Discrepancies in tumor mutation burden reporting from sequential endobronchial ultrasound transbronchial needle aspiration samples within single lymph node stations - brief report.

Introduction: Tumour Mutation Burden (TMB) is a potential biomarker for immune cancer therapies. Here we investigated parameters that might affect TMB using duplicate cytology smears obtained from endobronchial ultrasound transbronchial needle aspiration (EBUS TBNA)-sampled malignant lymph nodes. Methods: Individual Diff-Quik cytology smears were prepared for each needle pass. DNA extracted from each smear underwent sequencing using large gene panel (TruSight Oncology 500 (TSO500 - Illumina)). TMB was estimated using the TSO500 Local App v. 2.0 (Illumina). Results: Twenty patients had two or more Diff-Quik smears (total 45 smears) which passed sequencing quality control. Average smear TMB was 8.7 ± 5.0 mutations per megabase (Mb). Sixteen of the 20 patients had paired samples with minimal differences in TMB score (average difference 1.3 ± 0.85). Paired samples from 13 patients had concordant TMB (scores below or above a threshold of 10 mutations/Mb). Markedly discrepant TMB was observed in four cases, with an average difference of 11.3 ± 2.7 mutations/Mb. Factors affecting TMB calling included sample tumour content, the amount of DNA used in sequencing, and bone fide heterogeneity of node tumour between paired samples. Conclusion: TMB assessment is feasible from EBUS-TBNA smears from a single needle pass. Repeated samples of a lymph node station have minimal variation in TMB in most cases. However, this novel data shows how tumour content and minor change in site of node sampling can impact TMB. Further study is needed on whether all node aspirates should be combined in 1 sample, or whether testing independent nodes using smears is needed.

Whole Genome Sequencing in Advanced Lung Cancer can be Performed Using Diff-Quik Cytology Smears Derived from Endobronchial Ultrasound, Transbronchial Needle Aspiration (EBUS TBNA).

INTRODUCTION: Maximising alternative sample types for genomics in advanced lung cancer is important because bronchoscopic samples may sometimes be insufficient for this purpose. Further, the clinical applications of comprehensive molecular analysis such as whole genome sequencing (WGS) are rapidly developing. Diff-Quik cytology smears from EBUS TBNA is an alternative source of DNA, but its feasibility for WGS has not been previously demonstrated. METHODS: Diff-Quik smears were collected along with research cell pellets. RESULTS: Tumour content of smears were compared to research cell pellets from 42 patients, which showed good correlation (Spearman correlation 0.85, P < 0.0001). A subset of eight smears underwent WGS, which presented similar mutation profiles to WGS of the matched cell pellet. DNA yield was predicted using a regression equation of the smears cytology features, which correctly predicted DNA yield > 1500 ng in 7 out of 8 smears. CONCLUSIONS: WGS of commonly collected Diff-Quik slides is feasible and their DNA yield can be predicted.

Evaluating Diff-Quik cytology smears for large-panel mutation testing in lung cancer-Predicting DNA content and success with low-malignant-cellularity samples.

BACKGROUND: Cytology smears are commonly collected during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA) procedures but are rarely used for molecular testing. Studies are needed to demonstrate their great potential, in particular for the prediction of malignant cell DNA content and for utility in molecular diagnostics using large gene panels. METHODS: A prospective study was performed on samples from 66 patients with malignant lymph nodes who underwent EBUS TBNA. All patients had air-dried, Diff-Quik cytology smears and formalin-fixed, paraffin-embedded cell blocks collected for cytopathology and molecular testing. One hundred eighty-five smears were evaluated by microscopy to estimate malignant cell percentage and abundance and to calculate smear size and were subjected to DNA extraction. DNA from 56 smears from 27 patients was sequenced with the TruSight Oncology 500 assay (Illumina). RESULTS: Each microscopy parameter had a significant effect on the DNA yield. An algorithm was developed that predicted a >50-ng DNA yield of a smear with an area under the curve of 0.86. Fifty DNA samples (89%) with varying malignant yields were successfully sequenced. Low-malignant-cell content (<25%) and smear area (<15%) were the main reasons for failure. All standard-of-care mutations were detected in replicate smears from individual patients, regardless of malignant cell content. Tier 1/2 mutations were discovered in two cases where standard-of-care specimens were inadequate for sequencing. Smears were scored for tumor mutation burden. CONCLUSIONS: Microscopy of Diff-Quik smears can triage samples for comprehensive panel sequencing, which highlights smears as an excellent alternative to traditional testing with cell blocks.

