First evidence of sexual dimorphism in olfactory organs of deep-sea lanternfishes (Myctophidae).

Finding a mate is of the utmost importance for organisms, and the traits associated with successfully finding one can be under strong selective pressures. In habitats where biomass and population density is often low, like the enormous open spaces of the deep sea, animals have evolved many adaptations for finding mates. One convergent adaptation seen in many deep-sea fishes is sexual dimorphism in olfactory organs, where, relative to body size, males have evolved greatly enlarged olfactory organs compared to females. Females are known to give off chemical cues such as pheromones, and these chemical stimuli can traverse long distances in the stable, stratified water of the deep sea and be picked up by the olfactory organs of males. This adaptation is believed to help males in multiple lineages of fishes find mates in deep-sea habitats. In this study, we describe the first morphological evidence of sexual dimorphism in the olfactory organs of lanternfishes (Myctophidae) in the genus Loweina. Lanternfishes are one of the most abundant vertebrates in the deep sea and are hypothesized to use visual signals from bioluminescence for mate recognition or mate detection. Bioluminescent cues that are readily visible at distances as far as 10 m in the aphotic deep sea are likely important for high population density lanternfish species that have high mate encounter rates. In contrast, myctophids found in lower density environments where species encounter rates are lower, like those in Loweina, likely benefit from longer-range chemical or olfactory cues for finding and identifying mates.

Pharyngeal Constrictor Dose-Volume Histogram Metrics and Patient-Reported Dysphagia in Head and Neck Radiotherapy.

AIMS: Head and neck radiotherapy long-term survival continues to improve and the management of long-term side-effects is moving to the forefront of patient care. Dysphagia is associated with dose to the pharyngeal constrictors and can be measured using patient-reported outcomes to evaluate its effect on quality of life. The aim of the present study was to relate pharyngeal constrictor dose-volume parameters with patient-reported outcomes to identify prognostic dose constraints. MATERIALS AND METHODS: A 64-patient training cohort and a 24-patient testing cohort of oropharynx and nasopharynx cancer patients treated with curative-intent chemoradiotherapy were retrospectively examined. These patients completed the MD Anderson Dysphagia Inventory outcome survey at 12 months post-radiotherapy to evaluate late dysphagia: a composite score lower than 60 indicated dysphagia. The pharyngeal constrictor muscles were subdivided into four substructures: superior, middle, inferior and cricopharyngeal. Dose-volume histogram (DVH) metrics for each of the structure combinations were extracted. A decision tree classifier was run for each DVH metric to identify dose constraints optimising the accuracy and sensitivity of the cohort. A 60% accuracy threshold and feature selection method were used to ensure statistically significant DVH metrics were identified. These dose constraints were then validated on the 24-patient testing cohort. RESULTS: Existing literature dose constraints only had two dose constraints performing above 60% accuracy and sensitivity when evaluated on our training cohort. We identified two well-performing dose constraints: the pharyngeal constrictor muscle D63% < 55 Gy and the superior-middle pharyngeal constrictor combination structure V31Gy < 100%. Both dose constraints resulted in ≥73% mean accuracy and ≥80% mean sensitivity on the training and testing patient cohorts. In addition, a pharyngeal constrictor muscle mean dose <57 Gy resulted in a mean accuracy ≥74% and mean sensitivity ≥60%. CONCLUSION: Mid-dose pharyngeal constrictor muscle and substructure combination dose constraints should be used in the treatment planning process to reduce late patient-reported dysphagia.

Long-term ecological research in freshwaters enabled by regional biodiversity collections, stable isotope analysis, and environmental informatics.

Biodiversity collections are experiencing a renaissance fueled by the intersection of informatics, emerging technologies, and the extended use and interpretation of specimens and archived databases. In this article, we explore the potential for transformative research in ecology integrating biodiversity collections, stable isotope analysis (SIA), and environmental informatics. Like genomic DNA, SIA provides a common currency interpreted in the context of biogeochemical principles. Integration of SIA data across collections allows for evaluation of long-term ecological change at local to continental scales. Challenges including the analysis of sparse samples, a lack of information about baseline isotopic composition, and the effects of preservation remain, but none of these challenges is insurmountable. The proposed research framework interfaces with existing databases and observatories to provide benchmarks for retrospective studies and ecological forecasting. Collections and SIA add historical context to fundamental questions in freshwater ecological research, reference points for ecosystem monitoring, and a means of quantitative assessment for ecosystem restoration.

The role of surface tension on the growth of bubbles by rectified diffusion.

