MYO7A accounting for >50% of type 1 and USH2A contributing to approximately 80% of type 2 Usher syndrome. Variants in these genes can also cause non-syndromic RP and deafness. Genotype–phenotype correlations have been described for several of the Usher genes. Hearing loss is managed with hearing aids and cochlear implants, which has made a significant improvement in quality of life for patients. While there is currently no available approved treatment for the RP, various therapeutic strategies are in development or in clinical trials for Usher syndrome, including gene replacement, gene editing, antisense oligonucleotides and small molecule drugs.Usher syndrome has three subtypes, each being clinically and genetically heterogeneous characterised by sensorineural hearing loss and retinitis pigmentosa (RP), with or without vestibular dysfunction. It is the most common cause of deaf–blindness worldwide with a prevalence of between 4 and 17 in 100 000. To date, 10 causative genes have been identified for Usher syndrome, with  The Usher genes encode a variety of proteins that are expressed in the inner ear and retina where they perform essential functions in sensory hair cell development and function, and photoreceptor maintenance. While many promising treatments are under investigation, there is no approved treatment for this disease to date.Usher syndrome encompasses a group of inherited disorders characterised by dual sensory impairment of the auditory and visual systems, with a variable presentation of vestibular dysfunction in a proportion of cases. It is the most common cause of combined sight and hearing loss, accounting for more than half of deaf–blindness cases.5  is the most severe subtype in which patients exhibit severe to profound bilateral congenital sensorineural hearing loss , most frg screen , and whe11 The sensorineural hearing loss is typically described as sloping, mild to moderate in the low frequencies and severe to profound in the high frequencies ,19 which increased speech intelligibility, quality of life and communication, with similar outcomes to a control group of adults with post-lingual hearing loss. Vestibular function is intact in Usher 2 patients and reflected in normal motor milestones. However, one study found vestibular abnormalities in four out of five genetically confirmed Usher 2 patients.20 Episodes of vertigo were reported by patients although clinical balance was normal, and the authors suggested that subclinical changes in the vestibular system should be looked for.Usher syndrome type 2 (Usher 2) is the most common form of the disorder, representing over half of all cases.quencies .15 Heari21 and among Ashkenazi Jewish people.22 The audiovestibular features are the most variable of the Usher subtypes. Hearing loss is of post-lingual onset and usually detected in the first decade of life, although onset can be delayed until adult life. It is typically of a progressive nature, with audiograms showing high frequencies more affected or a U-shaped configuration. Vestibular abnormalities are present in approximately half of patients, although most report a normal age of independent walking.7 Hearing aids are of benefit early in the course of disease, but cochlear implants may be required with progressive hearing loss. Improved hearing and subjective benefit were demonstrated in Usher 3 patients with implants (mean age at implantation: 41 years) comparable with that observed in implanted patients without visual impairment.23Usher syndrome type 3 (Usher 3) is rare in most populations, accounting for approximately 2–4% of all cases, although it is particularly prevalent in FinlandGJB2 and mitochondrial m.1555A > G mutations and electrocardiography (ECG) in some patients, which is expected to be normal.24 Initial investigations for bilateral sensorineural hearing loss also include ophthalmological assessment, which in the early years can be normal depending on the Usher subtype. Diagnosis of Usher 1 should be suspected in any infant with bilateral profound sensorineural hearing loss and delayed motor milestones, even if the initial ophthalmology screen is normal. Usher 2 should be considered in patients with typical sloping configuration of hearing loss. Diagnosis of Usher 2 or 3 is made after visual symptoms or signs are detected through routine examination or electroretinography (ERG). A meta-analysis of next-generation sequencing (NGS) data in the United States indicates that 7.5% of patients with seemingly ‘isolated deafness’ have mutations in the Usher genes and may be at high risk of developing RP.25 With the availability of NGS, Usher genes are included in the ‘deafness’ and ‘retinal’ gene panels to help with early genetic diagnosis.In all types of Usher syndrome, audiological findings are detected/present prior to ophthalmological signs and symptoms. Children and adults with Usher syndrome do not have dysmorphism, and the disorder is commonly mistaken for a non-syndromic isolated sensorineural hearing loss. They will often have the initial aetiologic investigations for hearing loss, which include cytomegalovirus (CMV) testing and magnetic resonance imaging (MRI) scan, genetic testing for 19 The onset of RP has a significant impact on communication, as patients will have difficulty in lip reading and understanding gesture and sign, and on balance as visual compensation is compromised. Patients with Usher syndrome and bilateral vestibular areflexia should be advised about the risk of disorientation and potential drowning with underwater swimming, due to poor availability of visual and proprioceptive inputs.27Audiological rehabilitation in all forms of Usher syndrome is started soon after diagnosis by fitting of bilateral hearing aids. Hearing aid fitting in patients with Usher syndrome may need special considerations, especially with visual impairment.28 Visual prognosis also differs between the clinical types; Usher 1 patients generally show a more severe visual decline with age, reaching legal blindness on average 15 years earlier than patients with Usher 2.9 Typically, the first presenting symptom is night blindness  with progressive visual field loss beginning in the mid-periphery caused by rod photoreceptor degeneration. It eventually progresses to involve cone photoreceptors, resulting in central and colour vision loss. Patients are often registered severely sight impaired but there can be significant intra- and interfamilial phenotypic variability. Fundus features include variable amounts of bone spicule pigmentation, retinal pigment epithelium (RPE) atrophy or depigmentation, retinal arteriolar attenuation and optic disc pallor  and deep capillary plexus (DCP) in all patients compared with healthy age-matched controls.30 However, peripheral defects were detected in the choriocapillaris (CC) earlier and more severely in MYO7A patients compared with the USH2A group. It was suggested that this is because the MYO7A protein (myosin VIIa) is mainly expressed in the RPE, thus affecting the CC directly. In the same study, patients were tested for macular sensitivity (MS) using microperimetry with the Macular Integrity Assessment, and this detected decreased mesopic mean MS in all patients, especially in the periphery. Patients with MYO7A mutations had a slightly lower mean MS than patients with USH2A mutations; however, the differences were found not to be statistically significant (p = 0.66). Static and dynamic perimetry detects mid-peripheral visual field loss with progression to residual small central islands, with a small temporal peripheral field preservation in the most advanced stages of the disease. ERG measurement can show reduction and delay of amplitudes in the early stages of the disease, and is a useful test to perform in an infant born with profound deafness to determine the likelihood of underlying Usher 1, even before visual dysfunction is otherwise noted. Later, the full-field ERG is often non-recordable.Retinal imaging using fundus autofluorescence shows a ring of hyperautofluorescence in the macula , and spe32 Dual sensory clinics will promote faster accurate diagnosis through more extensive genetic testing and detection of visual symptoms and mental health issues at an earlier stage.Dual sensory clinics are now being established to improve the clinical pathways and experience of children with hearing and sight impairment. These clinics will provide access to the relevant multidisciplinary in one visit, hence reducing stress and the burden associated with numerous, separate medical appointments. Children with Usher syndrome have been reported to develop mental and behavioural disorders, including autism, conduct disorder, schizophrenia and learning difficulty. This could be multifactorial, due to sensory deprivation, stress, difficulty in diagnosis and a possible unproven genetic association.34 but this is time-consuming and costly, particularly for large patient cohorts. Microarray-based testing provided ~33% detection for Usher patients but can only screen for known mutations.36 NGS, including whole exome and genome sequencing, is now the method of choice with high efficiency offering the advantage of detecting a range of known and novel mutations, including large genomic DNA rearrangements. Targeted exome gene panel testing can reach diagnostic rates of around 70–80% in Usher families.3739All Usher syndrome types are inherited in an autosomal recessive manner. To date, at least 10 causative genes have been identified for the disease, which include six Usher 1 genes, three Usher 2 genes and one Usher 3 gene. Historically, traditional Sanger sequencing of all Usher gene exons was found to provide a genetic diagnosis for more than 80% of Usher families,MYO7A (USH1B),40USH1C (USH1C),42CDH23 (USH1D),43PCDH15 (USH1F),44USH1G (USH1G),45 and CIB2 (USH1J).46 Of these genes, MYO7A is the most frequent cause of Usher 1, accounting for more than half of cases.34 The USH1E, USH1H and USH1K loci have been mapped to chromosomes 21q21, 15q22–23 and 10p11.21–q21.1, respectively,4749 but the genes are yet to be identified. It is worth noting that CIB2 bi-allelic loss of function variants has been reported in patients with non-syndromic recessive hearing loss (DFNB48) but with no retinal symptoms.50Currently, there are nine loci (USH1B-J) known to be involved in Usher 1. The genes identified for six of these loci are as follows: USH2A (USH2A),51ADGRV1 (USH2C)52 and WHRN (USH2D).53USH2A mutations are the most common cause of Usher syndrome, accounting for around 80% of Usher 2 cases.34 In addition, PDZ domain-containing 7 (PDZD7) has been reported to act as a disease modifier and contributor to a digenic form of Usher 2.54Three genes underlying Usher 2 have been identified as CLRN1 (or USH3A) is the only gene currently confirmed to cause Usher 3,56 with two prevalent mutations, p.(Tyr176*) and p.