Prospective Optimization of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Lymph Node Assessment for Lung Cancer: Three Needle Agitations Are Noninferior to 10 Agitations for Adequate Tumor Cell and DNA Yield.

Introduction: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS TBNA) is an important means of obtaining a tissue for advanced lung cancer. Optimizing the EBUS TBNA needling technique is important to maintain procedural simplicity and maximize sample quality for emerging molecular diagnostics. Methods: We prospectively explored three versus 10 agitations of the needle in sequential passes into the lymph node using separate needles. Resulting Diff-Quik cytology smears were quantitatively assessed using microscopic (tumor cell cellularity, abundance scores, erythrocyte contamination) and DNA yields. Microscopy was reported by two cytopathologists, and an inter-rater assessment was made by four additional cytopathologists. Results: In 86 patients confirmed as having malignant disease by EBUS TBNA (45 males, 41 females), a mean of 5.3 smears were made per patient with a total of 459 smears scored by pathologists and 168 paired smears extracted for DNA. There was no significant difference between three versus 10 agitations for smear cellularity (p = 0.44), DNA yield (p = 0.84), or DNA integrity (p = 0.20), but there was significantly less contamination by erythrocytes from three agitations (chi-square p = 0.008). There was significantly more DNA in the first pass into the node using three agitations than with other passes and with 10 agitations (pass × agitations interaction, p = 0.031). Reviewing pathologists correctly classified smears as more than or equal to 25% cellularity 86.3% of the time (κ = 0.63 [95% confidence interval: 0.55-0.71]). Conclusions: Three agitations are noninferior to 10 agitations for overall abundance of malignant cells and DNA content on smears. A smear with adequate DNA for panel sequencing could almost always be made with the first needle pass using three agitations.

Environmentally Relevant Iron Oxide Nanoparticles Produce Limited Acute Pulmonary Effects in Rats at Realistic Exposure Levels.

Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeOxNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeOxNPs present in the environment. We, therefore, analysed the potential toxicity of FeOxNPs of different forms (Fe3O4, α-Fe2O3 and γ-Fe2O3) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeOx-mix). A preliminary in vitro study indicated Fe3O4 and FeOx-mix were more cytotoxic than either form of Fe2O3 in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague-Dawley rats, nose-only exposure, 50 µg/m3 and 500 µg/m3, 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects.

Conservation value of Codium tenue habitat for the endangered Knysna seahorse Hippocampus capensis.

We investigated the density and size structure of the endangered Knysna seahorse Hippocampus capensis in the macroalga Codium tenue at three sites in a residential marina estate in the Knysna Estuary, South Africa, over an 18 month period from March 2017 to August 2018. Seahorses were present in C. tenue throughout the year. Average (± SE) densities of 0.33 (± 0.03) and 0.23 (± 0.03) seahorses per kg of C. tenue were found for 2017 and 2018 respectively. Density did not vary across sites but varied significantly across months with a decreasing trend from summer to spring. The average (± SE) adult seahorse height was 75.16 mm (± 0.63 mm) and 69.09 mm (± 0.64 mm) and mass was 2.05 g (± 0.04 g) and 1.65 g (± 0.04 g) for males and females, respectively, and an even sex ratio was observed throughout the sampling period. Height varied across months, with significantly smaller seahorses found in June and August of 2017. Tail length was highly correlated with height and therefore can serve as a potential proxy for determining the height of H. capensis. This study showed that C. tenue is consistently utilised by Knysna seahorse and should therefore be considered an important habitat to protect and facilitate the ongoing conservation of this endangered seahorse species.

Pulmonary toxicity of inhaled nano-sized cerium oxide aerosols in Sprague-Dawley rats.