Rectified diffusion has wide and important applications in sonochemistry, ultrasonic cleaning and medical ultrasound. Recent experimental results have demonstrated that the addition of surfactant substantially enhances bubble growth rate. As a hypothesis, this was widely attributed to the acoustic microstreaming and mass transfer resistance caused by surfactants. In this research, the effects of the surfactant of sodium dodecyl sulphate on the rectification have been simulated by considering only the variation of the surface tension coefficient due to the surfactant. The computations are carried out using a newly developed tractable model based on the multi-scale method and the method of matched asymptotic expansions, which allows the prediction of bubble growth taking place over millions of oscillation cycles. The rate of bubble growth observed in the experiments is accurately predicted by our computations, for a range for bulk surfactant SDS concentrations less than or equal to 2.4 mM. Contrary to the widely held hypothesis in the published literature, this has demonstrated that the dominant physical mechanisms remain the shell and area effects in this range of bulk surfactant concentrations. The further enhancement of bubble growth rate provided by either acoustic microstreaming or the resistance to mass transfer is only evident at higher bulk surfactant concentrations. Therefore, the role of surface tension in rectified diffusion for aqueous surfactant solutions is more significant than previously understood. The new results also show that the bubble growth rate is sensitive to small changes in the bubble radius which may account for its unpredictability in applications of sonochemistry.

Simulating nitrogen balance in Canadian agricultural soils from 1981 to 2016.

Agriculture produces food, fiber and biofuels for the world's growing population, however, agriculture can be a major contributor of nitrogen (N) losses including emissions of ammonia (NH3), nitrous oxide (N2O) and nitrate (NO3-) leaching and runoff. A Canadian Agricultural Nitrogen Budget for Reactive N (CANBNr) model was developed to estimate the soil N balance in 3487 soil landscape of Canada polygons from 1981 to 2016. The CANBNr model integrates NH3 emission from fertilizers, manure from housing, storage and field, as well as direct/indirect N2O emissions from fertilizers, manures, crop residues and soil organic matter. The NO3- leaching is estimated based on the residual soil N (RSN) at harvest and drainage derived with the DeNitrification-DeComposition (DNDC) model. From 1981 to 2016, the N input from fertilizer and N fixation increased at a greater rate than N removal in harvested crops in all provinces of Canada, resulting in an increase in the RSN and N losses. In 2016, the Prairie provinces had lower N losses (11.7 kg N ha-1) from N2O, NH3 and NO3- compared with 43.2 kg N ha-1 in central Canada, and 76.5 kg N ha-1 in Atlantic Canada. However, the Prairie provinces had 84.3% of the total Canadian farmland (74.3% of the total Canadian N input), while central Canada had 12.9% of Canadian farmland (21.7% of the total Canadian N input). In the Prairie provinces, the total N2O loss from fertilizer N ranged 4.4-8.6 Gg N whereas NH3 loss ranged from 17.1 to 44.6 Gg N and these values were influenced by both emission intensity and total land area. Total N2O losses from manure were highest in Alberta, Ontario and Quebec resulting in 4.8, 4.4, and 3.4 Gg N and NH3 losses from manure were also highest in these 3 provinces at 61.1, 45.2 and 40.4 Gg N, respectively. Nitrate leaching was impacted by drainage volumes, soil type and N inputs. In the non-growing season, NO3- leaching losses (36-yr average) were 63.3 Gg in Ontario and 57.5 Gg N in Quebec compared with 20.8 Gg N for Ontario and 35.5 Gg N for Quebec in the growing season. In contrast, the Prairie provinces showed higher NO3- leaching in the growing season (23.1-37.4 Gg N) than in the non-growing season (10.4-13.7 Gg N). In summary, total fertilizer N increased the most over the 36 years in the Prairies which resulted in increased RSN and N leaching losses that will require further intervention.

Forages and pastures symposium: forage biodegradation: advances in ruminal microbial ecology.

