Background: Postexercise hypotension (PEH) is a common physiological phenomenon occurring immediately after endurance training (ET), resistance training (RT), and ET plus RT, also termed concurrent training (CT); however, there is little knowledge about the interindividual and magnitude response of PEH in morbidly obese patients.Aim: The aims of this study were (1) to investigate the effect of CT order (ET + RT vs. RT + ET) on the blood pressure responses; 2) characterize these responses in responders and nonresponders, and 3) identify potential baseline outcomes for predicting blood pressure decreases as responders.Methods: A quasi-experimental study developed in sedentary morbidly obese men and women  was assigned to a CT group of ET plus RT  or RT plus ET order group . Subjects of both groups received eight exercise sessions over four weeks. Primary outcomes include systolic (SBP), diastolic (DBP), mean arterial pressure [MAP], heart rate at rest [HR], and pulse pressure [PP] measurements before and after 10 min post-exercise. Secondary outcomes were other anthropometric, body composition, metabolic, and physical fitness parameters. Using the delta ∆SBP reduction, quartile categorization (Q) in “high” (Rs: quartile 4), “moderate” (MRs: quartile 3), “low” (LRs: quartile 2), and “nonresponders” (NRs: quartile 1) was reported.Results: Significant pre–post changes were observed in ET + RT in session 2 for SBP  and session 4 , while the RT + ET group showed significant reductions in session 4 . No significant differences were detected in the sum of the eight sessions for SBP (∑∆SBP) between ET + RT vs. RT + ET . Interindividual analyses revealed significant differences among frequencies comparing Q1 “NRs” , Q2 “LRs” , Q3 “MRs” , and Q4 “HRs” , p < 0.0001. Quartile comparisons showed significant differences in SBP changes (p = 0.035). Linear regression analyses revealed significant association between ∑∆SBP with body fat % , skeletal muscle mass , fasting glucose , triglycerides , and the 6-min walking test .Conclusion: The CT order of ET + RT and RT + ET promote a similar ‘magnitude’ in the postexercise hypotensive effects during the eight sessions of both CT orders in 4 weeks of training duration, revealing “nonresponders” and ‘high’ responders that can be predicted from body composition, metabolic, and physical fitness outcomes. Physical inactivity , CVD mortality (RR = 2.40), myocardial infarction , and all-cause mortality (RR = 1.58) , another complex cardiometabolic square that also implies obesity, poor glucose control, high blood pressure, dyslipidemia, and high triglycerides in plasma, exacerbates the risk for suffering from cardiovascular diseases, particularly to those with morbid obesity who are candidates for bariatric surgery . For exa = 1.58) . Patient = 1.58) . From th = 1.58) . Thus, a = 1.58) , being t = 1.58) .Henceforth, lifestyle interventions, such as exercise training, play a key role , and relOn the other hand, the main PEH studies report data in terms of ‘average data’, and it is well-known that there is a wide interindividual variability to exercise training , having 2; aged 30–55 years) who were assigned for convenience in 1:1 allocation to a CT group of ET plus RT  or a RT plus ET group . Patients were recruited in 2021 from the abovementioned non-governmental institution. All participants were part of the lifestyle program previous to the possibility of receiving the bariatric surgery benefit for this disease condition from the Health Ministry of Chile (MINSAL).This was a quasi-experimental study developed in patients with morbid obesity. The patients were invited for participating by a public invitation and directed to the Morbidly Obesity Association of Temuco, City, Chile (OBEMOB). After providing detailed information and feedback about the risks/benefits of the intervention, all participants signed an informed consent for participating in the study. We recruited diagnosed morbid obesity sedentary/physically inactive subjects  physical limitations to performing the physical test , 2) exercise-related dyspnea or respiratory alterations, and 3) chronic heart disease diagnosed during the time of morbid obesity diagnosed and the starting of the exercise intervention. In the enrolment stage, (n = 40) participants showed their intention for participating, being allocated to . However, after the screening of inclusion/exclusion criteria, only (n = 36) were monitored after eight sessions of intervention (during 4 weeks) of follow-up, with two participants being excluded from each [ET + RT (n = 2) and RT + ET (n = 2)] group due to exercise adherence of less than 80%. Thus, the final sample size was as follows:  and . The study was carried out following the Declaration of Helsinki (2013) and was approved by the Ethical Committee of the Universidad de La Frontera, Temuco, Chile (ACTA Nº 080_21). The trial registration was part of the ClinicalTrials.gov ID: NCT05504629. The study design is shown in The inclusion criteria were as follows: 1) age >18 and <60 years, 2) women or men, 3) medical authorization for physical tests, and 4) body mass index (BMI) ≥40 kg/mi.e., ET + RT, or RT + ET), each CT session had two sections, namely, RT and ET, which were applied two days per week, during eight sessions (4 weeks), and where the blood pressure changes were measured only in four opportunities . Thus, the CT included in each session had three stages: 1) callisthenics exercises, 2) main exercise (ET + RT or RT + ET), and 3) cool down exercises.Independent of the order ] that does not change based on the sex, age of the population, or the exercise modality that is performed . After t2). The BMI was determined to estimate the degree of obesity (kg/m2) using standard criteria for obesity and morbid obesity classification (Body mass (kg) was measured using a digital bioimpedance scale . Height (m) was measured using a SECA™ stadiometer , with subjects in light clothes and without shoes. The BMI was calculated as the body weight divided by the square of the height , and diastolic blood pressure (DBP) was measured in the sitting position after 10 min of rest before and 10 min after each exercise session, similar to the procedure in previous studies . When thAfter overnight fasting of 10 ± 2 h, all patients underwent a baseline assessment (pre-test) between 08:00 and 9:00 in the morning. All participants arrived at the health center for blood sample extraction of 5 ml to determine the plasma outcomes; fasting plasma glucose (FPG), high-density lipoprotein cholesterol (HDL-c), triglycerides (Tg), cholesterol total (Tc), and low-density lipoprotein cholesterol (LDL-c) were included. These measurements were taken in an external private laboratory, being applied the protocol in the same conditions as in previous reports .th percentile)’, ‘low’ (5th–25th percentile), “regular” (26th–50th percentile), “good” (51st–75th percentile), “excellent” (76st–95th percentile), and “superior” (>95th percentile)  was used to estimate cardiorespiratory fitness. Thus, according to the distance in meters, each subject was classified with a correlated cardiorespiratory fitness by the maximum oxygen consumption following the categories; ‘very low (<5centile) . This inHandgrip strength (HGS) was assessed using a digital dynamometer , which has been used in previous studies. Two attempts were made, measuring each hand, and the best result from each was selected. As mentioned previously, the mean value obtained was taken as the total score and implemented according to similar procedures .t-test differences between groups, where the non-parametric Wilcoxon test was optionally applied for non-parametric variables. The Cohen d effect size was obtained with threshold values at 0.20, 0.60, 1.2, and 