*Asn48Lys), accounting for most cases in Finnish and Ashkenazi Jewish patients, respectively.57 A homozygous missense variant in histidyl-tRNA synthetase (HARS) has also been reported in two patients with a phenotype compatible with Usher 3 (sometimes referred to as USH3B).5840), scaffolding proteins , cell adhesion/transmembrane proteins , an adhesion G-coupled receptor  and a calcium- and integrin-binding protein . Most are expressed as multiple splice and protein variants in a range of tissues,5964 but all of the Usher proteins are present in the inner ear and retina where most have been found to interact and form complexes that localise to subcellular locations in the ciliated sensory neurons, that is, inner ear hair cells and retinal photoreceptors.66 Myosin VIIA is also an essential RPE protein,6769 and evidence suggests that clarin-1 is restricted to the retinal Müller glia.70 Various studies have indicated the involvement of Usher proteins in a range of processes, including cohesion, mechanotransduction, synaptic maturation, and protein and organelle transport.The Usher genes encode a number of structurally and functionally distinct proteins; these include an actin-binding motor protein .82 It has been suggested that mutations that allow some residual motor protein function, for example, in-frame deletion c.5146_5148delGAG p.(Glu1716del), cause the milder non-syndromic phenotypes, whereas mutations associated with Usher 1, for example, c.1309G > A p.(Asp437Asn), have a more severe effect on protein function.83 A separate study of 33 USH1B patients proposed that null MYO7A alleles, that is, those with stop mutations within the motor domain coding region such as c.999T > C p.(Tyr333*), may cause milder visual dysfunction than missense variants owing to a lack of mutant protein contributing to disease pathology.10 Significant correlations have not been reported in other patient populations with MYO7A mutations.84 Mutations of MYO7A reported to cause DFNB2 are comparable with those causing Usher 1, leading to question whether this phenotype results from missed RP or whether there may be modifying factors which influence the phenotype.85MYO7A mutations have also been reported to cause a phenotype of unilateral auditory neuropathy in a Chinese family with Usher 186 and an Usher 2 phenotype,87 expanding its phenotypic spectrum.The Usher genes show vast clinical heterogeneity and different mutations in most Usher genes have been linked to non-syndromic cases of autosomal recessive RP, or autosomal dominant or recessive sensorineural hearing loss (annotated as DFNA or DFNB).CDH23 and PCDH15; missense variants are primarily associated with non-syndromic deafness (DFNB12 [OMIM #601386] or DFNB23 [OMIM #609533]) or more subtle RP symptoms, whereas frameshift, nonsense and splice site mutations cause Usher 1.8890 However, missense mutations in CDH23 can also cause milder or ‘atypical’ Usher 1, and genotype–phenotype correlations are not always predictable.88 Pathogenic USH1C variants, including a leaky splice site mutation IVS12 + 5G > C,73 have also been shown to cause recessive non-syndromic hearing loss (DFNB18 [OMIM #602092]), which has been proposed to be related to the less deleterious effect of the variants being located within alternatively spliced exons.91 A family with non-syndromic sensorineural hearing loss caused by compound heterozygous missense and frameshift mutations in USH1G has been described;78 missense mutations in USH1G that are expected to result in residual protein function have been reported in Usher 1 families, thus expanding the phenotypic heterogeneity of Usher 1G disease.Among the other Usher 1 genes, there is evidence of a genotype–phenotype correlation in both USH2A has a diverse mutation spectrum, which includes nonsense, frameshift, missense and splice-affecting mutations, as well as deletions and duplications. The most common mutation found in USH2A is a single base pair (bp) deletion in exon 13, c.2299delG p.(Glu767Serfs*21),92 which has been shown to be associated with exon splicing.93 This variant is predicted to produce a severely truncated protein and/or be subject to nonsense-mediated decay; however, transcript analysis showed that it caused skipping of exon 13 or exons 12 and 13.93 Mutations of USH2A are associated with up to 23% of non-syndromic RP cases,94 and specific mutant alleles are more frequent among such patients and families, the most common being missense variant c.2276G > T p.(Cys759Phe).96 Unusually, one patient with compound heterozygous USH2A mutations, c.1036A > C p.(Asn346His) and c.13316C > T p.(Thr4439Ile), was reported to have non-syndromic hearing loss, while their sibling harboured the same mutations and was diagnosed with typical Usher 2.97USH2A phenotype-genotype studies have been carried out to date. A survey conducted by Lenassi and colleagues of patients with USH2A-associated RP reported several ‘retinal disease-specific’ alleles that were rarely found in Usher 2 families, mostly missense variants that were likely to be less deleterious, while the Usher-associated variants mostly included those that were predicted to produce no viable protein (e.g. those causing premature truncation).96 They proposed an allelic hierarchy model in which the presence of at least one retinal disease-specific allele in a patient with USH2A-related retinopathy resulted in the preservation of hearing. While this has not been supported by subsequent studies,99 the same analysis on a different cohort100 combined with two large external cohorts101 found that the allelic hierarchy model was valid in 86% of individuals with non-syndromic USH2A-RP.100 In addition, it has been reported that Usher 2 patients with one copy of the p.(Cys759Phe) allele showed a later onset of RP and milder hearing loss compared with the general Usher 2 population,6 and the presence of the p.(Cys759Phe) variant in a homozygous state or in combination with other USH2A missense mutations has been associated with isolated RP or RP with late onset hearing loss.99 In contrast, the p.(Glu767Serfs*21) variant results in a more rapid deterioration and severe hearing threshold, heralding the need for careful audiological monitoring and consideration of cochlear implants.100 In general, severe hearing impairment has been associated with truncating variants in USH2A.102 Further investigations into USH2A genotype–phenotype correlations have reported that the presence of two truncating mutations, or two missense variants in the N-terminal laminin domain of the gene, were associated with Usher 2 patients and not those with non-syndromic RP,103 and the presence of at least one truncating mutation was related to earlier visual decline regardless of the phenotype.99Several WHRN is an additional Usher gene that causes non-syndromic deafness, which is related to the mutation location affecting the two predominantly expressed variants (long and short): N-terminal mutations that affect the long isoform are found in USH2D patients,104 whereas mutations in the C-terminal region manifest as DFNB31.10666 Most Usher mice display sensorineural hearing loss and vestibular phenotype reminiscent of their human counterparts, with only a limited number showing progressive retinal degeneration. Despite this, they have still aided in the assessment of potential treatments.While there is currently no available cure, there are numerous therapeutic strategies under development for Usher-related RP and other inherited retinal diseases (IRDs): these include gene replacement, gene editing, nonsense suppression and antisense oligonucleotide (ASO)–based approaches . The eyeMyo7a,107110Whrn131 and Clrn1,134 knockout mice via subretinal injection to restore expression of the wild-type Usher gene that was defective in each model. Dual overlapping AAV vectors have also been tested for MYO7A delivery as a potentially safer alternative for large genes with promising results, although they were not found to be as efficient.111 Alternatively, delivery of functional MYO7A to the USH1B mouse model retina via lentiviral-based vectors with larger carrying capacities (9 versus 4.7 kb for single AAV) proved successful,113 although it harbours the risk of insertional mutagenesis. Among the other Usher models, gene delivery using AAV vectors has also produced significant improvements in auditory and vestibular hair cell function in mouse models of USH1C,114 USH1G,124 USH2D,132 and USH3.135137 This was achieved by viral injection into the inner ear through the round window membrane135137 or posterior semicircular canal132 in postnatal neonatal mice.Gene replacement is an approach that has been shown to be effective in several Usher mouse models: adeno-associated virus (AAV) vectors have been used in 145 Comparatively, hearing in humans is fully mature at birth (onset of startle response at 19 weeks gestation). The hearing loss in Usher 1 is established at birth and it is not clear whether the hair cells in the human inner ear are a viable therapeutic target. In order to be effective, human intervention should be considered within the foetal stage, before establishment of hearing (at ~18 weeks of gestation).145 Hence, where therapeutic response in mice is successful when given soon after birth, it is questionable whether the same effect will be seen in patients with postnatal treatment. Overall, further studies in non-human primates will be useful in addressing some of these issues.66The mouse inner ear is immature at birth and continues to mature postnatally. The acquisition of hearing (measured by the onset of startle response) occurs 12 days postnatally in mice, providing a window of opportunity for effective intervention with gene therapy.146 This can be used to correct point mutations, small indels and splice site mutations, and is suitable for any gene size. Early investigations into the use of this strategy for Usher-directed treatment employed the use of two zinc finger nucleases to correct an USH1C point mutation (c.91C > T p.[Arg31*]) and induce full-length harmonin expression in cultured cells.115 In recent years, the CRISPR/Cas9 system has become highly popular for gene editing due to its efficiency and ease of use. This technique has been used for successful in vitro mutation repair in USH2A patient fibroblasts harbouring homozygous p.(Glu767Serfs*21) mutations,125 as well as patient-derived induced pluripotent stem cells (iPSCs) either homozygous for USH2A p.(Glu767Serfs*21) mutations or compound heterozygous for p.(Glu767Serfs*21) and p.(Cys759Phe).126 However, the efficiency of mutation correction was only 2.5% in fibroblasts125 and up to 3% in iPSCs,126 although the second study reported an 80% editing efficiency in the small number of homozygous USH2A clones that survived. Encouragingly, neither study reported off-target effects, which are unwanted mutations induced at DNA locations that show homology to the guide sequence. CRISPR/Cas9-based editing shows huge promise for the treatment of IRDs caused by a range of mutations; however, ensuring the absence of off-target effects and a high level of editing efficiency in retinal cells will be essential for future investigations.One alternative approach to gene replacement is gene editing, which involves cutting around genetic mutations through the use of nuclease enzymes and correcting the DNA error by homologous recombination with a DNA template containing the wild-type sequence.PCDH15 and USH1C nonsense mutations in vitro, in cell cultures and in retinal explants.121123 Furthermore, in vivo administration of NB54 and PTC124 was able to restore expression of full-length harmonin in mouse retinas transfected with Ush1c reporter constructs.117 For Usher 2 investigation, PTC124 was administered to a human embryonic kidney (HEK) cell model expressing a cDNA fragment of USH2A containing the c.11864G > A p.(Trp3955*) mutation and showed a substantial increase in USH2A expression compared with the control.127 Further studies demonstrated PTC124 efficacy in restoring USH2A protein expression and primary ciliogenesis capability in USH2A patient-derived fibroblasts with the c.9424G > T p.