Cerium oxide nanoparticles (CeO2NPs), used in some diesel fuel additives to improve fuel combustion efficiency and exhaust filter operation, have been detected in ambient air and concerns have been raised about their potential human health impact. The majority of CeO2NP inhalation studies undertaken to date have used aerosol particles of larger sizes than the evidence suggests are emitted from vehicles using such fuel additives. Hence, the objective of this study was to investigate the effects of inhaled CeO2NP aerosols of a more environmentally relevant size, utilizing a combination of methods, including untargeted multi-omics to enable the broadest possible survey of molecular responses and synchrotron X-ray spectroscopy to investigate cerium speciation. Male Sprague-Dawley rats were exposed by nose-only inhalation to aerosolized CeO2NPs (mass concentration 1.8 mg/m3, aerosol count median diameter 40 nm) for 3 h/d for 4 d/week, for 1 or 2 weeks and sacrificed at 3 and 7 d post-exposure. Markers of inflammation changed significantly in a dose- and time-dependent manner, which, combined with results from lung histopathology and gene expression analyses suggest an inflammatory response greater than that seen in studies using micron-sized ceria aerosols. Lipidomics of lung tissue revealed changes to minor lipid species, implying specific rather than general cellular effects. Cerium speciation analysis indicated a change in Ce3+/Ce4+ ratio within lung tissue. Collectively, these results in conjunction with earlier studies emphasize the importance of aerosol particle size on toxicity determination. Furthermore, the limited effect resolution within 7 d suggested the possibility of longer-term effects.

The Importance and Quantification of Plutonium Binding in Human Lungs.

Epidemiological studies have shown that the main risk arising from exposure to plutonium aerosols is lung cancer, with other detrimental effects in the bone and liver. A realistic assessment of these risks, in turn, depends on the accuracy of the dosimetric models used to calculate doses in such studies. A state-of-the-art biokinetic model for plutonium, based on the current International Commission on Radiological Protection biokinetic model, has been developed for this purpose in an epidemiological study involving the plutonium exposure of Mayak workers in Ozersk, Russia. One important consequence of this model is that the lung dose is extremely sensitive to the fraction (fb) of plutonium, which becomes bound to lung tissue after it dissolves. It has been shown that if just 1% of the material becomes bound in the bronchial region, this will double the lung dose. Furthermore, fb is very difficult to quantify from experimental measurements. This paper summarizes the work carried out thus far to quantify fb. Bayesian techniques have been used to analyze data from different sources, including both humans and dogs, and the results suggest a small, but nonzero, fraction of < 1%. A Bayesian analysis of 20 Mayak workers exposed to plutonium nitrate suggests an fb between 0 and 0.3%. Based on this work, the International Commission on Radiological Protection is currently considering the adoption of a value of 0.2% for the default bound fraction for all actinides in its forthcoming recommendations on internal dosimetry. In an attempt to corroborate these findings, further experimental work has been carried out by the US Transuranium and Uranium Registries. This work has involved direct measurements of plutonium in the respiratory tract tissues of workers who have been exposed to soluble plutonium nitrate. Without binding, one would not expect to see any activity remaining in the lungs at long times after exposure since it would have been cleared by the natural process of mucociliary clearance. Further supportive study of workers exposed to plutonium oxide is planned. This paper ascertains the extent to which these results corroborate previous inferences concerning the bound fraction.

The small airway epithelium as a target for the adverse pulmonary effects of silver nanoparticle inhalation.

Experimental modeling to identify specific inhalation hazards for nanomaterials has in the main focused on in vivo approaches. However, these models suffer from uncertainties surrounding species-specific differences and cellular targets for biologic response. In terms of pulmonary exposure, approaches which combine 'inhalation-like' nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterize alterations in mRNA and miRNA within the lung. Organotypic-reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark-generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid interface (AE-ALI) system. Adverse effects were characterized using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag+) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects.

Natural levels of polonium-210 in urine.

