RNA upon vRNP treatment, and the 5’-terminalCP-encapsulated RNA segments in vRNPs retain their integrity and translationalproperties during treatment with MN [Fig. 7B),as in the case of native PVX.Investigation of the interaction between magnetic nanoparticles and PVX vRNPsrevealed binding of MNPs to CP-free RNA (vRNP tails) and PVX and AltMV(Potexvirus genus) was mapped.During the TMV assembly, the CP in the form of 20S discs is known to interactwith an RNA site located at a distance of about 1,000 nucleotides from the3’-end of the molecule (assembly origin).At the