(Gly3142*) mutation.128 Overall, TRIDs show promise as a safe and effective strategy to treat a range of Usher-related nonsense mutations; however, these particular variants cause ~16% of USH2A-related RP and 13% of all IRD cases.128Small molecule-based methods for treatment of Usher syndrome have included the use of translational read-through-inducing drugs (TRIDs), which bind to the translational machinery and are able to induce insertion of an amino acid at the site of premature stop codons, allowing read-through of nonsense mutations. These small molecule drugs include ataluren (PTC124) and designer aminoglycosides (NB compounds such as NB54). Several TRIDs have been used to suppress Usher 1-associated CLRN1 missense variant p.(Asn48Lys).138 BF844 was shown to protect against progressive hearing loss when administered intraperitoneally to an Usher 3 knock-in mouse model.An additional small molecule that has been of interest for Usher syndrome treatment, known as BioFocus 844 (BF844), was identified through cell-based high throughput screening as capable for stabilising the defective Clarin-1 protein produced by the common Ush1c knock-in mice, which possess a cryptic splice site mutation that results in truncated harmonin protein.118120 Initially, peritoneal injection of ASOs in neonatal mice was shown to partially correct defective pre-mRNA splicing of mutant Ush1c transcripts; the same group delivered ASOs to Ush1c knock-in foetal mice in utero via transuterine injection into the amniotic cavity and observed partial correction of vestibular function and hearing in the mice postnatally,119 while most recently transuterine injection directly into the developing inner ear produced more substantial improvements in both hearing and vestibular function that sustained into adulthood.120 ASOs have also been used to correct a splicing defect caused by a deep intronic mutation in the USH2A gene (c.7595-2144A > G) which leads to insertion of a pseudoexon (PE40), in both patient-derived fibroblasts and a minigene splice assay.129A further therapeutic option is the use of ASOs, which are short synthetic modified nucleic acids that bind RNA through complementary base pairing. They can be designed to bind pre-mRNA at splice enhancer or silencer target sites, preventing or stimulating binding of the spliceosome thereby modulating pre-mRNA splicing. ASOs have been used to rescue both the hearing and vestibular defects in USH2A mutations,148 will further aid in the testing of novel treatments by providing the opportunity to demonstrate therapeutic potential in retinal-specific cells in vitro.Overall, there are a number of promising therapeutic strategies in the development for the Usher subtypes. The use of patient-derived retinal organoids, which have already been generated for Usher syndrome caused by MYO7A149 and USH2A150152 mutations using various clinical functional and structural measures, including visual acuity, perimetry, ERG, fundus autofluorescence and OCT-derived measurements.Although several treatment strategies are already under development for Usher syndrome and other IRDs, identifying metrics that display detectable changes within relatively short time periods (e.g. 1–2 years) in otherwise slowly progressive conditions will aid the assessment of therapeutic efficacy in clinical trials. This is especially important when the treatments are administered systemically, such as orally, and the untreated eye cannot be used as a control. As the hearing loss is congenital and relatively stable throughout the lifetime of Usher patients (aside from Usher 3), a number of natural history studies have focussed on the progress of the retinal disease in Usher patients; these have included longitudinal assessment of patients with USH2A c.2299delG mutation were studied and rod perimetry across the visual field was highlighted as a potential clinical measure for timely investigations, predicted to show detectable change within 1.4 years.150 Ellipsoid zone (EZ) line width, which is the inner/outer segment boundary measured from OCT scans, was an effective surrogate measure of central visual loss and was predicted to show a detectable decrease in 2.3 years. A recent investigation into an Usher 2 patient population carrying a range of USH2A variants used retrospective longitudinal data to identify suitable clinical outcome metrics.151 Both EZ line and hyperautofluorescent outer retinal ring area showed significant reductions within the follow-up period (2–5 years); however, there was considerable variability in the population. Visual acuity was not found to be a suitable measurement due to its slow decline, consistent with previous work.150 Furthermore, measuring retinal thickness from OCT images was confounded by the presence of macular oedema.For Usher 2, longitudinal data from a cohort of patients carrying the common USH2A-related Retinal Degeneration’  and the ‘Multicentre Longitudinal, Observational Natural History Study to Evaluate Disease Progression in Subjects With Usher Syndrome Type 1B’ (NCT03814499), in addition to the use of artificial intelligence–based methods will further aid in identifying suitable outcome metrics for clinical trials for Usher syndrome. This will likely be tailored to the mechanism (gain of function or slowing of disease progression) and target of treatment, that is, retina-wide or central retina. If gain of function is anticipated, trials could be relatively short (between 12 and 18 months) to arrive at an estimate of potential longevity.Data from ongoing longitudinal natural history studies, such as the ‘Rate of Progression in MYO7A cDNA (UshStat) for treating patients with MYO7A-related Usher 1 (NCT01505062).113 However, this phase I/IIA trial has been terminated by the sponsor Sanofi due to review of clinical development plans and priorities. A second trial is ongoing to assess long-term safety of patients who received UshStat (NCT02065011). A further clinical trial is being prepared using dual hybrid AAV vectors to deliver MYO7A to the retina of USH1B patients (https://cordis.europa.eu/project/id/754848/it).153 Considering the Food and Drug Administration and European Medicines Agency approval of Spark Therapeutics Luxturna gene therapy for patients with RPE65-related retinal disease, gene replacement therapy has become a more likely future option for the treatment of several Usher subtypes. However, such therapies are likely to be highly costly, and conventional viral methods are not appropriate for very large genes like USH2A (cDNA length >15 kb).Owing to the success of preclinical investigations, there are several completed and ongoing clinical trials for patients with Usher-related RP. For Usher-specific gene therapy, the first clinical trial evaluated subretinal injection of a recombinant equine infectious anaemia virus (EIAV)–based lentiviral vector for delivery of USH2A mature mRNA transcript; this has been shown preclinically to result in restoration of functional usherin protein.130 Considering that two of the most common pathogenic USH2A mutations occur in exon 13,96 if successful this treatment would be suitable for a large proportion of patients with USH2A-related Usher 2 and RP. The phase I/II clinical trial is currently ongoing for intravitreal injection of QR-421a in patients with USH2A exon 13 variants (NCT03780257).For ASO-based treatments, there is currently a trial sponsored by ProQR for an ASO candidate (QR-421a), which has been designed to exclude the whole exon 13 in the 141 CNTF has been found to prolong photoreceptor survival in mouse and rat models of retinal degeneration.143 Viral delivery of rod-derived cone viability factor (RdCVF) is also under investigation for the treatment of RP; RdCVF is a factor naturally secreted by rods to protect cone photoreceptors,154 and has been found to promote photoreceptor survival in mouse models of RP after viral-mediated expression in the retina.155 If such strategies are effective in humans, this could be suitable for the significant number of IRD patients without a confirmed molecular diagnosis.In addition to Usher gene-specific clinical trials, subretinal implantation of capsules containing human NT-501 cells that release ciliary neurotrophic factor (CNTF) has been trialled in patients with choroideremia and RP, including some with Usher 2 and Usher 3 .156 or retinal prosthesis157 may be the most feasible options. Advances in embryonic stem cell and iPSC technology make cell transplantation an ever-likely option for patients with late-stage retinal disease.156 Bone marrow–derived stem cells (BMSC) have been trialled in five ungenotyped patients with varying subtypes of Usher syndrome as part of the Stem Cell Ophthalmology Treatment Study .144 Each Usher patient received autologous BMSC through either retrobulbar, subtenons, intravitreal, subretinal or intra-optic nerve injections into both eyes, followed by intravenous injections. The average pre-treatment logarithm of the minimum angle of resolution (LogMAR) acuity was 0.635, and the average postoperative change was a gain of 0.18 LogMAR. In the murine retina, it has been found that endogenous BMSC migrate and fuse with Müller glia cells after damage has been inflicted.158 The resulting hybrids were found to contribute to the replacement of damaged neurons, demonstrating the regenerative potential of BMSC in the mammalian retina.When choosing the method of treatment for Usher syndrome and other IRDs, the stage of disease will be an important consideration. The strategies already described are likely to be only effective at a stage where retinal photoreceptors are still intact. At the advanced stages of retinal degeneration, cell replacement therapiesATOH1) cDNA for administration via intra-labyrinthine infusion in patients with severe to profound sensorineural hearing loss (NCT02132130). The results are yet to be published. The successful preclinical work with several Usher mouse mutants makes gene therapy a promising future option; however, as discussed, these studies have involved treatment administration in prenatal or neonatal mice when the inner ear is still developing, and the use of similar therapies in children or adults with Usher syndrome may not be able to achieve reversal of the congenital inner ear defects.Although there are no ongoing clinical trials for the Usher-specific hearing loss, there was a clinical trial for a recombinant adenovirus 5 (Ad5) vector containing the human atonal transcription factor (Usher syndrome is a disorder with vast clinical and genetic heterogeneity, typically resulting in significant dual sensory loss causing great impact on patient quality of life. More than ever, the prospect of an available treatment for at least some Usher subtypes looks promising. However, there are still obstacles to overcome in developing safe treatments that work for each gene size and mutation. In addition to the many gene- and mutation-specific treatments under investigation, finding universal treatments that use common mechanisms for the treatment of RP should be a priority for the many patients that remain without a molecular diagnosis. Further patient analysis is necessary to determine better genotype–phenotype correlations for each clinical subtype to predict prognosis; this will inform genetic counselling, preimplantation diagnosis and the choice of best outcomes for each treatment trial.