2.0 for small, moderate, large, and very large effects, respectively (p < 0.05) for all tests of statistical significance.Data are presented as the mean ± standard deviation (SD). Normality and homoscedasticity assumptions for all data were checked using the Shapiro–Wilk and Levene tests. Wilcoxon’s test was used for non-parametric data. After this, we performed 2-way ANOVA (group x time) to test SBP, DBP, MAP, HR, and PP outcomes in absolute values, where Sidac´s post hoc was applied to identify potential differences between ET + RT vs. RT + ET. Additionally, delta changes (∆) from pre–post were calculated to SBP, DBP, MAP, HR, and PP outcomes to test by the independent Student ectively . These aectively . Linear There were no baseline differences between groups in the anthropometric, body composition, cardiovascular, and metabolic and physical condition of co-variables assessed .p = 0.050) and session 2  (p < 0.001) (p = 0.032) (p = 0.007) (p = 0.018)  . The RT < 0.001) . In the = 0.032) , while t= 0.032) .). In MA= 0.007) , and the= 0.018) .p < 0.0001), session 2 , session 3 , and post-exercise session 4  (p = 0.0005), session 2 , session 3 , and post-exercise session 4  (p = 0.020) (p < 0.001)  . In the  0.0001) . In the = 0.020) , and in < 0.001) .p < 0.0001, respectively, to each session) , but no significant differences were observed comparing ET + RT vs. RT + ET in these four sessions . There wsession) . Overallsession) .i.e., into 4 weeks) (∑∆MAP), no significant differences were detected comparing ∑∆MAP of ET + RT vs. ∑∆MAP of RT + ET groups (−0.8 vs. −0.4 mmHg) , but comparing ∆DBP between ET + RT vs. RT + ET, no significant differences were detected in each session of measurement  . Overall.4 mmHg) .From pre to post each exercise session of measurement, there were different delta changes in both ET + RT and RT + ET at ∆HR , but no significant differences were detected comparing groups in each session 2, 4, 6, and 8 . Overalln = 8; 22.2%), Q2 “low responders” , Q3 “moderate responders” , and Q4 “high responders” , p < 0.0001  frequencies comparing Q1 “nonresponders” (< 0.0001 .p = 0.035), , “LRs” (Q2), “MRs” (Q3), and “HRs” (Q4) revealed significant differences in baseline SBP between Q4 vs. Q1, between Q4 vs. Q3, and between Q2 vs. Q1 ( 0.035), .R2 0.211 (21.1%), p = 0.031], skeletal muscle mass , fasting glucose , triglycerides , and 6Mwt   and the body fat percentage  .The aims of this study were (1) to investigate the effect of CT order (ET + RT vs. RT + ET) on the blood pressure responses; 2) characterize these responses in responders and nonresponders, and 3) identify potential baseline outcomes for predicting blood pressure decreases as responders. The main findings of the present study were as follows: 1) both ET + RT and RT + ET promote PEH that is expressed in a similar ‘magnitude’ after eight CT acute exercise sessions  , 2) the As we observed similar PEH in both groups reported by the mean of the four measurements that we registered in eight exercise sessions during 4 weeks , our findings are not minor. For example, a recent study developed in older adults of 60 years reported PEH monitoring 6 h after exercise cessation (by 24 h ambulatory blood pressure measurement). The authors showed significant decreases in SBP −13.5 and MAP −2.0 mmHg but not in ∆DBP . In otheOn the other hand, from our results using two different orders of CT in morbidly obese patients, we show in the present study during 4 weeks ∆SBP decreases of , and in ∆DBP  in both ET + RT and RT + ET, respectively . Followi, ET from 42 to 61%; HIIT from 54 to 61%)  that there was a significant trend for declining the ‘magnitude’ in the blood pressure decreases . In youn to 61%) .Using a similar pre vs. post-exercise method for testing PEH and the same protocol of measuring post 10 min post-exercise cessation, On the other hand, the interindividual response to the PEH in terms of reporting the NR prevalence has been poorly studied than the blood pressure changes from continuous exercise training programs from middle (few weeks) and long-term  exercise duration. For example, starting with normotensive samples, 6 weeks of HIIT decreased ∆SBP −10.0 and ∆DBP −15.0 mmHg but also reported NRs to and DBP (72.7%), where both outcomes showed increased ∆SBP +10 and DBP +2.0. In this study, the authors reported that subjects with higher baseline blood pressure also reported higher blood pressure decreases . In longR2: 0.77%), and until 24 h post-exercise cessation (R2: 0.67%) future blood pressure decreases with exercise training , being in other terms all the Q2 “LRs” , Q3 ‘MRs’ , and Q4 “HRs” , in total , participants that certainly respond to decrease SBP, but in different degrees. Following this, our linear regression analyses revealed that ∆SBP decreases were significantly predicted by outcomes 1) the body fat percentage (21.1%), skeletal muscle mass (12.5%), fasting glucose (7.8%), triglycerides (1.4%), and 6 Mwt (3.8%), where, from a healthy lifestyle perspective, it is very relevant for morbid obesity populations to consider the relevant increase in the skeletal muscle mass in order to ascertain their high impact on future body fat reduction in this disease.In terms of predicting exercise response, traditionally, when more baseline  outcomes of health-related parameters are shown by a participant, usually more exercise benefits are received after exercise. Thus, baseline conditions have been reported as an important exercise predictor . On the training , as welltraining . In the i.e., based on SBP chosen outcome), in which other LRs, MRs, and HRs (77.8% of the sample) elicited different SBP decreases  PEH occurs immediately after exercise cessation , 2) PEH ecreases . Thus, wecreases . Additioecreases . Thus, iecreases , 2) a reecreases , 3) a reecreases , 4) imprecreases , 5) an iecreases , 6) an iecreases , and alsecreases , and, thecreases .Our study is not far from limitations, 1) we do not control the intervention from the diet patterns; however, we remember each session to not change the regular nutrition behaviour, and smoking, caffeine, and other sympathetic stimulants were strictly avoided and recorded for each participant, and 2) our PEH was immediately recorded after each CT group, and after 10 min of exercise at rest, where we do not extend measurements for more hours after the exercise, 3) we did not include a strict control group, 4) we do not control the air temperature and relative humidity of the exercise room, and 5) we recruited only one man by each group. Some strengths of our work were that 1) we studied a poorly known morbid obesity sample and 2) we used the frequently PEH reported method of (PEH = postexercise blood pressure—pre-exercise blood pressure) being highly comparable to these results, 3) NRs were determined by quartile, where the minimum clinically benefit was −2 mmHg following clinical benefits, and 4) we reported the PEH in patients with morbid obesity that have been poorly studied.The CT order of ET + RT and RT + ET promote a similar “magnitude” in the post-exercise hypotensive effects during eight sessions of both CT orders in 4 weeks of duration, revealing “nonresponders” and “high” responders that can be predicted from body composition and metabolic and physical condition outcomes.Independent of the concurrent training order , concurrent training promotes the post-exercise hypotension effect in morbid obesity conditions and a different interindividual response.The postexercise hypotensive effect from both concurrent training orders promotes clinically blood pressure decreases, reducing the risk factors for metabolic syndrome and cardiovascular disease in morbid obesity patients and can be predicted from body fat and skeletal muscle mass baseline outcomes.