The rumen microbial ecosystem provides ruminants a selective advantage, the ability to utilize forages, allowing them to flourish worldwide in various environments. For many years, our understanding of the ruminal microbial ecosystem was limited to understanding the microbes (usually only laboratory-amenable bacteria) grown in pure culture, meaning that much of our understanding of ruminal function remained a "black box." However, the ruminal degradation of plant cell walls is performed by a consortium of bacteria, archaea, protozoa, and fungi that produces a wide variety of carbohydrate-active enzymes (CAZymes) that are responsible for the catabolism of cellulose, hemicellulose, and pectin. The past 15 years have seen the development and implementation of numerous next-generation sequencing (NGS) approaches (e.g., pyrosequencing, Illumina, and shotgun sequencing), which have contributed significantly to a greater level of insight regarding the microbial ecology of ruminants fed a variety of forages. There has also been an increase in the utilization of liquid chromatography and mass spectrometry that revolutionized transcriptomic approaches, and further improvements in the measurement of fermentation intermediates and end products have advanced with metabolomics. These advanced NGS techniques along with other analytic approaches, such as metaproteomics, have been utilized to elucidate the specific role of microbial CAZymes in forage degradation. Other methods have provided new insights into dynamic changes in the ruminal microbial population fed different diets and how these changes impact the assortment of products presented to the host animal. As more omics-based data has accumulated on forage-fed ruminants, the sequence of events that occur during fiber colonization by the microbial consortium has become more apparent, with fungal populations and fibrolytic bacterial populations working in conjunction, as well as expanding understanding of the individual microbial contributions to degradation of plant cell walls and polysaccharide components. In the future, the ability to predict microbial population and enzymatic activity and end products will be able to support the development of dynamic predictive models of rumen forage degradation and fermentation. Consequently, it is imperative to understand the rumen's microbial population better to improve fiber degradation in ruminants and, thus, stimulate more sustainable production systems.

Forage degradation in the rumen is critical to producing ruminant animals. For many years, scientists were limited to biochemical techniques to understand how ruminal microbes degraded forage, impairing our understanding of which microbes were involved with degrading which forage components. However, we have understood that as the ruminant opened up plant cells to microbial activity, a succession of microbes was involved in colonizing and breaking fiber into increasingly smaller pieces. The recent development of sequencing techniques has allowed a more detailed understanding of changes in the microbial population of the rumen during forage degradation and the types of degradative enzymes produced by this complex microbial ecosystem. We described the enzymes involved in the degradation of specific forage components, how their end products impact the microbial population through cross-feeding interactions, and how fermentation products can impact food animal production.

The potential effects of polyunsaturated ω-3 fatty acids on spinal cord injury: A systematic review & meta-analysis of preclinical evidence.

Polyunsaturated fatty acids (PUFAs) have received attention for their anti-inflammatory and antioxidant properties. Preclinical studies have investigated the efficacy of PUFAs in animal models of spinal cord injury (SCI) to determine if these properties can translate to neuroprotection and locomotor recovery. Findings from such studies have been promising, suggesting PUFAs as potential treatments against the neurological dysfunction induced by SCI. This systematic review and meta-analysis sought to investigate the efficacy of PUFAs for promoting locomotor recovery in animal models of SCI. PubMed, Web of Science and Embase (Ovid) were searched for relevant papers and those that examined the restorative effects of PUFAs on locomotor recovery in preclinical SCI models were included in our analysis. A random effects meta-analysis (restricted maximum likelihood estimator) was employed. A total of 28 studies were included and the results showed the claim that PUFAs have a beneficial therapeutic effect for promoting locomotor recovery (SMD = 1.037, 95% CI = 0.809-1.2644, p = <0.001) and cell survival (SMD = 1.101, 95% CI = 0.889-1.313, p = <0.001) in animal models of SCI. No significant differences for the secondary outcomes of neuropathic pain and lesion volume. Moderate asymmetry was observed in the funnel plots for locomotor recovery, cell survival and neuropathic pain measures, suggesting publication bias. Trim-and-fill analysis estimated 13, 3, 0 and 4 missing studies for locomotor recovery, cell survival, neuropathic pain, and lesion volume, respectively. A modified CAMARADES checklist was also used to assess risk of bias, showing that the median score for all included papers was 4 out of a possible 7.

Technical note: Investigating the suitability of existing facilities for a new Lu-177 prostate-specific membrane antigen therapy program.