Interrelationship between growth habit and flowering played a key role in the domestication history of pulses; however, the actual genes responsible for these traits have not been identified in Indian bean. Determinate growth habit is desirable due to its early flowering, photo-insensitivity, synchronous pod maturity, ease in manual harvesting and short crop duration. The present study aimed to identify, characterize and validate the gene responsible for growth habit by using a candidate gene approach coupled with sequencing, multiple sequence alignment, protein structure prediction and binding pocket analysis.PvTFL1y locus of common bean. Gene prediction revealed that the length of the third and fourth exons differed between the two alleles. Allelic sequence comparison indicated a transition from guanine to adenine at the end of the third exon in GNIB 21. This splice site single-nucleotide polymorphism (SNP) was validated in germplasm lines by sequencing. Protein structure analysis indicated involvement of two binding pockets for interaction of terminal flowering locus (TFL) protein with other proteins.Terminal flowering locus was amplified from GPKH 120 (indeterminate) and GNIB-21 (determinate) using the primers designed from TFL locus is responsible for the transformation of shoot apical meristem into a reproductive fate in the determinate genotype GNIB 21. The splice site SNP leads to absence of 14 amino acids in mutant TFL protein of GNIB 21, rendering the protein non-functional. This deletion disturbed previously reported anion-binding pocket and secondary binding pocket due to displacement of small β-sheet away from an external loop. This finding may enable the modulation of growth habit in Indian bean and other pulse crops through genome editing.The splice site SNP present at the end of the third exon of  Lablab purpureus (L.) Sweet) is a short-day vegetable as well as a split pulse crop of tropical countries, viz., India and Africa. It is also utilized as cover and forage crops especially in drought-prone areas due to its inherent drought tolerance. It also fixes atmospheric nitrogen, hence is popular as an intercrop to enrich soil fertility. Although with immense genetic and morphological variabilities present for pod length, pod aroma, pod color, pod shape, and pod fiber, this orphan legume has not been given due emphasis. Few high-yielding determinate and indeterminate varieties have been produced which has changed the pattern of Indian bean cultivation from intercropping to monoculture. These released varieties are almost uniform considering pod qualities and unable to cater the needs of consumer preferences. Consumer preference varies from small, tubular, curved, light green-seed-filled pods to long, flat, strait, dark green/pigmented pods. Besides, this crop is predominantly used as an intercrop or kitchen garden crop. One can find the vines of this crop on almost every hut of tribal area or in the kitchen garden of urban homes. Despite its importance as a protein-rich vegetable in daily diet, the crop has not been given much importance as far as crop improvement and molecular breeding are concerned.Indian bean  gene has a unique effect on shoot apex architecture through different developmental stages [TFL1 acts as a repressor for floral initiation and maintains the inflorescence meristem through suppression of the expression of APETALA1 (AP1) and LEAFY (LFY) [. Antagonistically, FLOWERING LOCUS T (FT) interacts with Basic Leucine Zipper Domain (bZIP) transcription factor FLOWERING LOCUS D (FD) to form a heterodimer that binds to the promoter of APETALA1 (AP1) which activates flowering initiation [PvTFL1y is a homologue of Arabidopsis TFL controlling growth habit in common bean [PvTFL1y was validated as the homologue of TFL1 through functional complementation [PvTFL1y locus, a retrotransposon and splice site mutation. Many homologues for growth habit have been identified in soybean, GmTFL1 [PsTFL1a, PsTFLb and PsTFL1c [VfTFL1 [CsTFL [. The structures of AtFT and AtTFL proteins have been previously compared to determine the cause of their opposite actions and their interactions with other proteins [AtFT and AtTFL, of these, FT protein structure has been extensively studied. Yet, the actual mechanism responsible for their opposite actions and key binding pocket(s) involved in their interaction with other proteins are unclear. However, the protein structures of wild type and natural mutants of TFL have not been compared.Gene discovery using public sequence databases of model plants is useful especially for crops that do not have this information . In the l stages . TFL1 acFY (LFY) –13. Antaitiation , 15. Regitiation , 17. PvTmon bean . PvTFL1y, GmTFL1 ; in pea  PsTFL1c ; in faba [VfTFL1  and in c1 [CsTFL . The strproteins –28. ImpoPvTFL1y-specific primers showed the association of this marker with growth habit [Study in the Indian bean with th habit . The preTFL homologue . The primers were designed in two frames from the sequence of PvTFL1y to isolate its homologue in Indian bean, expected to give an amplification of 700 and 800 bp fragments . PCR mixture prepared in 200 μl contained approximately 100ng genomic DNA, 200 μM of dNTPs, 10 pmol of forward and reverse primers, standard Taq buffer (Mg2+ plus) and 1 unit of Taq DNA polymerase in total volume of 25 μl reaction. The PCR cycle involved 7 min of 95 °C initial denaturation and 35 cycles for 30 s at 94°C, for 45 s at 56°C and finally for 1 min at 72°C followed by 10 min of extension at 72 °C.To identify homologous sequence of ian bean . Basic LAmplified fragments from GNIB 21 and GPKH 120 were sequenced. The sequences obtained in two frames were merged, and overlapping sequences were identified in both directions using Bioedit Sequence Alignment Editor . Merged Taq DNA polymerase . PCR mixture and cycle set up are the same as previously described. Whole TFL locus was amplified and sequenced from these genotypes. Gene prediction was done by GeneMark, and multiple sequence alignment of DNA as well as predicted protein sequences was carried out using ClustalW. The sequences have been submitted to NCBI database with GeneBank accession number MT230590 to MT230597.Genomic DNA was extracted from the young fresh leaves with CTAB method with some modifications from eight other indeterminate genotypes of Indian bean. Targeted fragment was amplified by using polymerase chain reaction (PCR) with Arabidopsis TFL1 protein) as reference. Quality of modelled protein was judged on the basis of GMQE  and QMEAN  Z-score parameters [Z-score provides an estimate of the “degree of nativeness” of the structural features observed in the model on a global scale. QMEAN Z-scores around zero indicate good agreement between the model structure and experimental structures. Scores of − 4.0 or below are an indication of models with low quality. The quality of the resulting models was also monitored with PROCHECK [The predicted protein sequences of GNIB 21 and GPKH 120 were subjected to SWISS-MODEL homology modelling online tool . Proteinrameters , 33. ThePROCHECK . GeometrPROCHECK . MoleculPROCHECK .Lablab purpureus TFL) and yielded expected amplicons of 700 and 800 bp in GNIB 21 and GPKH 120, respectively  with a zero The results indicated probable splice site variation at the junction of the third exon and third intron, which altered the length as well as the start and end points of the fourth and third exons, respectively. The sequences obtained from both the parents were compared using Bioedit tool for allelic characterization which revealed transition of G → A at the end of the third exon Figs. , Table 2TFL protein non-functional  and QMEAN Z (GPKH 120: 0.11 and GNIB 21 : − 1.57) scores indicated that these models possessed reliable and good quality. The quality of the resulting models was monitored with PROCHECK. Ramachandran plot analysis revealed that 87.7% of the non-glycine residues in the GPKH 120 TFL protein structure fell within the most favoured regions, with a further 12.3% in the additionally allowed region  encoded by the fourth exon  in TFL protein of GPKH 120 are ASP71, VAL74, HIS85, HIS87, GLU109, ILE110, LYS112, PRO113, ASN114, HIS118 and PHE120  gene and PvTFL1c as the Late Flowering (LF) gene [PvTFL1y locus in common bean is related with growth habit and cosegregated with determinacy locus [TFL1a, GmTFL1a and GmTFL1b, were isolated from the soybean through molecular dissection; mapping analysis indicated that GmTFL1b was a candidate for Dt1 [. Cosegregation of the marker and TFL locus indicated that LprTFL is a homologue of the TFL1 in Indian bean [Due to unavailability of genome sequence database, not a single economically important gene has been identified in Indian bean. Comparative gene mapping indicated that genomic structure of related plant species has been conserved in context to genetic content, order and function . Phylogel stages . In pea,LF) gene . PvTFL1ycy locus . Two ort for Dt1 . Cosegreian bean .Arabidopsis TFL1 gene belongs to CETS  family which has an important role in the transformation of vegetative shoot apex into inflorescence morphology [LprTFL is found to be responsible for growth habit difference in the present study. This loss of function splice site variation is SNP, created due to transition from guanine to adenine which results into determinate growth habit  [LprTFL might be a very important component of this molecular pathway.A linkage relationship between growth habit and flowering homologues has been reported in soybean , 53, pea soybean . The pos soybean . GmCOL1amologues . Availab (PEBPs) . This inTFL with other regulatory protein involved in plant growth architecture. This potential anion-binding site has been previously proposed to play a key role in interaction of TFL protein with phosphorylated proteins [TFL protein structure of GPKH 120 with anion-binding pocket undisturbed . FD is involved in both positive and negative regulation of flowering through formation of phosphorylation dependent complex with FT or TFL1 [TFL1 protein is capable of interacting with the FD transcription factor, tfl1 mutants flower early and their shoot apical meristem is converted into a terminal flower [TFL protein’s interaction with bZIP transcription factor FD [We envision that a loop present on segment B and small β-sheet are together involved in the interaction of proteins . Both thentified , 15, 58.entified . One maj or TFL1 . TFL1 prl flower . The binactor FD –28, 57 oactor FD –28 or anactor FD , 60 or oTFL locus is responsible for determinate growth habit in GNIB 21. This splice site variation result into variation in the length of the third and fourth exons which eventually leads to deletion of 14 amino acids in final protein sequence. This structural anomaly disturbs previously reported anion-binding pocket and secondary binding pocket due to displacement of small β-sheet away from external loop rendering TFL protein non-functional. Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated targeted mutagenesis of GmFT2a has been demonstrated in soya bean which delayed flowering time [Allelic characterization of genes responsible for growth habit and photoperiod-responsive flowering may throw deeper insight into molecular mechanisms responsible for shaping plant architecture and enable modulation of these traits through genome editing. A splice site SNP in ing time . Our stu


A 31-year-old G5P1 patient with unremarkable past medical history at 29 weeks of gestation was diagnosed with a gigantic left frontotemporal brain mass. Initial clinical management as an inpatient achieved an improvement in the symptoms. The patient and surgical team agreed to schedule a cesarean delivery at 32 weeks of gestation if no neurological deterioration was observed. Intraoperative course with general endotracheal anesthesia and bilateral transversus abdominis plane block was uneventful and promoted efficient postoperative pain control. Seven days after delivery, the patient underwent craniotomy for brain tumor resection. This report describes the anesthetic management of a patient with an intracranial tumor during pregnancy. Central nervous system (CNS) tumors present in the general population at a low incidence and are generally classified as either 1) primary or metastatic and 2) nonmalignant or benign and malignant. The most prevalent primary CNS tumors in adults are meningiomas, pituitary adenomas, gliomas, Schwannomas, and pineal tumors in order of occurrence [ primary  nonmaligThis case report obtained written permission from the patient to publish, and all authors either participated in the care of the patient or substantively helped to prepare this manuscript.Here, we present successful anesthetic management of a patient with an intracranial tumor who underwent cesarean delivery (CD). We highlight the utility of transversus abdominis plane (TAP) blocks with liposomal bupivacaine in the provision of postcesarean analgesia when intrathecal analgesia is contraindicated. A 31-year-old gravida 5, para 1 (G5P1) with an unremarkable past medical history, at 29 weeks gestation was diagnosed with a left frontotemporal brain mass measuring 7 × 7 × 5.5 cm. Four months prior, the patient began therapy for migraine headaches that were occasionally accompanied by episodes of word-finding difficulty. After acute worsening and persistence of symptoms including nausea, appetite loss, and vision blurriness, the patient underwent magnetic resonance imaging (MRI) of the brain, which demonstrated the expansive intracranial process as well as subfalcine herniation  due to iDiagnosis of an intracranial tumor may be delayed in pregnancy, given that intracranial expansive-associated symptoms  are frequent during pregnancy. Physiological changes of pregnancy, primarily related to increased levels of progesterone, may precipitate growth of intracranial tumors as well as trigger flaring of symptoms . AssisteThis case demonstrates the effectiveness of US-guided TAP block following CD, mainly when neuraxial analgesia cannot be performed. It is indicated for postoperative pain control, increasing patient satisfaction, and reducing postoperative opioid consumption and its undesired side effects.