Cleisomeria lanatum (family: Orchidaceae) has been investigated for its phytochemical, antioxidant, anti-inflammatory, and thrombolytic properties. All phytochemicals studied were identified in varying degrees during qualitative screening. In quantitative screening, a maximum of 106.02 ± 0.08 mg/g alkaloids (root), 179.67 ± 8.83 mg/g phenols (stem), 17.34 ± 0.88 mg/g flavonoids (stem), 73.67 ± 1.76 mg/g tannins (stem), and 180.04 ± 0.02 μg/mL proteins (root) were detected. Antioxidant, anti-inflammatory, and thrombolytic properties were investigated using a free-radical scavenging assay, heat-induced albumin denaturation assay, and blood clotting inhibition assay. The root extracts had the most effective antioxidant (IC50 = 67.98 μg/mL) and anti-inflammatory (IC50 = 60.86 μg/mL) properties, whereas the stem extracts had the most effective thrombolytic property (IC50 = 163.8 μg/mL). The bioactivities studied also had a significant positive relationship  with the amount of phenolics and tannins.For the first time,  Orchids are the most numerous and diversified species of flowering plants, which were originally grown for their lovely flowers . AlthougCleisomeria has ethnomedicinal potential as well as bioactive properties [Cleisomeria lanatum are still unknown. Therefore, in light of the foregoing discussion, C. lanatum, a promising member of the Cleisomeria genus, was chosen to be studied with its phytochemical screening and bioactivity  analysis.Orchids have long been prized for their medicinal properties and as a source of pharmaceuticals , 5. Arunoperties . Bioactioperties . Accordioperties , 12, thePlants were collected from Cox's Bazar, Chittagong, Bangladesh. The collected plants were carefully examined and authenticated by Professor Dr Muhammed Kamrul Huda, Department of Botany, University of Chittagong. The fresh, disease-free plants were selected for experiments. The leaves, root, and stems of the studied orchid samples were used for the qualitative and quantitative estimation of secondary metabolites and their bioactivity tests.For further analysis, 50 g of powdered samples from each part was taken. In a conical flask, 100 ml of methanol was added to 50 g of the sample. Then, it was shaken very well for 30 minutes and kept overnight; after that, it was shaken again and sonicated for 10 minutes and filtered using the Whatman No.1 filter paper.Following established methods, alkaloids , flavonoStandard methods were also used to quantify alkaloids , phenolsC. lanatum and the standard antioxidant ascorbic acid were assessed on the basis of the free-radical scavenging effect of the 1, 1-diphenyl1-picrylhydrazyl (DPPH) free-radical activity in accordance with the method described by the standard method with a slight modification [A represents the absorbance of the control (DPPH solution without the sample) and B represents the absorbance of the DPPH solution in the presence of the sample (extract/ascorbic acid).The antioxidant activities of the methanolic crude extracts of the leaves, stems, and roots of fication . The absC. lanatum was studied by using inhibition of the albumin denaturation technique, which was studied according to the standard method [μg/mL) concentrations and 5% aqueous solution of egg albumin, and the pH (5.6 ± 0.2) of all reaction mixtures was adjusted by 1N HCl. The sample extracts were incubated at 37°C for 20 min and then heated to 51°C for 20 min, and after cooling the samples, the turbidity was measured at 660 nm using the spectrophotometer. The experiment was performed in triplicate [The anti-inflammatory activity of d method , 28. Theiplicate .μL/tube) and incubated at 37°C for 45 minutes. After clot formation, serum was completely removed (aspirated out without disturbing the clot formed). Each tube having a clot was again weighed to determine the clot weight. Each Eppendorf tube containing the clot was properly labelled, and 100 μL of the plant extract was added to the tubes. All tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, the fluid obtained was removed and the tubes were again weighed to observe the difference in weight after clot disruption. The difference obtained in weight taken before and after clot lysis was expressed as the percentage of clot lysis. Streptokinase and water were used as positive and negative (nonthrombolytic) controls, respectively. The experiments were repeated several times with the blood samples of different volunteers.The experiment for clot lysis was carried out as per the standard method  with a sAll experiments were performed in triplicate. The obtained data were analyzed using Microsoft Office Excel 2010 for statistical analysis. Pearson's correlation coefficient analysis was used to find the correlation between quantitative phytochemicals with the bioactivities previously mentioned .Qualitative screening revealed the presence of alkaloids, flavonoids, saponins, tannins, phenols, terpenoids, steroids, glycosides, cardiac glycosides, quinines, coumarins, proteins, and resins in varying degrees, as given in (1) Total alkaloid. The total alkaloid content of C. lanatum leaf, stem, and root methanolic extracts recorded was 98.23 ± 0.22, 101.78 ± 1.15, and 106.02 ± 0.08 mg/g, respectively. In comparison to leaf and stem extracts, the root extract had the most alkaloids (106.02 ± 0.08 mg/g).(2) Total flavonoid. Methanolic extracts of C. lanatum leaf, stem, and root produced 3.2 ± 0.3, 17.34 ± 0.88, and 0.45 ± 0.15 mg flavonoids per gram, respectively. In comparison to leaf and root extracts, stem extracts contained the most flavonoids (17.34 ± 0.88 mg/g).(3) Total phenol. The total phenolic content in the methanolic crude extracts of the leaf, stem, and roots of C. lanatum was estimated using the Folin Ciocalteu's reagent (FCR) and compared with the gallic acid equivalent standard curve equation y = mx + c . In comparison to leaf and root extracts, stem extracts contained the most phenols 179.67 ± 8.83 mg/g.(4) Total protein. The results of the total protein contents of leaf, stem, and root methanolic crude extracts of C. lanatum were found as 133.37 ± 0.75, 84.20 ± 0.23, and 180.04 ± 0.02 μg/mL, respectively. The highest amount of protein was found in root extracts 180.04 ± 0.02 μg/mL, equivalent to bovine serum albumin, in comparison with leaf and stem.(5) Total tannin. In this study, tannic acid was used as the standard, and the total tannin content was expressed as tannic acid equivalents (TAEs). Absorbance was measured at 700 nm. From the quantitative estimation of tannins, the results of total tannin contents of leaf, stem, and root methanolic crude extracts of C. lanatum were found as 40.33 ± 1.33, 73.67 ± 1.76, and 52.33 ± 11.31 mg g−1, respectively. The highest amount of tannin found in the stem extract was 73.67 ± 1.76 mg/g, in comparison with leaves and roots.C. lanatum is shwon in The overall result of the quantitative phytochemical study of C. lanatum was tested by DPPH-scavenging activity. The percentage of scavenging activity was subjected to regression and correlation to establish the IC50 value . A free-radical scavenging assay was used to assess the antioxidant activity of leaf, stem, and root extracts with IC50 values of 92.58, 97.10, and 67.98 μg/mL, respectively, have the highest antioxidant activity when compared to the standard (20.33 μg/mL) . The anti-inflammatory efficacy was determined using the heat-induced albumin denaturation assay. The leaf, stem, and root extracts had IC50 values of 79.37, 71.13, and 60.86 μg/mL, respectively, with the root extract having the best anti-inflammatory effect when compared to the standard (58.46 μg/mL).As part of the anti-inflammatory investigation, the protein denaturation ability of leaves, bulbs, and roots of  Figures  and 5. TDendrobium macrostachyum, with IC50 values of 114.13 and 135.818 μg/mL, respectively [The current study is consistent with the work on the anti-inflammatory activity of ectively . This leC. lanatum, leaves, roots, and stems were assessed with the clot lysis activity . The blood clotting inhibition method was used to determine the thrombolytic activity of the leaf, stem, and root extracts, yielding IC50 values of 173.7, 163.8, and 190.9 μg/mL, respectively, with stem exhibiting the best thrombolytic activity when compared to the standard (100 μL).To discover the thrombolytic activity of  Figures  and 7. TCamellia sinensis where 90.34% clot lysis activity was found in leaves at a concentration of 800 μg/mL [This study can also be supported by the work on 00 μg/mL .Finally, total phytochemicals  were statistically examined for their effect on bioactivity  in the study. For this, regression analysis and Pearson's correlation coefficient analysis were performed by using Microsoft Excel 2010.r) were calculated as 0.9 for