This paper presents a review and analysis of available data on background levels of polonium-210 in urine. It was established that 819 measurements could be considered to correspond to natural background levels, excluding a large number of values identified by the original investigators as potentially due to an artificial source or due to recognised enhancement of dietary intake. Almost 550 measurements were extracted from studies reported in the literature; additional measurements were contributions of previously unpublished data by authors of more recent papers. The majority of the measurements (462) were single samples from individuals but 357 values were repeat measurements provided by 81 subjects and for these the mean value for each subject was used. The final dataset consisted of measurements from 543 individuals. Analysis of the measurements confirmed the data was log normally distributed with mean and median values of 15.5 mBq d-1 and 11 mBq d-1 in urine samples, respectively. While the overall range was from 0.3-111 mBq d-1, almost 90% of the measurements were less than 30 mBq d-1, 95% were less than 45 mBq d-1, and 99% less than 70 mBq d-1. Separate analysis of data for smokers and non-smokers suggested a modest increase in smokers of up to 5 mBq d-1. Perhaps reflecting the importance of dietary differences such as seafood consumption, a marked difference between countries was observed in the range of results. While for most countries, 95% or more of results were below 30 mBq d-1, China and Italy were notable exceptions, with greater than 20% of values above this level.

Collateral contamination concomitant to the polonium-210 poisoning of Mr Alexander Litvinenko.

Mr Litvinenko died on 23 November 2006, having been poisoned with polonium-210 on 1 November, with evidence of a previous poisoning attempt during October 2006. Measurements of 210Po in urine samples were made for a large number of people to determine whether they may have been contaminated. In the majority of cases, measured levels were attributable to the presence of 210Po from normal dietary sources. For a small number of cases, elevated levels provided evidence of direct contamination associated with the poisonings. For one individual, while estimated doses were below thresholds for irreversible organ damage, a notably increased risk of cancer can be inferred. The use of the chelating agent, unithiol, to increase 210Po excretion in this case was only moderately effective in reducing doses received.

Slow lung clearance and limited translocation of four sizes of inhaled iridium nanoparticles.

BACKGROUND: Concerns have been expressed that inhaled nanoparticles may behave differently to larger particles in terms of lung clearance and translocation, with potential implications for their toxicity. Studies undertaken to investigate this have typically involved limited post-exposure periods. There is a shortage of information on longer-term clearance and translocation patterns and their dependence on particle size, which this study aimed to address. METHODS: Rats were exposed (<3 h) nose-only to aerosols of spark-generated radioactive iridium-192 nanoparticles of four sizes: 10 nm, 15 nm, 35 nm and 75 nm (count median diameter) (aerosol mass concentrations 17, 140, 430, and 690 µg/m3, respectively). The content of iridium-192 in the whole animal, organs, tissues, and excreta was measured at various times post-exposure to ≥ 1 month. Limited toxicological investigations were undertaken for the 10 nm aerosol using bronchoalveolar lavage fluid. Elemental maps of tissue samples were produced using laser ablation inductively coupled plasma mass spectrometry and synchrotron micro-focus x-ray fluorescence. The chemical speciation of the iridium was explored using synchrotron micro focus x-ray near-edge absorption spectroscopy. RESULTS: Long-term lung retention half-times of several hundred days were found, which were not dependent on particle size. There was significant variation between individual animals. Analysis of bronchoalveolar lavage fluid for the 10 nm aerosol indicated a limited inflammatory response resolving within the first 7 days. Low levels of, particle size dependent, translocation to the kidney and liver were found (maximum 0.4% of the lung content). Any translocation to the brain was below the limits of detection (i.e. < 0.01% of the lung content). The kidney content increased to approximately 30 days and then remained broadly constant or decreased, whereas the content in the liver increased throughout the study. Laser ablation inductively coupled plasma mass spectrometry analysis indicated homogeneous iridium distribution in the liver and within the cortex in the kidney. CONCLUSIONS: Slow lung clearance and a pattern of temporally increasing concentrations in key secondary target organs has been demonstrated for inhaled iridium aerosol particles < 100 nm, which may have implications for long-term toxicity, especially in the context of chronic exposures.

Pulmonary effects of inhalation of spark-generated silver nanoparticles in Brown-Norway and Sprague-Dawley rats.