Also, we found evidence of positive selection on at least three codon positions, encoded by NADH dehydrogenase genes (ND4 and ND5). The accelerated evolutionary rates found for sea turtles on COX2, ND1 and CYTB and the molecular footprints of positive selection found on ND4 and ND5 genes may be related to mitochondrial molecular adaptation to stress likely resulted from a more active lifestyle in sea turtles. Our study provides insight into the adaptive evolution of the mtDNA genome in sea turtles and its implications for the molecular mechanism of oxidative phosphorylation.Sea turtles are the only extant chelonian representatives that inhabit the marine environment. One key to successful colonization of this habitat is the adaptation to different energetic demands. Such energetic requirement is intrinsically related to the mitochondrial ability to generate energy through oxidative phosphorylation (OXPHOS) process. Here, we estimated Testudines phylogenetic relationships from 90 complete chelonian mitochondrial genomes and tested the adaptive evolution of 13 mitochondrial protein-coding genes of sea turtles to determine how natural selection shaped mitochondrial genes of the Chelonioidea clade. Complete mitogenomes showed strong support and resolution, differing at the position of the Chelonioidea clade in comparison to the turtle phylogeny based on nuclear genomic data. Codon models retrieved a relatively increased  The mitogenome is composed of 13 genes encoding OXPHOS proteins, 2 rRNAs (12S rRNA and 16S rRNA), and 22 tRNAs3, and is thought to evolve under continuous purifying selection for coding regions5. However, as mitochondrial genes respond to changes in energy requirements, extreme environments may favor positive selection, driving adaptations in different mitogenome genes for some lineages8.How different environmental conditions shape mitochondrial DNA evolution has become a common question in the field of evolutionary biology, revealing more about adaptive patterns in organisms facing ecological changes than expected. Mitochondria is a cellular component directly involved in oxygen use, metabolism, and energy production, and hence plays an important role in aerobic respiration through oxidative phosphorylation (OXPHOS)12. Among extreme environmental conditions, research on the molecular evolution of mtDNA related to high altitude has received special attention16, mainly because evaluating selective pressures of environmental temperature and oxygen availability on mtDNA molecular changes could provide key insights on mitogenome adaptive evolution14. Aquatic environments present similar challenging conditions (hypoxia and low temperatures), and accordingly, also demands mitogenome adaptations in vertebrates, which were reported for killer whales17, river dolphins18, and penguins11. Despite most researches on the role of mtDNA selection in endothermic organisms evolution, recently there has been an increased interest in ectothermic species which are highly dependent on environmental conditions. For instance, Escalona et al.19 studied mitochondrial evolution on softshell turtles revealing positively selected sites in complex I genes for Trionychidae clade and Carettochelys insculpta, suggesting convergent evolution of OXPHOS genes in response to a long-lasting aquatic lifestyle in both lineages.Selection in mtDNA genes has been linked to environmental temperature, high demanding metabolism, altitude and oxygen availability in several species20. Among chelonians, several lineages independently adapted to the marine environment and only the representatives of the Chelonioidea clade (extant sea turtles) have survived to the present (see Evers and Benson21). Sea turtles comprise seven extant species grouped into two sister families, Dermochelyidae (one species) and Cheloniidae (six species)22. These species present adaptations to marine environmental challenges, such as flippers, high salt excretion by modified lachrymal glands23, hydrodynamic shells, and cardiorespiratory adaptations to deliver O2 to tissues during dives24. In addition, sea turtles have an active lifestyle, being diving organisms, highly migratory, and with high fecundity rates26. Marine turtles are among the fastest moving extant reptiles and, since energy demand for locomotion is considered the primary determinant of metabolic rate for marine organisms27, the active lifestyle of marine turtles is expected to affect their energetic demands, resulting in larger metabolic rates compared to other reptiles28. Therefore, it is reasonable to hypothesize that mitochondrial gene adaptations may have had an important role in the adaptive success of sea turtles lineages in the marine environment, leaving molecular footprints in their mitogenomes.Chelonians are a group with more than 300 species distributed in diverse ecological niches around the world, including rivers, lakes, forests, deserts and oceans29. Here we aimed to investigate the evolutionary patterns of sea turtle mitogenomes and address the possible role of mtDNA evolution in the adaptation to metabolic energetic high demands on these species. We provided a comprehensive phylogenetic analysis of mitogenome evolution in Testudines and investigated the evolutionary rates and molecular signatures of natural selection for all 13 mtDNA protein-coding genes for sea turtles, in a phylogenetic comparison against lineages of non-marine turtles and other non-avian reptiles.Despite previous works on the characterization of sea turtles mitogenomes, mainly used in phylogenetic and phylogeographic approaches, only exploratory analysis has examined potential adaptations to the marine environment in these species at the molecular levelND5 translocation in the Platysternidae family  followed by NADH dehydrogenase genes with ND6 (π = 0.133) presenting higher diversity. The cytochrome c oxidase genes  were the least diverse among the mtDNA genes  families  and an alignment of the 13 concatenated PCGs for the 110 species (dataset II) with high bootstrap (BP > 50%) and posterior probabilities (PP > 90%) support for most branches   for all 13 PCGs, suggesting different evolutionary rates among lineages included in our dataset. Values of age Fig.  online aND4 and ND5 genes   while ND6 with the lowest number (10.92%). Only two positions were assigned with diversifying positive selection with FUBAR tests: the codon 3 from ND5 (PP = 0.92) and the codon 169 from ND4 (PP = 0.95)  Fig. . MEME rech Table  online. 5) Table  online.Tp < 0.001) for ND1 gene  and in one physicochemical property for CYTB gene   Fig. . FunctioDermatemys mawii mitogenome is important for achieving a complete mitochondrial phylogeny. Besides the lack of this mitogenome, the phylogenetic relationships we recovered were congruent with a recent chelonian phylogeny based on 57 complete mitochondrial turtle genomes19 but with incongruences regarding the positions of Chelydridae + Dermatemyidae + Kinosternidae lineage and the sea turtles lineage (Cheloniidae + Dermochelyidae) when compared to recent phylogenies using nuclear genes31  sex-biased disparities on dispersion and on differential introgression susceptibility of mitochondrial versus nuclear alleles, resulting in mitochondrial capture35 and (3) selection, causing trait convergence or diversifying adaptive sites37. The discordance between trees recovered from mitogenomes and nuclear genes was already reported for other animal groups. For example, Li et al.38 recovered different topologies using mtDNA and nuDNA markers in felid phylogeny and focused on the second hypotheses to explain that difference, in which historical admixture and mitochondrial capture may have occurred between cat ancestor’s lineages which had their divergences around 11 million years ago (MYA)39. Here we found widespread signature of purifying selection across chelonian mitogenomes, consistent with the previous statement that purifying selection acts constraining the mitogenome evolution to conserve OXPHOS proteins functionality40 but we also found evidence for positive selection on OXPHOS genes, revealed by codon-based test when comparing sea turtle lineages with other chelonian lineages. Moreover, some OXPHOS genes seem to be under intensified selection based on RELAX results (K > 1) and codeML results (ω higher in relation to other genes due to less conservative evolutionary constraints), suggesting that in sea turtles, these genes experienced an acceleration on their evolution and that selection may be a factor that could explain discordance among mitochondrial and nuclear gene trees. However, we cannot rule out possible ancient introgression and incomplete lineage sorting (ILS) as plausible sources for this divergence. Although hybridization and ILS effects are known to be stronger in the case of groups with considerably recent divergence times33, microevolutionary processes can also impact deep divergences 41 as the divergence of the ancestral lineage of sea turtles from their non-marine sister group41, which dates around 66 MYA  42. Moreover, ancient mitochondrial capture has already been suggested as an explanation for conflicting topologies of the well supported nuclear and mitochondrial trees for other groups of turtles45 and the use of historical type specimens sequencing can be effective to highlight this issue for sea turtles.In this study we present the most comprehensive analysis of mitogenome evolution in Testudines, including data for 90 species, focusing on the molecular evolution of OXPHOS genes in sea turtles. As the data we compiled is derived from publicly available sources, no representative of the Dermatemydidae family was included in the phylogenetic analyses. Thus, the generation of the COX2, CYTB, and ND1) specifically for the sea turtles clade over other chelonian species, although there is no statistically significant evidence for positive selection acting on a greater proportion of these genes. Also, our analysis showed a faster rate of evolution for all turtles to the ATP8 gene, when compared to the other genes, evidenced by the analysis of genetic diversity and the high estimated ω values. PAML results, together with the selection intensification signals indicated by the RELAX tests, suggest that marine lineages have accumulated a greater number of non-synonymous amino acid changes than the other testudines lineages on these genes.PAML analysis showed higher ω on three OXPHOS genes  , suggesting a physiological impact on this gene in sea turtles.The use of distinct methods and assumptions allowed us to better explore patterns of molecular evolution in sea turtles. Taken together, all selection tests were able to identify 5 sites evolving under positive selection in four genes  Tables , 3. MoreCOX2—complex IV, CYTB—complex III and ND1—complex I). This result reveals that these genes have an excess of non-synonymous replacements than expected for the sea turtle lineage, differentiating the evolution rate of these genes for the sea turtle lineage from the rest of the turtles. Many studies have investigated the role of selection on mitochondrial genes in organisms that inhabit high altitudes16, but few focused on organisms with a more active lifestyle. For instance, studies comparing flying versus non-flying species in groups of bats46 and grasshoppers13 found seven positively selected genes  related to flight in grasshoppers, while ND2, ND3, ND4L, ND4, ND5, ND6, and COX2 genes were found to have higher values of ω in bats46. Our results, combined with these studies, suggest that complex I and IV genes may be important candidates to be impacted by the action of positive selection related to a more active lifestyle. On the other hand, as with other studies, COX1 and COX3 genes showed the lowest ω values and highest signs of purifying selection47.The three genes that showed evidence of accelerated evolution encode subunits of three separate complexes of the electron transport chain  more specialized metabolic