phenolics, as 0.7 for flavonoids, as 0.51 for proteins, as 0.58 for alkaloids, and as 0.9 for tannins, which indicated a strong positive correlation for phenolics as well as tannins, whereas moderate positive correlation for flavonoids, proteins, and alkaloids.In this analysis , the corThis study was supported by the work on statistical analysis , 39 wherAs a strong positive correlation between phenolics and tannins in bioassays was found, both were further statistically analyzed for checking the significance level.p value; hence, it can be said that it has statistically significant correlations with antioxidant, anti-inflammatory, and thrombolytic effects, which accorded with the finding of a similar work [Phenols and tannins of the plant were found to have (significance F) 0.043130 < 0.05 (phenol) and 0.04473 < 0.05 (tannin) lar work .C. lanatum were reported. According to the phytochemicals and bioactivities studied, the root of C. lanatum has the greatest antioxidant and anti-inflammatory potential, with the highest alkaloid and protein content, while the stem has the greatest thrombolytic activity, with the highest phenol, flavonoid, and tannin contents. Further research is recommended to fractionate and purify the extract to find the bioactive compounds responsible for the antioxidant, anti-inflammatory, and thrombolytic activities.For the first time, the phytochemical, antioxidant, anti-inflammatory, and thrombolytic potentials of


Plasmodium knowlesi has become a major public health concern in Sabah, Malaysian Borneo, where it is now the only cause of indigenous malaria. The importance of P. knowlesi has spurred on a series of studies on this parasite, as well as on the biology and ecology of its principal vector, Anopheles balabacensis. However, there remain critical knowledge gaps on the biology of An. balabacensis, such as life history data and life table parameters. To fill these gaps, we conducted a life table study of An. balabacensis in the laboratory. Characterising vector life cycles and survival rates can inform more accurate estimations of the serial interval, the time between two linked cases, which is crucial to understanding and monitoring potentially changing transmission patterns.An. balabacensis were collected in the field in Ranau district, Sabah to establish a laboratory colony. Induced mating was used, and the life history parameters of the progeny were recorded. The age-stage, two-sex life table approach was used in the analysis. The culture conditions in the laboratory were 9 h light:15 h dark, mean temperature 25.7 °C ± 0.05 and relative humidity 75.8% ± 0.31.Individuals of mx) was 142 at age 59 days. The gross reproductive rate or number of offspring per individual was about 102. On average, each female laid 1.81 ± 0.19 (range 1–7) batches of eggs, with 63% of the females producing only one batch; only one female laid six batches, while one other laid seven. Each batch comprised 159 ± 17.1 eggs (range 5–224) and the female ratio of offspring was 0.28 ± 0.06. The intrinsic rate of increase, finite rate of increase, net reproductive rate, mean generation time and doubling time were, respectively, 0.12 ± 0.01 day−1, 1.12 ± 0.01 day−1, 46.2 ± 14.97, 33.02 ± 1.85 and 5.97 days.The eggs hatched within 2 days, and the larval stage lasted for 10.5 days in total, with duration of instar stages I, II, III and IV of 2.3, 3.7, 2.3, 2.2 days, respectively. The maximum total fecundity was 729 for one particular female, while the maximum female age-specific mean fecundity (An. balabacensis are lower than those of other species in published studies. Our results can be used to improve models of P. knowlesi transmission and to set a baseline for assessing the impacts of environmental change on malaria dynamics. Furthermore, incorporating these population parameters of An. balabacensis into spatial and temporal models on the transmission of P. knowlesi would provide better insight and increase the accuracy of epidemiological forecasting.Both the net reproductive rate and intrinsic rate of increase of  Plasmodium coatneyi, Plasmodiuminui, Plasmodiumfieldi, Plasmodiumcynomolgi and Plasmodiumknowlesi, have been reported  was 31 days Table . The oviThe stage-specific mortality rates (or the probability that a newborn will die at stage j) are 0% (egg), 2.5% (stage I larva), 1.7% (stage II larva), 3.3% (stage III larva), 0.8% (stage IV larva), 1.7% (pupa), 55.8%  and 54.2% . The daily survival rate at the adult stage ranged from 0.008 to 0.44 for females and 0.008–0.46 for males, with the rates decreasing with age.eij), which is the time that an individual of age i and stage j is expected to live, appears to be almost the same for female and male adults, as indicated by their longevity  and the finite rate of increase (λ) are, respectively, 0.12 day−1 and 1.12 day−1, while the net reproductive rate (R0), the mean generation time and the doubling time are 46.2, 33 days and 6 days, respectively , while An. balabacensis has the lowest (46), which is less than half of that of An. gambiae (105). For An. arabiensis, R0 was higher in the dry season (70) than in the rainy season (54), and lower under simulated spring conditions (51) compared to summer conditions (80). The value of r is lowest for An. balabacensis (0.12 day−1) and highest for An. albimanus (0.32 day−1), followed by that for A. gambiae (0.29 day−1). The generation time is shortest for An. gambiae (5.4 days), and when coupled with its high R0 (105), might indicate potentially high population growth in this species. All these species show characteristics of r-strategists, in that the adults have relatively high r and R0 but a short generation time, while the immature stages grow rapidly and have high mortality.A range of values of bothles spp. , 30, 36  R0 and r are good indicators of population growth, and in insects are affected by environmental variables such as temperature, shade, amount of available food, type of food, and intraspecific competition due to crowding, etc. For example, a study conducted in houses within deforested sites showed that the larval-to-adult survivorship of An. arabiensis increased to 65–82% [ R0 and r values of An. arabiensis in deforested areas were 2.7–3.6 and 1.4–1.5 times more than those in forested areas [ r, λ, R0 and T, which were respectively 0.12 day−1, 1.12 day−1, 46.2 and 33 days, seem to indicate good population growth. We do not have data from the field, but nevertheless would expect lower values there. Future studies could also assess the impacts of microclimatic conditions of different habitat types.Botho 65–82% , and theo 65–82% . Similared areas . HoweverAn. balabacensis as it considers the age-stage structure of a population, and thus keeps track of the overlap between stages, as depicted in the graph in which sxj is plotted  on the population size and stage structure of  R0, r, etc.). As noted by the authors [P. knowlesi; better estimates of mosquito survival rates enable assessment of the time scale and likelihood of the parasite developing in a mosquito and being transmitted to produce a secondary case.A Ross–MacDonald-type model has been published that compares the plausibility of transmission scenarios with variable rates of human–vector–human transmission . The mod authors , this maAn. balabacensis are reported here, to the best of our knowledge, for the first time. These estimates of mosquito survival rates enable assessment of the duration and likelihood of the parasite’s development in a mosquito and its transmission to give rise to a secondary case. They can provide comprehensive information on the growth rate of a population and can be important tools in evaluating and comparing population fitness under differing conditions when using modelling. Incorporating the population parameters of An. balabacensis into spatial and temporal models on the transmission of P. knowlesi would provide better insight and increase the accuracy of epidemiological forecasting.Life table parameters of