BACKGROUND: The increasing use of silver nanoparticles (AgNPs) in consumer products is concerning. We examined the potential toxic effects when inhaled in Brown-Norway (BN) rats with a pre-inflammatory state compared to Sprague-Dawley (SD) rats. METHODS: We determined the effect of AgNPs generated from a spark generator (mass concentration: 600-800 µg/mm(3); mean diameter: 13-16 nm; total lung doses: 8 [Low] and 26-28 [High] µg) inhaled by the nasal route in both rat strains. Rats were sacrificed at day 1 and day 7 after exposure and measurement of lung function. RESULTS: In both strains, there was an increase in neutrophils in bronchoalveolar lavage (BAL) fluid at 24 h at the high dose, with concomitant eosinophilia in BN rats. While BAL inflammatory cells were mostly normalised by Day 7, lung inflammation scores remained increased although not the tissue eosinophil scores. Total protein levels were elevated at both lung doses in both strains. There was an increase in BAL IL-1ß, KC, IL-17, CCL2 and CCL3 levels in both strains at Day 1, mostly at high dose. Phospholipid levels were increased at the high dose in SD rats at Day 1 and 7, while in BN rats, this was only seen at Day 1; surfactant protein D levels decreased at day 7 at the high dose in SD rats, but was increased at Day 1 at the low dose in BN rats. There was a transient increase in central airway resistance and in tissue elastance in BN rats at Day 1 but not in SD rats. Positive silver-staining was seen particularly in lung tissue macrophages in a dose and time-dependent response in both strains, maximal by day 7. Lung silver levels were relatively higher in BN rat and present at day 7 in both strains. CONCLUSIONS: Presence of cellular inflammation and increasing silver-positive macrophages in lungs at day 7, associated with significant levels of lung silver indicate that lung toxicity is persistent even with the absence of airway luminal inflammation at that time-point. The higher levels and persistence of lung silver in BN rats may be due to the pre-existing inflammatory state of the lungs.

Rapid cytological analysis of endobronchial ultrasound-guided aspirates in sarcoidosis.

Rapid on-site evaluation (ROSE) of endobronchial ultrasound-guided transbronchial needle aspirates (EBUS-TBNA) has not been compared to final detailed cytological analysis in patients with suspected sarcoidosis. To assess the diagnostic accuracy of EBUS-TBNA with ROSE in patients with suspected sarcoidosis, a prospective two-centre study performed EBUS-TBNA with ROSE of cellular material followed by transbronchial lung biopsy (TBLB) and endobronchial biopsy (EBB). The diagnostic accuracy of EBUS-TBNA with ROSE was compared to the final cytological assessment and to TBLB and EBB. Analysis confirmed 49 out of 60 cases of sarcoidosis. ROSE sensitivity was 87.8% (specificity 91%, positive predictive value 97.7%). ROSE slide interpretation in combination with the final fixed slide and cell block preparations had a sensitivity of 91.8% (specificity 100%, positive predictive value 100%). 67% of patients were confirmed as having sarcoidosis on TBLB and 29% on EBB. Interobserver agreement between cytotechnologists and pathologists was very good (κ=0.91, 95% CI 0.80-1.0 and κ=0.91, 95% CI 0.79-1.0, respectively). EBUS-TBNA with ROSE has high diagnostic accuracy and interobserver agreement and informs the bronchoscopist in theatre whether additional diagnostic procedures need to be undertaken. EBUS-TBNA with ROSE should therefore be considered as the first-line investigation of sarcoidosis.

Structure and formation of the unusual sperm of Patelloida latistrigata (Mollusca : Patellogastropoda): implications for fertilization biology.

The structure of the spermatozoa and spermatogenesis of the lottiid limpet Patelloida latistrigata is described by transmission electron microscopy. Although the lengths of the spermatozoa (about 60 µm) and their head region (about 12 µm) are similar to those of other patellogastropods, the structure of the sperm head and midpiece are very different. The head consists of an unusually large acrosome (about 11-µm long) with a broad posterior invagination that houses the relatively small nucleus. The midpiece mitochondria, which are rather elongate with large folded tubular cristae, are housed in a cytoplasmic sheath posterior to the nucleus. The proximal centriole is unusually elongate (about 2-µm long). The axoneme that emerges from the distal centriole is surrounded anteriorly by the cytoplasmic sheath in which the cytoplasmic side of the plasma membrane has electron-dense material. The flagellum is enlarged at its terminal end. Spermatogenesis is similar to that described for other patellogastropods. Patelloida latistrigata, therefore, has spermatozoa that seem to meet the morphological criteria of ent-aquasperm, which raises the question of whether fertilization is truly external in this limpet. However, it is also possible that the modifications to the sperm are linked to unknown specializations of the egg or egg envelope.