requirements, such as elephants and their large body size49, dugong, sloth, and pangolin, and their low energy diet50, (b) an increased tolerance in thermal range in cetaceans and penguins17, (c) flying in bats46, and (d) living at high altitudes in galliniform birds16 and alpine pheasants15. Compared to these studies, the mitogenome of sea turtles showed less evidence of the pervasive action of natural selection. This pattern was expected because, despite the deep divergence among marine species (at least 30 MYA)29, previous studies support a slower evolutionary ratio and a slower molecular clock for the chelonian mitogenome52. Moreover, sea turtles, as ectothermic reptiles, are considered to have a low metabolic rate, using their energy reserves more slowly than endothermic species53. Also, these poor thermogenic abilities generally confine sea turtles, except for D. coriacea, to shallow tropical waters53, reducing the selective pressure of temperature change, one of the main hypotheses used to explain their patterns of mitochondrial genome evolution11. These characteristics may explain the fewer adaptive molecular footprints in this chelonian system compared to the numerous molecular changes already described for endothermic organisms.TreeSaap analysis of these genes indicated that regions with non-synonymous substitutions correspond to radical amino acid changes, although not showing significant positive selection signals. These radical changes occur when the altered residue does not share similar physicochemical properties with the ancestral residue indicating significant functional impact on the protein55. Hence, research considering their nuclear components can contribute to fully understand the evolution of OXPHOS genes in sea turtles59. In this scenario, investigating all OXPHOS system in turtles is important mainly due to the high incidence of hybridization among sea turtle species67. Compatibility between the nuclear and mitochondrial components of the OXPHOS system is of utmost importance for metabolic and energetic optimal fitness55, and the impact of this phenomenon could be investigated on hybrid turtles. Moreover, approaches integrating population genetic studies with biochemical and physiological experiments can represent a next step57 to understand the evolution of the OXPHOS system in these organisms.OXPHOS genes comprise numerous subunits that are encoded by both the mitogenome and the nuclear genomeND4 and ND5 genes for different sea turtles species, highlighting a site within a functional domain of the ND4 gene with selection signal shared by all species of the Chelonioidea clade. Although the active lifestyle of sea turtles does not seem to exert strong selective pressure on the mitochondrial genes of the OXPHOS system, the few genes with higher ω values compared with other chelonians and the greater fixation of non-synonymous mutations in these genes found for the sea turtle lineage may be responsible for the incongruencies between mitochondrial or nuclear marker inferred topologies. Our results emphasize the importance of using different analyses when assessing selection at the mitogenome level. Also, our study provides first insights into the adaptive evolution of the mtDNA genome in sea turtles, which may have facilitated the successful radiation and diversification of turtle species into the marine environment.In summary, here we investigated evolutionary patterns and footprints of selection in sea turtles OXPHOS genes under the hypothesis that the more active lifestyle of sea turtles could be exerting greater selective pressure on these genes. We found evidence for positive selection at the coding level for several sites in Dermatemys mawii mitogenome on public databases, the only living species in this family. We also retrieved 20 reptile mitogenomes encompassing Squamata, Aves, and Crocodylia orders to be used as outgroups  prior to alignment, as some species with frameshifting insertions and/or deletions (indels) of 1 or 2 bp in ND3 gene were observed by71. Due to an extensive translocation of the gene cluster trnH/trnS1/trnL1/nad5 on Platysternon megacephalum72 and the usual overlapping sites between ATP8 and ATP6 genes found in turtles mitogenome19, we extracted individual genes and non-coding regions for all species, based on the genome annotations in GenBank and on sequence alignments to keep the correct genes size and correctly infer substitution models. All sequences for ND6 were reverse complemented due to their encoding by the reverse strand of the mitogenome. These regions were aligned individually and then concatenated, resulting in 39 partitions. Four different datasets were used in different analytical steps: (I) complete mitogenome for 110 species, (II) only protein-coding genes (PCGs) aligned separately and then concatenated for 110 species, (III) PCGs aligned individually for 110 species, and (IV) PCGs aligned individually only for the seven marine species.We retrieved the whole mtDNA genome sequences of 90 turtles from NCBI database, comprising 13 of the 14 Testudines families  and nucleotide diversity (π) using the DNAsp v. 6.12.0374 to reconstruct maximum likelihood (ML) trees for all datasets, with 1000 ultrafast bootstrap replicates to assess nodal support75. Only nodes with support values ≥ 80 were considered robust. We also estimated Bayesian trees for datasets I and II using MRBAYES v. 3.2.676, applying the partitioned models estimated with PARTITION FINDER v. 2.1.177 , in a Bayesian framework using FUBAR from HyPhy package v. 2.179, a ML framework using codeML program from PAML v. 4.9 h package80, FITMODEL v. 0.5.3 software81, MEME and RELAX from HyPhy package v. 2.182 and in a joint approach of ML and counting methods in SLAC, also from HyPhy package83. See Spielman et al.84 for a detailed comparison of HyPhy's methods.In order to test a possible effect of topology on the inference of sites under selection, we also performed the selection analyses using the topology inferred with genomic data. Since there was no difference, only the results with the topology inferred with the mitochondrial data are shown. We performed selection analyses using an alignment of the aligned PCGs for the 110 species (dataset III) and an alignment of the aligned PCGs only for the 7 species of the Chelonioidea clade (dataset IV) alignments after conversion into codon alignments on PAL2NAL program85. codeML groups several different models, that vary in terms of their assumptions about how ω varies across branches of the phylogeny (branch models), across the sequence (site models), and across both (branch-site model)86. For branch models, we first estimated a unique ω value for all branches along the tree with the one-ratio model. Then, using the free-ratio model, we assumed an independent ω for each branch. Finally, we estimated one ω for sea turtle lineage and another for the rest of the phylogeny applying a two-ratio model. We labeled the superfamily Chelonioidea (lineage of sea turtles) as the foreground branch in each phylogenetic tree generated for each PCG in separate analyses, for two-ratio model and all branch-site models described below. The target clade for these analyses is represented in bold clade in Fig. To test if the ω in sea turtles were different from the rest of the tree we used codeML branch models on dataset III, which allows ω to vary among branches in the phylogeny87. On codeML we used model A versus their null model. FITMODEL is well suited for exploratory analysis and we tested if our data fitted to the nested codon-substitution models M0 and M387. While model M0 assumes that all sites in a sequence alignment are subject to the same selection process, the M3 model assumes variation in selective constraint across sites and is modeled as three rate ratio classes with ω1, ω2, and ω3. FITMODEL allows site-specific switches between different values of the nonsynonymous/synonymous rate ratio81 and we tested the switching models M3 + S1 and M3 + S2. Under the Switching test on FITMODEL, a time-reversible Markov process with three additional parameters is modeled: the overall rate of interchange among rate ratio classes (δ), a coefficient for shifts between ω1 and ω3 (α), and a coefficient for shifts between ω2 and ω3 (β). The S1 model imposes equal switching rates among ω1, ω2 and ω3 rate ratio classes (α = β = 1), while the S2 model allows α and β to vary freely accounting for unequal rates of switches between selection classes81. Finally, we applied a third branch-site test using aBSREL (“adaptive branch-site random effects likelihood”) from HyPhy88 using dataset III. Different from codeML and FITMODEL, rates are calculated for every branch in aBSREL, allowing positive selection on background branches. To avoid overparameterization aBSREL infers, using small-sample Akaike Information Criterion correction (AICc), the optimal number of rate categories per branch, rather than assuming that each branch should be fitted with three rate classes. Also, p values obtained from individual tests for multiple comparisons were corrected by aBSREL using the Bonferroni–Holm procedure to control family-wise false-positive rates88.Branch-site models were used to determine if some proportion of sites is subject to positive selection along Chelonioidea lineage. Therefore, for branch-site analysis on codeML and FITMODEL we also divided the tree inferred from dataset III into foreground branch (Chelonioidea clade), where sites may be evolving under positive selection, and background branches , where positive selection is absent89 to get an overview of the 13 PCGs selection signatures for sea turtles. SLAC infer dN and dS rates on a per-site basis using a combination of ML and counting approaches. We used a mixed-effects model of evolution (MEME)90 to detect sites evolving under positive selection inside the Chelonioidea branch. Similar to FITMODEL, MEME applies a branch-site random effects phylogenetic framework allowing the distribution of ω to vary from site to site as well as from branch to branch, which allows MEME to identify instances of both episodic and pervasive positive selection. We also used FUBAR software to estimate the number of nonsynonymous and synonymous substitutions at each codon on 13 PCGs for sea turtles phylogeny, providing the posterior probability of every codon belonging to a set of classes of ω 91. Significance was assessed by posterior probability (PP) > 0.95.We also applied site-model analysis inside the Chelonioidea phylogeny using dataset IV. We used Single-Likelihood Ancestor Counting (SLAC)p < 0.05.For all selection analyses based on ML, nested models were compared using the likelihood ratio test (LRT) and their results were evaluated against 92 inside the Chelonioidea phylogeny (dataset IV). TreeSAAP relies on the MM01 model implemented in baseML from PAML package85 and uses a phylogeny to reconstruct the most likely ancestral states for the gene sequences under investigation. This software assigns weight values to the nonsynonymous codon changes, for which overall physicochemical effects are assessed using a model with 31 physicochemical amino acid properties. The magnitude of the change is rated from 1 (most conservative) to 8 . A significant deviation from neutral evolution is tested via a z-score and interpreted as a result of positive selection. A highly significant z-score calculated in TreeSAAP  indicates more non-synonymous substitutions than assumed under the neutral model92. To ensure conservative calling of positively selected codon sites only amino acid changes with a score between 6 and 8 and with a positive z-score < 0.001 were used92. Finally, we employed functional analysis of PCGs using InterPro 76.0 web resource93, to predict protein domains and investigate whether sites identified under positive selection are present within or near functional regions.Genes detected with higher ω values in codeML tests were then analyzed in TreeSAAP v. 3.2Supplementary information.