Marine carbonate, an important CO2 reservoir, is continuously sent to the Earth's deep interior at subduction zones, forming an essential part of the global carbon cycle. The pros and cons of using calcium isotope compositions to trace marine carbonates recycled into the mantle are discussed in this Perspective. Marine carbonates, such as sedimentary carbonates and secondary carbonates formed during marine alteration processes, are returned to the mantle at subduction zones, and their fates regulate the global carbon cycle. Some carbonates are destroyed at the subduction zones, contributing to arc volcanism ,2. Howev3 with ∼40% CaO while the Earth's mantle typically has only 3.5% CaO. If subducted carbonates have a different Ca isotope composition, measured as δ44/40Ca (= [(44Ca/40Ca)sample/(44Ca/40Ca)standard – 1] × 1000), compared to the mantle [Fig. 44/40Ca can be a ‘smoking gun’ for tracing recycled carbonates. This idea led Huang et al. [44/40Ca to estimate the contribution of recycled carbonate in the mantle source of Hawaiian shield lavas.Marine carbonates are mostly CaCOet al. [44/40Ca values  for a global sampling of carbonatites (up to 3 Ga). They argued that the low δ44/40Ca reflects contributions from surface carbonates recycled into the mantle sources of these carbonatites. However, a subsequent study found that global carbonatites, after screening for alteration, have the same δ44/40Ca range as that shown in silicates [44/40Ca similar to that in unmetasomatized peridotites [et al. [44/40Ca, but some young carbonatites (<300 Ma) have significantly low δ44/40Ca value .Recently, Amsellem et al.  reportedidotites . In cont [et al.  found th44/40Ca variations, and they largely overlap with each other , peridotites and chondrites, ranging from 0.94 to 1.05  within the garnet stability field. The low-pressure mineral-melt Ca isotope fractionation factors inferred by Zhang et al. [ab initio calculations before making full use of δ44/40Ca data to trace recycled carbonates.Second, partial melting effects on δg et al.  showed tg et al.  found th results . Wang et [et al.  also infic rocks . Howeverg et al.  should net al. [44/40Ca, down to −0.1, in some peridotite xenoliths from the North China Craton, and they attributed the low δ44/40Ca to the melt-rock reaction that occurred at the base of the lithospheric mantle. Specifically, lighter Ca isotopes are preferentially incorporated into clinopyroxene during the melt-mantle reaction. Antonelli et al. [44/40Ca in both natural and experimentally grown plagioclases, and they attributed the low δ44/40Ca in plagioclases to kinetic effects during magmatic plagioclase crystallization. Additional geochemical and isotopic tracers must be used together with δ44/40Ca to distinguish between recycled carbonates, metasomatized mantle and plagioclase cumulates.Last, kinetic processes can also fractionate Ca isotopes. Zhao et al.  found vei et al.  reported44/40Ca variation in carbonates compared to the mantle, and the large CaO concentration difference between marine carbonates and the mantle, Ca isotope composition is a potentially powerful tool for tracing the recycling of carbonates into the mantle, especially when used in combination with other geochemical and isotopic tracers for carbonates . However, there remain some issues that should be addressed: (i) large inter-laboratory differences in Ca isotopic measurements, and (ii) a better understanding of Ca isotopic effects during igneous, metasomatic and metamorphic processes, including under high pressures, using analytical, experimental and theoretical approaches.In summary, because of the large δ


Aspergillus niger has been shown to be effective in producing β-glucosidase, which could degrade CNglcs. However, whether A. niger could remove CNglcs under ensiling conditions is still unknown.Cyanogenic glycosides (CNglcs) are bioactive plant products involving in plant defense against herbivores by virtue of their abilities to release toxic hydrogen cyanide (HCN). A. niger.In this study, we first investigated the HCN contents in ratooning sorghums for two years, then the sorghums were ensiled with or without the addition of A. niger could produce β-glucosidase over a range of pH and temperature, which degraded the CNglcs and removed the hydrogen cyanide (HCN) at early days of ratooning sorghum fermentation. The addition of A. niger (2.56 × 107 CFU/ml) altered the microbial community, increased bacterial diversity, improved the nutritive qualities, and reduced the HCN contents in ensiled ratooning sorghum lower than 100 mg/kg FW after 60 days of fermentation. Overall, the addition of 150 ml A. niger + 50 ml sterile water per 3 kg silage could efficiently remove CNglcs from ratooning sorghum silage.Two years’ investigation indicated that the contents of HCN in fresh ratooning sorghum were larger than 801 mg/kg FW (fresh weight), which could not be reduced by silage fermentation under safety threshold (200 mg/kg FW). A. niger could produce β-glucosidase which degraded the CNglcs during the early days of fermentation, benefiting the ensiling process and improving the utilization of ratooning sorghum.In conclusion,  Howevervestock) . This imAspergillus niger is a haploid filamentous fungus widely distributed in various environments  to prove the efficiency of A. niger addition in removing CNglcs; (2) to elucidate the mechanism of A. niger in removing CNglcs; and (3) to evaluate the potential utilization of ratooning sorghum as safe feeds.To fill the gap between the removal rate of CNglcs and the addition of to soils , or usedto soils . Droughtto soils . Therefo2.2.1.The experiment was conducted at Jiangxiaobai Red Sticky Sorghum Planting Demonstration Base , located in Chongqing, China. The area has a mean temperature of 17.9°C, and a mean annual rainfall of 1,100 mm during the last 30 years . The soil was a neutral yellow soil with a pH 7.5, alkali-dispelled nitrogen 96.15 mg/kg, available phosphorus (Olsen-P) 17.83 mg/kg, and available potassium 90.15 mg/kg.Sorghum bicolor cv. Chuannuoliang 2 is the principal variety planting in this base for Baijiu (distilled liquor) production. Sorghum-Canola rotation system is used in this base. Generally, the sorghum will be sown in May after the canola is harvested, and be harvested in August, then the canola will be sown in October. From August to October, there is about 2 months’ fallow period, which is suitable for the regrowth of the harvested sorghum, called ratooning sorghum. Currently, the ratooning sorghum is chopped and returned to soils or discarded. In this study, on 2th October, 2020 and 10th October, 2021, the ratooning sorghum at their heading and flowering stage were harvested for whole plant silage (above 5 cm from the ground). These ratooning sorghums have the potential to be used as fodder or silage.The red sticky A. niger was bought from Beijing Beina Chuanglian Biotechnology Research Institute. Before used as additives, the A. niger was cultured on Potato Dextrose Agar medium (PDA) at 28°C for 72 h, then, the activated A. niger was inoculated into Potato Dextrose Broph liquid medium (PDB) at 32°C for 3 days at 170 ~ 180 r/min , with adding 1 g/kg of cellulase, and with adding 1 g/kg of xylanase. The samples were packed into polyethylene plastic bags (18 cm × 26 cm), vacuum sealed, and stored at room temperature (~25°C) for 7, 15, 30 and 60 days. Each silage bag contained 500 g samples and each treatment replicated three times.In experimental one, the harvested ratooning sorghums (2020) were kept under room temperature for 12 h until the water contents reached A. niger + 150 ml sterile water / 3 kg (AN50), with 100 ml A. niger + 100 ml sterile water / 3 kg (AN100), with 150 ml A. niger + 50 ml sterile water / 3 kg (AN150), and with 200 ml A. niger/3 kg (AN200). The silage bags were placed at room temperature (~ 25°C) for 48 h before sealing, to provide oxygen for A. niger propagation  were used. The whole plants were chopped and ensiled with 200 ml sterile water / 3 kg (Control), with 50 ml pagation . The staA. niger was inoculated into PDB at 32°C for 3 days at 170 ~ 180 r/min. When the concentration of A. niger solution reached 2.56 × 107 CFU/mL, the A. niger was added into fermentation medium. A single-factor experiment was designed with different inoculation concentrations of A. niger , pH levels (3.0–9.0), and temperatures . Each treatment replicated four times.In experimental three, the activated 2.4.ca. 250 mL. Then, 10 mL distillate were taken and 1 mL of 10 g/mL sodium hydroxide solution and a drop of phenolphthalein indicator were added, and adjusted with acetic acid solution until the red color fades. Next, 5 mL phosphoric buffer were added and bathed at 37°C for 10 min, and then 0.25 mL chloramine T solutions were added and stand for 5 min. Finally, 5 mL isonicotinic-pyrazolone solution were added to a final volume of 25 mL, water bathed at 37°C for 40 min, and then the OD value was measured at 638 nm ; 10 is reaction time; N is dilution multiple of original enzyme solution; 0.1 is take 0.1 ml of enzyme solution for reaction; C is corresponded to the value on the p-nitrophenol optical density curve.Note: 2.6.To analyze fermentation characteristics of silages, 20 g wet samples were taken and homogenized in 180 mL sterile water at 4°C for 24 h . The mixThe rest silage samples were dried at 105°C for 30 min and then at 65°C for 48 h to measure the DM content. Prior to the chemical analysis, the dried samples were ground into powder with grinder through a 2 mm screen. Specifically, the