BACKGROUND: 177 Lu prostate-specific membrane antigen (PSMA) therapy prolongs survival for some prostate cancer patients. To adopt this technique, institutions may need to evaluate the suitability of existing infrastructure. PURPOSE: Develop a methodology to determine whether existing facilities can accommodate a 177 Lu-PSMA therapy program. METHODS: Room suitability is defined by both the ability to accommodate 177 Lu-PSMA therapy workflow and to provide appropriate radiation shielding. Two methods of shielding calculation were performed: (1) National Council on Radiation Protection and Measurements report 151 (NCRP-151), with workload defined in terms of the activity of 177 Lu administered, and (2) using the RadPro shielding calculator. This methodology was applied to 131 I therapy, PET-CT uptake, PET-SPECT injection, and orthovoltage therapy rooms. RESULTS: 131 I therapy rooms were found to meet both shielding and workflow requirements. The shielding was found to be adequate for orthovoltage and PET-SPECT facilities, neglecting patient transit between external washrooms. The workflow was the limiting factor for these rooms due to the requirement of dedicated washrooms that shield the patient and contain possible contamination. The PET-CT facility did not meet either criteria. The NCRP-151 method generally predicted a higher dose rate on the other side of shielding than did the RadPro calculator. The dose rate on the other side of concrete shielding as predicted by the NCRP-151 method increased relative to the dose rate predicted by the RadPro calculator as shielding thickness increased. For lead shielding, the dose rate predicted by the NCRP-151 method decreased relative to the result predicted by the RadPro calculator with increasing material thickness. CONCLUSIONS: 131 I therapy, PET-CT uptake, PET-SPECT injection, and orthovoltage therapy rooms were considered. The 131 I treatment rooms were the best candidate for 177 Lu-PSMA therapy, due to their shielding and capability to accommodate the necessary workflow.

A human donor (cadaveric) observation study of three needle placements in the piriformis muscle.

OBJECTIVE: To compare the safety and accuracy of three dry needling locations in the piriformis muscle using human donors. DESIGN: Observational dissection study of embalmed human donors. METHODS: A licensed physical therapist of 17 years clinical experience and 5 years teaching dry needling placed three needles in a medial, midpoint, and lateral location of 14 piriformis muscles of seven embalmed human donors. Block dissection allowed for observation of tissues the needles traversed and recording of the structures that the needles pierced. RESULTS: The lateral needle pierced piriformis in 3/14 trials, and contacted sciatic nerve in 0/14 trials. The medial needle pierced both piriformis and sciatic nerve in 11/14 trials. The midpoint needle pierced the piriformis in 11/14 trials, and contacted sciatic nerve in 3/14 trials. Fisher's Exact test (p < 0.001) found a nonrandom association between dry needle placement, and dry needle contact. CONCLUSIONS: When dry needling the piriformis, a lateral approach can avoid the sciatic nerve, but cannot accurately pierce the piriformis tendon. Furthermore, while a midpoint and medial approach finds the piriformis muscle with the same accuracy, the midpoint location avoided the sciatic nerve more often.

Splitting of the distal median nerve.

Variations of the nerves of the forearm can lead to unexpected clinical findings during physical examination. Additionally, surgery in this region might encounter and potentially damage the nerve in such patients. Here, we present a case of a high split of the median nerve and discuss the findings of the case as well as review salient reports in the literature. Knowledge of such a variation can be important in patient diagnosis and treatment.

Distinct Phenotypic Consequences of Pathogenic Mutants Associated with Late-Onset Retinal Degeneration.

A pathologic feature of late-onset retinal degeneration caused by the S163R mutation in C1q-tumor necrosis factor-5 (C1QTNF5) is the presence of unusually thick deposits between the retinal pigmented epithelium (RPE) and the vascular choroid, considered a hallmark of this disease. Following its specific expression in mouse RPE, the S163R mutant exhibits a reversed polarized distribution relative to the apically secreted wild-type C1QTNF5, and forms widespread, prominent deposits that gradually increase in size with aging. The current study shows that S163R deposits expand to a considerable thickness through a progressive increase in the basolateral RPE membrane, substantially raising the total RPE height, and enabling their clear imaging as a distinct hyporeflective layer by noninvasive optical coherence tomography in advanced age animals. This phenotype bears a striking resemblance to ocular pathology previously documented in patients harboring the S163R mutation. Therefore, a similar viral vector-based gene delivery approach was used to also investigate the behavior of P188T and G216C, two novel pathogenic C1QTNF5 mutants recently reported in patients for which histopathologic data are lacking. Both mutants primarily impacted the RPE/photoreceptor interface and did not generate basal laminar deposits. Distinct distribution patterns and phenotypic consequences of C1QTNF5 mutants were observed in vivo, which suggested that multiple pathobiological mechanisms contribute to RPE dysfunction and vision loss in this disorder.

Toward the fabrication of a 5-µm-resolution Wolter microscope for the National Ignition Facility (invited).