Drosophila melanogaster lines selected for postponed reproductive senescence and unselected control (B) lines. We quantified the productivity of the O and B lines and found that reproductive senescence is maternally controlled. We therefore chose 57 candidate genes that are expressed in ovaries, 49 of which have human orthologs, and assessed the effects of RNA interference in ovaries and accessary glands on lifespan and reproduction. All but one candidate gene affected at least one life history trait in one sex or productivity week. In addition, 23 genes had antagonistic pleiotropic effects on lifespan and productivity. Identifying evolutionarily conserved genes affecting increased lifespan and delayed reproductive senescence is the first step toward understanding the evolutionary forces that maintain segregating variation at these loci in nature and may provide potential targets for therapeutic intervention to delay senescence while increasing lifespan.Limited lifespan and senescence are near-universal phenomena. These quantitative traits exhibit variation in natural populations due to the segregation of many interacting loci and from environmental effects. Due to the complexity of the genetic control of lifespan and senescence, our understanding of the genetic basis of variation in these traits is incomplete. Here, we analyzed the pattern of genetic divergence between long-lived (O)  Limited lifespan and senescence, the post-reproductive decline in survival and fertility with advancing age, are near-universal phenomena. They are quantitative traits that exhibit variation in natural populations due to the segregation of many different interacting loci and environmental effects .Drosophila melanogaster provide experimental support for these predictions as there is increased genetic variance in mortality , C. elegansQuantitative trait loci (QTL) affecting lifespan have been mapped in D. melanogaster lines selected for postponed reproductive senescence and five unselected control (B) lines  B) lines  and the P{KK} RNAi lines are from the same genetic background, contain upstream activating sequence UAS-RNAi constructs for each candidate gene at the same locus on the second chromosome, and have no known off-target effects on other loci  with the model Y = µ + Gm + Gf + W + Gm×W + Gf×W + Gm×Gf + Gm×Gf×W + ε, where Gm is the fixed effect of the mother’s genotype, Gf is the fixed effect of the father’s genotype, W is the fixed effect of week, and ε is the error, using JMP Pro 14 . Reduced model ANOVAs of form Y = µ + Gm + Gf + Gm×Gf + ε were also run for each week, and Tukey’s tests were performed to determine significant differences between each cross at each week.We assessed the productivity of females from the five O (O1 – O5) and five B (B1 – B5) lines crossed to males from their own lines, and productivity of O and B females crossed to males from the other selection lines  as described in To identify genomic intervals that contained fixed or nearly fixed variants between the O and B lines, we first identified variants with allele frequency differences greater than 0.8 between the O and B lines. Next, we merged any adjacent variants whose allele frequency differences were greater than 0.8. These intervals were often interrupted by small numbers of variants that did not meet this stringent allele frequency difference threshold. These interruptions were merged with their flanking intervals if they contained fewer than three variants and if the minimum allele frequency differences were greater than 0.5. We calculated both the length in bp of these merged intervals  and the GAL4-c825 males and six P{KK} or P{GD} UAS-RNAi females to mate and lay eggs in vials containing 10 mL culture medium for two days. The GAL4-c825 driver is expressed in amniosera, adult female ovary, and adult male accessory glands and seminal vesicles. Two control genotypes were also used: F1 progeny of v60100, which is the isogenic strain in which the P{KK} UAS-RNAi lines were constructed with the empty PhiC31 vector, crossed to GAL4-c825; and F1 progeny of v60000, which is the isogenic strain in which the P{GD} UAS-RNAi lines were constructed with a modified pUAST vector pMF3, crossed to GAL4-c825. Of the F1 progeny from each mating, 3 males and 3 females were sorted into vials containing 5 mL culture medium with 48 replicates for each genotype. Three females per vial were allowed to lay eggs for 24 hr on 10 mL culture media once a week for their entire lifespan, and the average number of F2 offspring per female in each vial was recorded until 16 days after the initial egg laying for 13 of the 48 replicates. Flies were transferred without anesthesia to new vials containing 5 mL culture media every 1-3 days to provide fresh food and minimize bacterial and fungal infections. Dead flies were removed, and their deaths recorded. The sum of the average number of offspring across all weeks determined the lifetime productivity for each vial. The mixed factorial ANOVA model Y = µ + S + G + S×G + Rep(G) + S×Rep(G) + ε was used to partition variation in lifespan between the fixed main effects of Sex (S), Genotype  and their interaction, and the random effect of replicate vial (Rep), nested within genotype. Reduced model ANOVAs of form Y = µ + G + Rep(G) + ε were also run for males and females separately. The full fixed effect factorial ANOVA model for lifetime productivity, which is the summation of all individual weeks, was Y = µ + G + ε; where G is Genotype. The full fixed effect factorial ANOVA model for weekly productivity was Y = µ + W + G + W×G + ε; where W is Week and G is Genotype. Reduced models of form Y = µ + G + ε were run for each week. These analyses were performed in six blocks with different genes in each block.The first generation of experimental flies were produced by allowing six L×G) + ε, where L indicates the RNAi or control line, G is the gene tested, R is biological replicate, nested within line and gene, and ε is the residual  variance. Reduced models of form Y = µ + L + Rep(L) + ε were run for each gene. Effect and p-Value summaries were performed to determine significant differences of the RNAi line from the control line. All analyses were performed using JMP Pro 14 .The magnitude of RNAi-mediated suppression of gene expression was assessed using quantitative PCR (qPCR) of female ovaries and male accessory glands for a subset of the RNAi and control genotypes. Ovary tissue was dissected from females and accessory glands were dissected from males at 3-5 days old, with 10 ovaries and 10 accessory glands in each of two biological replicates per genotype, and RNA was extracted. cDNA was synthesized from 120 ng of total RNA using the iScript cDNA synthesis kit (Bio-Rad). We performed qPCR for each of three technical replicates per biological replicate using Maxima SYBR Green (Thermo Scientific). GAPDH was the internal control. The levels of expression for each gene were normalized against the internal control and compared to the RNAi line control to determine differences in gene expression of the RNAi mutants. We first assessed overall differences in gene expression using the ANOVA model Y = μ + L + G + L×G + Rep of lifespan for GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies. Supplementary Table 9 gives the analyses of variance (ANOVAs) of lifetime productivity for GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies. Supplementary Table 10 gives the analyses of variance (ANOVAs) of weekly productivity for GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies. Supplementary Table 11 gives the analyses of variance (ANOVAs) of ovary qPCR for GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies. Supplementary Table 12 gives the analyses of variance (ANOVAs) of accessory gland qPCR for GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies. Supplementary Table 13 is a summary of genes for which RNAi affects lifespan and/or productivity. Supplementary Figure 1 shows the results of ovary qPCR for the difference in expression between GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies. Supplementary Figure 2 show the results of accessory gland qPCR for the difference in expression between GAL4-c825 × UAS RNAi and GAL4-c825 × control F1 flies.. Supplemental material available at figshare: https://doi.org/10.25387/g3.11503278.The DNA sequencing raw data are publicly available on GEO under SRA Project ID PRJNA286855. Supplementary Table 1 gives the raw data for the four week productivity assay of the pure and reciprocal corsses between the O and B lines. Supplementary Table 2 gives the analyses of variance (ANOVAs) of O and B line productivity. Supplementary Table 3 shows the genomic regions containing genetically divergent SNPS. Supplementary Table 4 gives the list of 57 candidate genes tested, including their FlyBase ID, gene name, gene, symbol, and the Vienna stock number used in the RNAi assay. Supplementary Table 5 gives the raw lifespan data for P = 0.012) and female genotype by male genotype (P = 0.013) interaction.In order to assess whether the net effects of selection on reproductive senescence were maternally or paternally controlled, we quantified productivity for females of each of the O and B lines at one, two, three and four weeks of age. We assessed productivity for O females crossed to males of the same O line, O females crossed to B males; B females crossed to males from the same B line, and B females crossed to O males. We found little difference in the total number of viable offspring between any of these crosses for young flies, but divergence between the O and B genotypes at later ages  allele frequency differences, nearly 300 of which showed gene expression changes consistent with delayed senescence between young and old flies of the O and B lines. There were some large regions of genetic divergence between the O and B lines in this study, especially on the X chromosome. We re-analyzed these data, imposing the more stringent criterion that genetically divergent variants were those for which the average difference in allele frequency between the O and B lines was greater than 0.8. This analysis identified individual variants as well as intervals, some of which were quite large, containing multiple genetically divergent SNPs  the candidate gene is expressed in ovaries and accessory glands since the productivity assay of the O and B lines implicated a strong maternal and weaker paternal effect, and selection was on reproductive senescence; and (3) viable RNAi stocks with no off-target effects were available. We assessed lifespan (Supplementary Table 5), average lifetime reproduction (Supplementary Table 6), and average weekly reproduction (Supplementary Table 7) for the F1 progeny of UAS-RNAi lines crossed to GAL4-c825, and F1 progeny of the crosses of an appropriate control line without the UAS transgene and the GAL4 driver. GAL4-c825 is expressed in amniosera, adult female ovary, and adult male accessory glands and seminal vesicles and is therefore expected to reduce gene expression specifically in these tissues.We chose 57 candidate genes (Supplementary Table 4) that were either located in the intervals with high SNP divergence between the O and B lines (Supplementary Table 3), or that had low capu, Cdc7, CG13369, CG3326, Dredd, egh, lama, MED22, Nmd3, Pgant1). The effects on lifespan were highly sex-specific: seven genes increased lifespan in both sexes and six decreased lifespan in both sexes; eight genes increased lifespan in only one sex and 15 decreased lifespan in only one sex.RNAi of 47 of the 57 genes tested affected lifespan G0004, ND-ACP and RpL22 RNAi decreased total productivity G0004 and Fdx1 all decrease productivity; babos and sov have increased productivity early in life and decreased productivity later in life; and piwi has increased productivity at an early age. RNAi of blw, boi, CG12018, CG14969, CG16868, CG17777, CG4452, CG5254, crn, CycE, Cyp4g1, dx, ND-ACP, pn and RpL22 had deleterious effects on lifespan in at least one sex and productivity in at least one week; and RNAi of CG3071, sgg and zf30c increased both lifespan and productivity in at least one sex and at least one week. RNAi of dnc had no effects on either trait.Given these data on lifespan and productivity, we can now assess the extent to which RNAi of these candidate genes gives the antagonistic pleiotropic effects between