water-soluble carbohydrate (WSC) was extracted by using anhydrous ethanol and analyzed using the anthrone method. Total nitrogen (TN), ether extract (EE) and crude ash (Ash) were determined according to the procedure described by 2.7.Total genomic DNA of surface bacteria of fresh and ensiled sorghum fermented for 0, 7, 15, 30, and 60 days were extracted using a DNA isolation kit. The DNA quality and quantity were assessed by the NanoDrop2000. Afterwards, DNA samples were stored at-80°C until used. The V3-V4 region of the bacterial 16S rRNA gene was amplified with the common primer pair  combined with adapter and barcode sequences. The PCR amplification was performed as follows: 4 μL buffer, 0.4 μL Q5 High-Fidelity DNA Polymerase, 10 μL High GC Enhancer, 2 μL dNTP, 0.8 μM of each primer, 10 ng genome DNA, and sterilized deionized water were mixed to make the total volume to 20 μL. The fungus ITS gene was amplified with the common primer pair  combined with adapter and barcode sequences. The PCR amplification was performed as follows: 10 μL Taq, 0.8 μM of each primer, 10 ng/μL Template DNA, and sterilized deionized water were mixed to make the total volume to 20 μL. Thermal cycling conditions included: an initial denaturation at 95°C for 3 min, followed by 27 cycles  and a final extension at 72°C for 10 min. The original sequences were processed by Meiji Bio Cloud Platform. The RNA-Seq reads were deposited and available at the database Sequence Read Archive at NCBI (PRJNA921848).2.8.p < 0.05. The figures were made using origin 2021 . After comparison with the NCBI 16S rRNA database (classified at a bootstrap threshold of 0.8) using RDP classifier software, sample ordination based on beta diversity was examined using principal coordinate analysis (PCoA) with phylogeny-based (UniFrac) unweighted and weighted distance. Heat map analysis was used to confirm the relationship between the microbial species, fermentation quality and HCN contents in ratooning sorghum silage. The high throughput sequencing data were analyzed on the online platform of Majorbio Cloud Platform.The data were expressed as “mean ± standard deviation.” One-way ANOVA was applied to analyze the influence of additives on HCN contents and the parameters of silage quality using SPSS 23.0 . Significance was tested using Tukey’s multiple range test at 3.3.1.After two months’ regrowth, the ratooning sorghums were in their heading and flowering stages, with plant height reaching 157.56 cm, stem diameter 1.77 cm, and stem/leaf ratio 4.45 in year 2020 . Similar3.2.p < 0.05), and the lowest was observed 60 days after ensiling . The HCN removal rate did not increase with the increase of the concentrations of A. niger. Among the concentrations of A. niger, 150 ml A. niger + 50 ml sterile water per 3 kg was the optimal concentration for ratooning sorghum, with HCN removal rate reaching 93.63%.Though the HCN removal rate increased with the increase of fermentation time, the highest HCN amount removed from silage was observed at Day 7 for silages added with 3.4.A. niger could produce β-glucosidase under lower pH levels in ensiled ratooning sorghum, the β-glucosidase production characteristics of A. niger were measured under different inoculation amounts, pH levels and temperatures  (p > 0.05) (A. niger concentrations (A. niger concentrations increased above 150 A. niger + 50 ml sterile water per 3 kg after15 days of fermentation (p < 0.05)   , whereas > 0.05) ,D, with trations . For exa < 0.05) . Overall < 0.05) . No sign3.6.A. niger decreased the alpha diversity before ensiling .Using high-throughput sequencing, the bacterial community was analyzed. The coverage values of all samples were above 99%, indicating that most bacteria were detected. With the prolongation of fermentation, the number of OUT, index of Shannon, ace and chao reduced, while Simpson index increased , resultiA. niger showed no obvious difference in succession. In PCoA analysis, the microbial diversities were grouped into three clusters, 0 day, 7 and 15 days, and 30 and 60 days. Overall, the microbial diversity of silage at 0 day was obviously separated from other silages.Based on the community dissimilarities calculated by unweighted and weighted UniFrac distances, principal coordinates analysis (PCoA) indicated that the bacterial species had a significant succession with the prolongation of fermentation time ,B. HowevL. plantarum, Lactococcus_lactis, Weissella_paramesenteroides and unclassified_Raoultella , Enterobacter (r = 0.58), Enterococcus (r = 0.82), Weigella (r = 0.82) and Lactobacillus (r = 0.81) in silage with A. niger were positively correlated with the pH value  and Weigella (r = 0.83) were positively correlated with pH value . The HCN contents in silage added with A. niger (r = −0.67) was negatively correlated with the abundance of A. niger  and temperatures (20°C to 40°C), contributing to the degradation of CNglcs. A study on Prunus armeniaca has also shown that the β-glucosidase exhibited higher activities under low pH levels (4 to 6), and the optimum temperature for the activity of this enzyme was 35°C , whereas Lactobacillus was dominant 7 days after silage. In this study, we also found that the dominant LAB species changes with the fermentation, and L. plantarum and L. brevis dominated the microbial community after 60 days of fermentation. These results indicated that the predominant microbes in silage of ratooning sorghum were similar with those of other sorghum silages  starts LA fermentation at the early stage of silage  in cassava leaf was achieved under Lactobacillus plantarum fermentation  can be found below: BioProject, PRJNA921848.JZ: investigation, software, data curation, formal analysis, and writing. BW: software, data curation, formal analysis, and writing. YS: investigation, methodology, visualization, and validation. JY, TW, and WZ: investigation, methodology, visualization, and validation. JZ: investigation, methodology, and visualization. CQ: investigation, methodology, and visualization. YG: conceptualization, data curation, funding acquisition, project administration, supervision, validation, and writing – review and editing. All authors contributed to the article and approved the submitted version.This work was funded by Chongqing Technology Innovation and Application Development Project (cstc2021jscx-gksbX0014), “First Class Grassland Science Discipline” program in Shandong Province, China, and Special Fund Project of Provincial Science and technology innovation development in agricultural high district of Yellow River Delta (2022SZX32).JZ was employed by Chongqing Jiangxiaobai Farm Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ostreopsis comprises twelve described species, of which seven are potentially toxic and produce compounds presenting a threat to human and environmental health. In this study, isolates previously identified as “Ostreopsis sp. 3” were sampled from the area where it was first reported, Rarotonga, Cook Islands, and have been taxonomically and phylogenetically characterised as Ostreopsis tairoto sp. nov. Phylogenetically, the species is closely related to “Ostreopsis sp. 8”, O. mascarenensis, “O. sp. 4”, O. fattorussoi, O. rhodesiae and O. cf. siamensis. Previously, it was considered a part of the O. cf. ovata complex but can be distinguished from O. cf. ovata based on the small pores identified on this study, and from O. fattorussoi and O. rhodesiae based on relative lengths of the 2′ plates. No known palytoxin -like compounds were detected in strains investigated in this study. Strains of O. lenticularis, Coolia malayensis and C. tropicalis were also identified and described. This study advances our knowledge of biogeography, distribution, and toxins of Ostreopsis and Coolia species.It is important to decipher the diversity and distribution of benthic dinoflagellates, as there are many morphologically indistinct taxa that differ from one another in production of potent toxins. To date, the genus  Efforts to understand the changing distributions of HAB species in response to ocean climate change are hampered by unclear identifications of taxa and the presence of cryptic and/or pseudo-cryptic diversity amongst several culprit genera7. The cosmopolitan HAB genus Ostreopsis Schmidt, occurs worldwide, but mainly in warm waters, and is associated with a variety of benthic/epiphytic habitats9. Ostreopsis was first described by Schmidt in 1901 from the Gulf of Thailand, with O. siamensis as the type species10. Traditionally, nine Ostreopsis species had been described based on morphology and thecal plate pattern, namely: O. siamensis Schmidt (1901), O. lenticularis Y.Fukuyo and O. ovata Y.Fukuyo (1981), O. heptagona D.R.Norris, J.W.Bomber & Balech (1985), O. mascarenensis J.P.Quod (1994) emend. Chomérat & J.