Advancements in computer-controlled polishing, metrology, and replication have led to an x-ray mirror fabrication process that is capable of producing high-resolution Wolter microscopes. We present the fabrication and test of a nickel-cobalt replicated full-shell x-ray mirror that was electroformed from a finely figured and polished mandrel. This mandrel was designed for an 8-m source-to-detector-distance microscope, with 10× magnification, and was optimized to reduce shell distortions that occur within 20 mm of the shell ends. This, in combination with an improved replication tooling design and refined bath parameters informed by a detailed COMSOL Multiphysics® model, has led to reductions in replication errors in the mirrors. Mandrel surface fabrication was improved by implementing a computer-controlled polishing process that corrected the low-frequency mandrel figure error and achieved <2.0 nm RMS convergence error. X-ray tests performed on a pair of mirror shells replicated from the mandrel have demonstrated <10 µm full-width at half-maximum (FWHM) spatial resolution. Here, we discuss the development process, highlight results from metrology and x-ray testing, and define a path for achieving a program goal of 5 µm FWHM resolution.

AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension.

Purpose: Ocular hypertension is a significant risk factor for vision loss in glaucoma caused by the death of retinal ganglion cells (RGCs). We investigated whether small heat shock proteins (sHsps) expressed in RGCs protect those cells against ocular hypertension in mice. Methods: AAV2 vectors encoding genes for one of the following four human sHsps: HSPB1, HSPB4, HSPB5, or HSPB6 were constructed for RGC-specific expression. Ischemia/reperfusion was induced by elevating the intraocular pressure (IOP) to 120 mm Hg for one hour, followed by a rapid return to normal IOP. Microbeads (MB) were injected into the anterior chamber of mice to induce ocular hypertension. RGC death and glial activation were assessed by immunostaining for Brn3a, RBPMS, Iba1, and glial fibrillary acid protein in retinal flat mounts. RGC axonal defects were evaluated by anterograde transport of intravitreally injected cholera toxin-B. RGC function was assessed by pattern electroretinography. Results: Among the sHsps, HspB1 offered the best protection against RGC death from ischemia/reperfusion injury in the mouse retina. Intravitreal administration of AAV2-HSPB1 either two weeks before or one week after instituting ocular hypertension resulted in significant prevention of RGC loss. The MB-injected mice showed RGC axonal transportation defects, but AAV2-HSPB1 administration significantly inhibited this defect. AAV2-HSPB1 prevented glial activation caused by ocular hypertension. More importantly, a single injection of AAV2-HSPB1 protected RGCs long-term in MB-injected eyes. Conclusions: The administration of AAV2-HSPB1 inhibited RGC death and axonal transport defects and reduced glial activation in a mouse model of ocular hypertension. Translational Relevance: Our results suggested that the intravitreal delivery of AAV2-HSPB1 could be developed as a gene therapy to prevent vision loss on a long-term basis in glaucoma patients.

Photochemical Dearomative Cycloadditions of Quinolines and Alkenes: Scope and Mechanism Studies.

Photochemical dearomative cycloaddition has emerged as a useful strategy to rapidly generate molecular complexity. Within this context, stereo- and regiocontrolled intermolecular para-cycloadditions are rare. Herein, a method to achieve photochemical cycloaddition of quinolines and alkenes is shown. Emphasis is placed on generating sterically congested products and reaction of highly substituted alkenes and allenes. In addition, the mechanistic details of the process are studied, which revealed a reversible radical addition and a selectivity-determining radical recombination. The regio- and stereochemical outcome of the reaction is also rationalized.

Gene Therapy for Rhodopsin Mutations.

Mutations in RHO, the gene for rhodopsin, account for a large fraction of autosomal-dominant retinitis pigmentosa (adRP). Patients fall into two clinical classes, those with early onset, pan retinal photoreceptor degeneration, and those who experience slowly progressive disease. The latter class of patients are candidates for photoreceptor-directed gene therapy, while former may be candidates for delivery of light-responsive proteins to interneurons or retinal ganglion cells. Gene therapy for RHO adRP may be targeted to the mutant gene at the DNA or RNA level, while other therapies preserve the viability of photoreceptors without addressing the underlying mutation. Correcting the RHO gene and replacing the mutant RNA show promise in animal models, while sustaining viable photoreceptors has the potential to delay the loss of central vision and may preserve photoreceptors for gene-directed treatments.

Phylogenetics of Archerfishes (Toxotidae) and Evolution of the Toxotid Shooting Apparatus.