different fitness traits predicted by evolutionary theory . RNAi ofabo, CG13760, CG7971, fs(1)N, G9a, and Klp67A had increased lifespan in at least one sex and decreased early productivity. RNAi of CG3704, msn and sra decreased lifespan in at least one sex and increased early productivity. RNAi of Cdc7, CG3326, Dredd, egh and lama decreased productivity but had sexually antagonistic effects on lifespan; whereas RNAi of CG32809, CG4281, CG4554, DAAM, and wds increased lifespan in at least one sex and decreased early and increased late productivity. RNAi of Eip75B decreased female lifespan and had increased early and decreased late productivity. Finally, RNAi of capu and MED22 had increased early and decreased late productivity and decreased female and increased male lifespan; while RNAi of CG13369 reduced early and increased late productivity and increased female and decreased male lifespan.RNAi of the remaining 23 genes had antagonistic pleiotropic effects on lifespan and/or productivity (abo in accessory glands) had lower point estimates of expression in the RNAi lines relative to the control, and the combined analyses across all genes showed significant effects of gene . Based on the large standard errors, we infer the qPCR experiment to quantify gene expression differences between the RNAi lines and the control were underpowered, and that the number of biological samples would need to be increased to detect small changes in gene expression for some of these genes. In contrast, the phenotypic data were based on large sample sizes and are thus reliable read-outs of the effects of small differences in gene expression in reproductive tissues.We assessed the effectiveness of the RNAi knockdown for 16 candidate genes in female ovaries and RNAi knockdown of 12 genes in male accessory glands using quantitative PCR (qPCR) . At the individual gene level, nine of RNAi lines tested in females and six of the RNAi lines tested in males had significantly reduced expression compared to the control. However, all genes  and rapid evolution of long-lived strains by selecting for increased age of reproduction from several different geographical populations (P < 10−3) genes based on analyses of individual variants. Here, we re-analyzed these data for signatures of local selective sweeps involving multiple variants and identified 1,071 genes in these intervals. Clearly, the genetic architecture of natural variation in lifespan is highly polygenic.Natural ulations . PreviouP-values and for which RNAi reagents with no off-target effects were publicly available. In addition, we assessed whether the difference in reproductive senescence between the O and B lines was due to maternal genotype, paternal genotype, or both. We found that late life reproductive capacity is primarily determined by the female genotype since O line females are on average more productive in weeks three and four than are B line females. However, the male genotype is also significant at weeks one, three and four, such that the productivity of O females crossed to B males is greater than that of O females crossed to O males. One plausible explanation for the paternal effect may be differences in the accessary gland products between the O and B males. Male accessory gland products evolve rapidly and have profound effects on female life history traits, increasing egg production, decreasing lifespan and reducing the probability of re-mating .These data can be used to begin to address two fundamental questions regarding naturally occurring genetic variation for lifespan and reproductive senescence: what are the causal genes, and what is the distribution of direct and pleiotropic effects of causal variants on each trait? However, not all of the candidate genes implicated by the divergence analyses are likely to be causal, since selection will cause local linkage disequilibrium between the focal variants and closely linked variants. We chose a subset of candidate genes to evaluate with low divergence dunce affected lifespan and/or productivity (Supplementary Table 13). RNAi of six genes increased lifespan and/or reproduction, suggesting that normal levels of expression of these genes in ovaries and accessory glands inhibit extended lifespan and/or reproduction. RNAi of CG3071, sgg and zf30c results in increased lifespan and productivity; RNAi of Lztr1 and pcx increases lifespan with no deleterious effects on productivity; while piwi RNAi increases productivity with no deleterious effects on lifespan. Thus, increasing lifespan does not necessarily occur at the cost of reduced reproductive capacity . piwi and sgg are highly pleiotropic genes known to affect oogenesis; in addition, both genes have been independently associated with naturally occurring genetic variation in lifespan G0004, ND-ACP) has been independently associated with variation in lifespan  and response to selection for delayed reproductive senescence , as would be expected if normal levels of expression of these genes in ovaries and accessory glands are required for average lifespan and productivity. This suggests there may be naturally occurring variation in levels of expression of these genes in these tissues, and selection resulted in their increased expression in ovaries and accessory glands in the O lines. Regardless of whether this testable hypothesis is true, variation in nine of these genes  with antagonistic pleiotropic effects between the sexes and/or between lifespan and productivity were associated with variation in lifespan or response to selection for delayed reproductive senescence in independent populations .RNAi of the remaining 27 genes in ovaries and accessory glands resulted in antagonistic pleiotropic effects that varied in their complexity (Supplementary Table 13): antagonistic pleiotropy between lifespan and reproduction; sexual antagonistic pleiotropy for lifespan with no effects on productivity; antagonistic pleiotropic effects on early and late reproduction with no effects on lifespan; sexual antagonistic pleiotropy for lifespan and antagonistic pleiotropy between lifespan and reproduction for one sex; antagonistic pleiotropy between early and late reproduction and antagonistic pleiotropy between lifespan and early reproduction; and sexual antagonistic pleiotropy for lifespan, antagonistic pleiotropy between early and late reproduction, and antagonistic pleiotropy between lifespan and reproduction. Variation in genes exhibiting antagonistic effects between female lifespan and productivity affects alternative resource allocation strategies in terms of reproduction and somatic cell maintenance. Variation in genes with sexually antagonistic effects on lifespan affects variation in alternative life history strategies in the two sexes. All sources of antagonistic pleiotropy can lead to the maintenance of genetic variation for lifespan and reproductive senescence at intermediate frequencies in natural populations, depending on the model assumptions . Seven gpiwi and sgg, 10 of the candidate genes also affect oogenesis, spermatogenesis or another aspect of reproduction N, MED22, sra) (Supplementary Table 13)  for which prior information on their function was lacking.In addition to able 13) . Many offication , carbohyKlp67A has increased female lifespan, but not the tightly linked genes CG4452, Fdx1 and Hsp22, which all have decreased lifespan and/or productivity; and Nmd3 has increased female lifespan but the adjacent gene pn has decreased female lifespan (Supplementary Table 13). On the other hand, fs(1)N and DAAM are adjacent; as are CG13760, wds and egh: RNAi of all of these genes causes increased lifespan (Supplementary Table 13). Therefore, some large divergence intervals may actually be due to selection of several closely linked genes with effects in the same direction.This study is the first step to ‘reverse engineer’ the genomic response to selection for delayed reproductive senescence and the accompanying correlated response of increased lifespan from variation segregating in a natural population. We have shown that an RNAi screen targeting knockdown of gene expression in reproductive tissues in both sexes for genes that exhibit genetic divergence in replicate selection lines successfully identified genes affecting lifespan and/or reproduction. Further, the effects of RNAi knockdown are consistent with predictions of evolutionary theory, in that RNAi knockdown can result in complex sexually antagonistic pleiotropic effects on lifespan and antagonistic pleiotropic effects between lifespan and productivity – such alleles would remain segregating at intermediate frequencies in nature. RNAi resulting in increased or decreased lifespan (and/or productivity) may mimic alleles remaining at intermediate frequency due to antagonistic pleiotropic effects on other fitness traits not assessed in his study. However, it is important to note that many genes for which RNAi caused decreased lifespan and/or productivity may not be direct targets of selection, but in linkage disequilibrium with the true causal loci. Several of our candidate genes are very closely linked, and only one of the genes in a high divergence interval has increased lifespan and/or productivity. For example, The genes for which RNAi causes increased lifespan in at least one sex are excellent candidates for the next step of the reverse engineering paradigm, which is to identify the actual variants that cause increased lifespan. Are there single variants in these genes that have sexually antagonistic effects on lifespan or antagonistic pleiotropic effects on productivity, or are different, closely linked variants in these genes independently causing these effects? What are the mechanisms by which these variants affect lifespan, and how do variants in different genes interact? Are their effects the same in short-lived and long-lived genetic backgrounds? Recent advances in gene editing technology will facilitate these future studies.


Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination. Autophagy  is an intracellular degradation system that maintains cellular homeostasis and is associated with many pathophysiological phenomena, including cancer and neurodegenerative diseases , 2. AutoThere are two types of autophagy: non-selective and selective. Non-selective autophagy engulfs cytoplasmic solution non-selectively, which includes soluble proteins among others. The diameter of non-selective autophagosomes is thought to be relatively uniform (~ 1 μm). Selective autophagy engulfs diverse substrates of various sizes while large autophagosomes enwrap large autophagic substrates such as mitochondria and bacteria –9. The sThe endoplasmic reticulum (ER) is an organelle that plays diverse roles, such as the synthesis of secreted proteins and membrane proteins , proteinThe architecture of the ER is composed of a tubular network and sheet like structures; recent advances in super-resolution microscopy imaging techniques have revealed that the ER sheet structure consists of a dense assembly of ER tube structures  Fig. 1)1). The tThe intracellular site where autophagosome formation takes place had earlier been the subject of controversy; however, it is now clear that it occurs near the ER subdomain called the omegasome  Fig. 1)1). DoublRecent advances have helped in understanding the relation between ER and autophagosome formation. Several reports have demonstrated the critical role of Atg2 in the connection between ER and autophagosomes both in mammalian and yeast cells , 35. AtgThe lipid molecules that flow into the autophagosomes may not be preexisting ER constituents. Phospholipid synthases like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1), accumulate in the ER subdomain and promote local lipid synthesis and elongation of the tube structure . The smaAre these Atg2-mediated lipid transfers the sole lipid source of autophagosome membrane formation? Coat protein complex II (COPII) vesicles, which bud from the ER, are also suggested to be involved in autophagosome formation . Atg9 isThe ERdj proteins are a family of proteins harboring the DNAJ domain that cooperate with the molecular chaperones and play diverse roles in the ER –56. ReceIn this review, we have discussed how the ER is related to autophagosome formation. In this regard, current studies are expected to shed light on the mechanisms that regulate the size of the autophagosomes.