-P.Quod (2020), O. labens M.A.Faust & S.L.Morton (1995), O. belizeana M.A.Faust, O. caribbeana M.A.Faust and O. marina M.A.Faust (1999). Only O. fattorussoi Accoroni, Romagnoli & Totti (2016), O. rhodesiae Verma, Hoppenrath & Murray (2016), and now the emended O. mascarenensis, O. lenticularis and O. siamensis have been described with both molecular and morphological information19.Dinoflagellates have been studied extensively due to their potential to synthesize potent toxic molecules and forming harmful algal blooms (HABs), thereby impacting coastal resources and human healthOstreopsis species are known to co-occur with other epi-benthic genera, such as Coolia Meunier that has a cosmopolitan distribution ranging from tropical to temperate waters26. Coolia species show homology with genus Ostreopsis in thecal plate tabulation, and at one time C. monotis Meunier (1919), the only species described until 1995, was proposed for transfer to Ostreopsis as O. monotis Lindemann (1928)27. It was later moved to Glenodinium Ehrenberg based on hypotheca tabulation28. A detailed morphological study conducted by Balech reinstated Coolia as a recognised genus29. Since then seven species have been described, namely: C. tropicalis M.A.Faust (1995), C. areolata Ten-Hage,Turquet, Quod & Couté (2000), C. canariensis S.Fraga (2008), C. malayensis Leaw, P.T.Lim & Usup (2010), C. palmyrensis Karafas, Tomas, York (2015), C. santacroce Karafas, Tomas, York (2015) and C. guanchica H.David, Laza-Martínez, F.Rodríguez & S.Fraga (2020)34.19. In the case of Ostreopsis, the morphological variation derived from culture  conditions increase the uncertainty. Tentative classifications such as Ostreopsis cf. ovata and O. cf. siamensis and undescribed ribotypes or clades (Ostreopsis sp. 1–9) emerged, the latter from phylogenetic studies37. Several such ‘ribotypes’ are morphologically indistinguishable and remain undescribed. Amongst Coolia spp., molecular data are available for all presently described species, except for C. areolata, but this species can be easily identified using morphological features32. Taxonomic identifications are important for species of these genera, as some Ostreopsis species are known to produce numerous palytoxin (PLTX) -like compounds and form HABs, which are known to cause human poisonings and the mortality of benthic invertebrates45. Also, four of the eight Coolia species, namely C. tropicalis, C. malayensis, C. palmyrensis, and C. santacroce are known to be toxic49.The combination of genetic and morphometric tools has aided in the description of several new species from these genera, but the highly variable morphology of these species and lack of strains from type locality of certain species has also caused inaccurate or ambiguous species delimination50). Chomérat et al.18 examined material from the type locality of Ostreopsis lenticularis and concluded that it was morphologically similar to the original description by Fukuyo13 and genetically similar to “Ostreopsis sp. 5”37, determining that “Ostreopsis sp. 5” is O. lenticularis. Phylogenetic and morphological studies of samples from Réunion Island, the type locality of O. mascarenensis by Chomérat et al.16 confirmed that O. mascarenensis forms a new lineage among Ostreopsis species, and it does not correspond to any of the unidentified ribotypes shown in previous studies. However, its relationship with O. fattorussoi, O. rhodesiae, and ribotypes “Ostreopsis sp. 3” and “Ostreopsis sp. 4” remained unclear. Chomérat et al.51 provided morphological and genetic support to separate the “temperate O. cf. siamensis” as a species distinct from “Ostreopsis sp. 6”. Later, Nguyen-Ngoc et al.19 concluded that “Ostreopsis sp. 6” is identical to the original description of O. siamensis, designated an epitype and presented an emended description of the species from Vietnamese waters.One approach for resolving these taxonomic issues is to isolate strains from type localities, and to obtain extensive data from them including molecular genetic sequences , 1000 km northeast of Aotearoa New Zealand52. The central aim of this study is to investigate “Ostreopsis sp. 3” from the location where it was first reported, the Cook Islands, and other strains isolated from Niue, also located in the South Pacific Ocean  and internal transcribed spacer regions and 5.8S rRNA gene (ITS/5.8S rDNA) regions. In addition, co-occurring O. lenticularis, C. malayensis and C. tropicalis were also identified and described from these samples. The toxin production of these isolates was investigated using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Ostreopsis strains were screened for PLTX-like compounds and Coolia strains were screened for gambierone and 44-methyl gambierone (44-MG). Altogether, the morphological, phylogenetic, and toxicological data allowed to describe “Ostreopsis sp. 3” as a new species, Ostreopsis tairoto sp. nov, and updated the current knowledge of biodiversity and distribution of potentially toxic benthic dinoflagellates in sub-tropical reef systems.However, there are still several undescribed ribotypes that need to be assigned taxonomically. Amongst them, “Ostreopsis and six Coolia isolates were established in this study  and ventrally tapering Fig. , variablHolotype: SEM-stub (designation CEDiT2022H147) prepared from strain O1C6 and deposited at Senckenberg am Meer, German Centre for Marine Biodiversity Research, Centre of Excellence for Dinophyte Taxonomy, Germany. Cells from the holotype are shown in Supplementary Fig. Reference material: Lugol-fixed subsample of strain O1C6 (designation CEDiT2022I148) deposited at the Senckenberg am Meer, German Centre for Marine Biodiversity Research, Centre of Excellence for Dinophyte Taxonomy, Germany.Type locality: Titikaveka Beach  Rarotonga, Cook Islands.Etymology: Tairoto is the Cook Islands Māori term for ‘from/of a wide lagoon’, i.e. isolated/ inhabitant of sea inside the lagoons of Rarotonga, Cook Islands, which is the type locality of this species.The photosynthetic cells were anterio-posteriorly flattened, lenticular, broadly ovate (drop-shaped) to almost round and ventrally tapering in apical and antapical views Fig. . The fieThe cells were globular Fig. , 22.5–30The cells were globular to subspherical, 35.3–51.2 μm  long, 32.5–40.2 μm  wide, and 26.2–29.1 μm  deep; containing chloroplasts (not shown). The thecal plate pattern was: APC 3′ 7′′ ?c ?s 5′′′ 2′′′′ Fig. . The firOstreopsis strains isolated in this study, with additional reference sequences from Genbank, and identified 14, 12 and 13 strongly supported clades based on ITS/5.8S, D1-D3 and D8-D10 rDNA analyses respectively     Table .Ostreopsis morpho-species identification is often difficult, as many species show significant intra-specific morphological plasticity and a lack of striking species-specific characters – suggesting they are cryptic or semi-cryptic species18. A given species can also encompass a wide genetic variability that is often linked to biogeography (cryptic intra-specific diversity recognized as ribotypes)53. The new species Ostreopsis tairoto sp. nov. belongs to a clade including the morphologically characterized taxa O. cf. ovata, O. cf. siamensis (sp. 9), and O. rhodesiae, as well as “ribotypes sp. 1, sp. 2, sp. 7, and sp. 8”. The closest sister lineage was O. fattorussoi . Ostreopsis rhodesiae cells can be of the same size as Ostreopsis tairoto and O. fattorussoi cells and it will be nearly impossible to identify such a cell by its morphology. The Po lengths can also overlap but the relative lengths of the 2′ plates might distinguish Ostreopsis tairoto from O. fattorussoi and O. rhodesiae  phenotypic traits. Hoppenrath67 discussed both the technical and conceptual the problems of one species concept for dinoflagellates. As discussed for protists in general by Boenigk et al.68, also for dinoflagellates, a consensus approach for species definitions may emerge within groups, but one approach is unlikely to encompass the whole dinoflagellate lineage.A curved suture between plates 1′ and 3′ was described as characteristic distinguishing feature of to Figs. A, 3A. ThO. cf. siamensis and O. cf. ovata, David et al.69 found that the main differential feature between them was the presence of thecal pores of two size classes in the former and only one in the latter. A great variety of cell sizes and shapes were observed for both species69. Cells of O. cf. siamensis were reported to be more flattened than cells of O. cf. ovata71. Ostreopsis tairoto cells seem to overlap in size with O. cf. ovataO. cf. siamensis populations . Whether O. cf. ovata really has only one size class of pores is uncertain as Aligizaki and Nikolaidis70 documented a very wide range of thecal pore sizes (0.07–0.32 µm) and