Synopsis: Archerfishes (Toxotidae) are variously found in the fresh- and brackish-water environments of Asia Pacific and are well known for their ability to shoot water at terrestrial prey. These shots of water are intended to strike their prey and cause it to fall into the water for capture and consumption. While this behavior is well known, there are competing hypotheses (blowpipe vs. pressure tank hypothesis) of how archerfishes shoot and which oral structures are involved. Current understanding of archerfish shooting structures is largely based on two species, Toxotes chatareus and T. jaculatrix. We do not know if all archerfishes possess the same oral structures to shoot water, if anatomical variation is present within these oral structures, or how these features have evolved. Additionally, there is little information on the evolution of the Toxotidae as a whole, with all previous systematic works focusing on the interrelationships of the family. We first investigate the limits of archerfish species using new and previously published genetic data. Our analyses highlight that the current taxonomy of archerfishes does not conform to the relationships we recover. Toxotes mekongensis and T. siamensis are placed in the synonymy of T. chatareus, Toxotes carpentariensis is recognized as a species and removed from the synonymy of T. chatareus, and the genus Protoxotes is recognized for T. lorentzi based on the results of our analyses. We then take an integrative approach, using a combined analysis of discrete hard- and soft-tissue morphological characters with genetic data, to construct a phylogeny of the Toxotidae. Using the resulting phylogenetic hypothesis, we then characterize the evolutionary history and anatomical variation within the archerfishes. We discuss variation in the oral structures and the evolution of the mechanism with respect to the interrelationships of archerfishes, and find that the oral structures of archerfishes support the blowpipe hypothesis but soft-tissue oral structures may also play a role in shooting. Finally, by comparing the morphology of archerfishes to their sister group, we find that the Leptobramidae has relevant shooting features in the oral cavity, suggesting that some components of the archerfish shooting mechanism are examples of co-opted or exapted traits. Sinopsis Malay: Pelbagai jenis Ikan Sumpit (Toxotidae) dapat dijumpai di persekitaran air tawar dan payau di Asia Pasifik dan mereka terkenal dengan kebolehan mereka menembak air ke arah mangsa di darat. Tembakan air ini bertujuan untuk menyerang mangsa mereka dan menyebabkan mereka jatuh ke dalam air untuk ditangkap dan dimakan. Walaupun tingkah laku ini diketahui umum, terdapat hipotesis yang bersaing (hipotesis sumpitan vs. tangki tekanan) tentang cara ikan sumpit menembak dan struktur mulut yang terlibat. Pemahaman semasa tentang struktur menembak ikan sumpit adalah sebahagian besarnya berdasarkan dua spesies, Toxotes chatareus dan T. jaculatrix. Kami tidak pasti sama ada semua ikan sumpit mempunyai struktur mulut yang sama untuk menembak air, jika variasi anatomi terdapat dalam struktur mulut ini, atau bagaimana ciri-ciri ini telah berkembang. Tambahan pula, terdapat sedikit maklumat tentang evolusi Toxotidae secara keseluruhan, dengan semua penyelidikan sistematik sebelum ini memfokuskan pada hubungan saling keluarga. Kami pada mulanya mengkaji had spesies ikan sumpit ini menggunakan data genetik baharu dan yang diterbitkan sebelum ini. Analisis kami menunjukkan bahawa taksonomi semasa ikan sumpit tidak mematuhi hubungan yang kami perolehi. Toxotes mekongensis dan T. siamensis diletakkan bersama kesinoniman T. chatareus, Toxotes carpentariensis yang diiktiraf sebagai satu spesies dan dikeluarkan daripada kesinoniman T. chatareus, dan genus Protoxotes yang diiktiraf untuk T. lorentzi adalah berdasarkan hasil analisis kami. Kemudian kami mengambil pendekatan integratif, menggunakan analisis gabungan karakter morfologi tisu keras dan lembut diskret dengan data genetik, untuk membina filogeni Toxotidae. Menggunakan hipotesis filogenetik yang terhasil, kami kemudian mencirikan sejarah evolusi dan variasi anatomi dalam ikan sumpit. Kami membincangkan variasi dalam struktur mulut dan evolusi mekanisme berkenaan yang berkaitan dengan ikan sumpit, dan mendapati bahawa struktur mulut ikan sumpit menyokong hipotesis sumpitan tetapi struktur mulut tisu lembut juga mungkin memainkan peranan dalam cara menembak. Akhir sekali, dengan membandingkan morfologi ikan sumpit kepada kumpulan saudara mereka, kami mendapati bahawa Leptobramidae mempunyai ciri penangkapan yang relevan dalam rongga mulut mereka, menunjukkan bahawa beberapa komponen mekanisme penangkapan ikan sumpit merupakan contoh ciri-ciri yang diikut-sertakan atau diguna semula.