Polypropionates from the South African marine mollusk Siphonaria oculus.

Three new polypropionate metabolites, 6Z,8E-Δ(8)-siphonarienfuranone (1), 6E,8E-Δ(8)-siphonarienfuranone (2), and 6E,8E-3-hydroxy-4,6,8,10,12-pentamethylpentadeca-6,8-dien-5-one (3), and the known polypropionate siphonarienfuranone (4) were isolated from the intertidal South African marine mollusk Siphonaria oculus. Evidence is presented to suggest that 1, 2, and 4 may cyclize from an acylic precursor on chromatographic workup of the acetone extract of this mollusk.

Review of the role of EBUS-TBNA for the pulmonologist, including lung cancer staging.

This review focuses on the role of endobronchial ultrasound-guided transbronchial needle aspiration in day-to-day pulmonology practice. Case examples are given of the common indications for endobronchial ultrasound-guided transbronchial needle aspiration which are: (i) lung cancer staging; (ii) confirming a diagnosis of malignancy in thoracic lymph nodes; (iii) diagnosing central pulmonary masses; (iv) sarcoidosis; and (v) inflammatory/benign thoracic lymph nodes. The technique is widely used, and after appropriate training by experienced bronchoscopists can be easily integrated into a bronchoscopy service.

A simple method for assessing that internal doses are below action levels.

The accidental or deliberate dispersal of radioactive aerosols into the public environment may require urgent assessments of radiation dose to be made to aid decisions on whether treatment should be implemented, or to reassure the public that doses are acceptably low. However, rapid assessments will be difficult due to lack of information on factors such as the particle size distribution and biokinetic characteristics of the aerosol. A procedure is described that relates the amount of activity of a radionuclide in the body and excreta to time after intake for a specified dose, taking into account the likely variations in aerosol size and differences in the biokinetic behaviour of the same or different chemical forms of the radionuclide. The implementation of the procedure for an intake of caesium-137 and a dose level of 1 mSv is described and the information presented graphically. Figures for other specified dose levels can be produced by simply scaling the data by an appropriate factor. The figures can also be used to assess the most appropriate monitoring procedure and indicate the uncertainty in the assessed dose according to the parameter and parameter values used. This approach is proposed for rapid decisions on public reassurance when potentially large numbers of people are involved. It is not intended as a substitute for individual dose assessment.

Sperm morphology in five species of cicadettine cicadas (Hemiptera: Cicadomorpha: Cicadidae).

Mature spermatozoa from five species of cicadas of the subfamily Cicadettinae (Quintilia wealei, Melampsalta leucoptera, Stagira simplex, Xosopsaltria thunbergi and Monomatapa matoposa) were examined by light and electron microscopy. In each species sperm are elongate, aggregated into organized bundles with their heads embedded in a homogenous matrix to form spermatodesmata, and exhibit polymegaly. The head of the sperm consist of an anteriorly positioned conical acrosome that has a tubular substructure and a deep, posterior invagination that forms the subacrosomal space (eccentrically positioned anteriorly). The acrosome is flattened anteriorly; posteriorly it extends along either side of the nucleus as two tubular processes that gradually decrease in diameter. The filiform nucleus tapers anteriorly and intrudes into the subscrosomal space. Posteriorly the nucleus has a lateral invagination that houses material of the so-called centriolar adjunct. Posterior to the centriolar adjuct and the nucleus are two crystalline mitochondrial derivatives and a centriole, respectively, the latter giving rise to the axoneme, which has a 9 + 9 + 2 arrangement of microtubules. In these respects the sperm are similar to those of platypleurine cicadas. However, some features seem unique to cicadettines, including the structural organization of an enlarged centriolar adjunct and the dimensions of the tails. The enlarged centriolar adjunct has a lamella-like substructure and can be considered a synapomorphic character in the Cicadettinae. It is, therefore, potentially useful in the separation of this subfamily from the Cicadinae. In addition, the great length of the sperm nucleus of long-headed sperm in M. matoposa could be a synapomorphy of this genus and related taphurine and cicadettine species.