labelled a minute pore on their Fig. Ostreopsis sp. 1” was described as O. cf. ovata from Korea72. Cells were in the width range of Ostreopsis tairoto but less deep. The Po plate was shorter (4.8–6.8 µm) and plate 2′ only about 1.25 times the Po length , so that the species now can be clearly identified. The problems related to past identifications and probable synonymy, were discussed in detail by Chomérat et al.18. The morphological features observed in this study were generally in agreement with the species description18, except of the recognition of a third size class of pores. These medium sized pores were not visible on published scanning electron micrographs18. But their numbers were few and they were irregularly scattered so that it is possible that they were identified as the smallest pores of the large size class in previous studies. Oblong or kidney-shaped large pores were described for strains isolated from the South China Sea in Zhang et al.54 and interpreted as possible culturing plasticity by Chomérat et al.18.Coolia malayensis the nucleus was originally described to be located in the hyposome31. Its position was interpreted to be in the episome in the present study . Plate 2′′′′ was in contact with 1′′′′ and 2′′′ in agreement with the original description20, the re-description30 and further reports , but not as shown in Hoppenrath et al.3, Fig. 31.For udy Fig. F. The thudy Fig. A,B,L witOstreopsis species in the samples studied from Rarotonga, Cook Islands and Niue. Our molecular phylogenies show a similar topology for ITS/5.8S, D1–D3 and D8–D10 of the LSU rDNA regions, albeit with different support. The phylogenetic analyses revealed the discovery of O. tairoto, previously an undescribed clade referred to as “Ostreopsis sp. 3” by Sato et al.35 and subsequent authors, that diverged from other clades with full nodal support. Isolate CAWD184 was originally reported in Sato et al.35 and was labelled “Ostreopsis sp. 3” with the report of sequences from the ITS/5.8S and D8-D10 LSU rDNA regions. Major sampling trips were carried out around the lagoons of Rarotonga , and O. lenticularis  which was also isolated in this study.Results from microscopic observations and phylogenetic analyses confirm the presence of two O. tairoto strains in the ITS/5.8S and LSU rDNA regions were observed, with strains isolated from the same island location clustering together  confirmed that PLTX and related structural analogues were below the LOD. All strains of Ostreopsis tairoto that have been screened to date have not produced PLTX-like compounds above the LOD of the LC–MS/MS method80. Numerous PLTX-like compounds  have been described from several Ostreopsis species to date84. However, the LC/MS–MS approach used in our study monitors sub-structures generated by the oxidative cleavage of large PLTX-like compounds and cannot differentiate between many of the toxin analogues. However, all PLTX-like compounds have a common sub-structure that is monitored during the analysis. The absence of this sub-structure in the Ostreopsis isolates tested during this study supports the statement that these strains do not produce any PLTX-like compounds.Strains of 80 Table . ChoméraC. tropicalis strains analysed in this study. However, the C. malayensis strain tested in this study did not produce 44-MG. 44-MG has been detected from several Gambierdiscus species/strains, namely G. australes, G. belizeanus, G. caribaeus, G. carpenteri, G. cheloniae, G. holmesii, G. honu, G. lapillus, G. lewisii, G. pacificus and G. polynesiensis85. The compound has also been detected in Fukuyoa species, C. malayensis and C. tropicalis85. Toxicity within the genus Coolia was first reported in the early 1980s, based on hemolytic activity via in vitro assays, although no toxicity to mice and fish was registered for the same methanol extract86. Since then, this genus of benthic dinoflagellates has been considered potentially toxic3. According to Boente-Juncal et al.87, 44-MG exhibits similar biological activities to gambierone and CTX-3B, although at a much lower rate, leading to the decreased viability of undifferentiated neuroblastoma (N2a) cells and modified expression of excitatory neurotransmitter receptor subunits. However, in contrast, the study by Murray et al.85 demonstrated 44-MG had an LD50 by intraperitoneal injection to mice of 20–38 mg/kg, which is essential non-toxic. This finding has been supported by a recent study that Stuart et al.88 conducted, where 44-MG was assessed using the N2a cell-based assay and only displayed a minor toxic effect when dosed at the highest concentration trialled (4.8 µg/mL).44-MG was detected in three out of the five 89  with 1 mL medium, and transported back to the laboratories at University of Technology Sydney, Australia and Cawthron Institute, New Zealand. On return to the labs, cultures were grown at 25 °C at a salinity of 35, with 40–100 μmol photons m−2 s−1 irradiance (12:12 h L:D). Cultures were maintained in the same conditions thereafter and subcultured once every 3–4 weeks.Samples were collected from several sites at Rarotonga, Cook Islands and Niue between November 2014 and July 2019 during research expeditions as described in Smith et al.Ostreopsis species were stained with Solophenyl Flavine 7GFE 500  as described by Chomérat et al.91 and thecal plates visualized by epifluorescence microscopy. Cell dimensions were measured under 400 × using a calibrated eyepiece of Eclipse TS100 inverted microscope with bright field optics . Cells were harvested from the culture medium via centrifugation and fixed in 1% Lugol solution (Sigma-Aldrich) to measure the depth = dorso-ventral diameter (DV) and transdiameter width (W) using ImageJ v1.4892.Living and fixed cells were picked under a Leica DMIL inverted microscope , placed on an object slide and observed with a Leica DMRB  equipped with differential interference contrast optics at 400 × and 640 × magnification with oil immersion objectives. Digital photos were taken using a Leica DFC420C camera . Fixed cells of the new For SEM, the cultures and field samples were fixed in 1% Lugol solution and stored in the dark. Cells were placed on a 5 µm Millipore filter, rinsed in distilled water, and dehydrated in a series of increasing ethanol concentrations , followed by chemical drying with hexamethyldisilazane at room temperature for 20 min and finally at 50 °C in a drying oven for 5 min. The sample/filter was mounted on a stub and sputter coated with gold–palladium . Cells were observed using a Tescan VEGA3 microscope  at 15 kV using the SE detector.89, i.e. dinoflagellate cultures were centrifuged  and DNA extracted using PowerSoil DNA isolation kit (Qiagen) as per manufacturer’s instructions. DNA from cultures maintained at UTS was extracted using modified 3% Cetyltrimethyl ammonium bromide (CTAB) buffer as described in Verma et al.12. Briefly, cell pellets were harvested via centrifugation  and lysed using 500 µL of buffer on a heat block at 68 °C for 2 h. The aqueous layer was separated using 24:1 chloroform: isoamyl alcohol (Sigma-Aldrich) and precipitated in 2-propanol (Sigma-Aldrich) and 3 M sodium acetate (pH 5.2) (Sigma-Aldrich). The DNA pellet was washed with 70% ethanol (Sigma-Aldrich) and vacuum dried to remove any traces of ethanol. Sterile Milli-Q water was added to DNA pellets and were stored at 20 °C until further analysis.Genomic DNA from cultures held at Cawthron Institute, New Zealand was extracted according to the protocols described in Smith et al.93  and the internal transcribed spacer regions and 5.8S rRNA gene (ITS/5.8S) were amplified and sequenced using primers and protocols described in Verma et al.94. The obtained sequences were aligned with reference sequences retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov). Multiple sequence alignments were performed using ClustalW v1.6 program as implemented in MEGA v795. Substitution models were selected for each dataset based on lowest Bayesian information criterion (BIC) as a measure of the relative quality of the models using MODELTEST96. Phylogenetic analysis was performed using both a maximum likelihood (ML) and Bayesian inference (BI) approach. ML trees were produced in MEGA v7 using general time reversible (GTR) + gamma (G) + invariant sites (I) with 5 gamma categories substitution model for all sequence analyses. Nodal support of the ML tree was estimated via bootstrap algorithm with 1,000 replications. Bayesian analysis was performed using MrBayes v3.2.297 as implemented in Geneious Prime v2020.0.5 using general time reversible model (GTR) + G model for all analyses. Four independent Markov Chain Monte Carlo (MCMC) simulations were run simultaneously for 2 × 106 generations. Trees were sampled every 1 × 103 generations and 1 × 103 trees were discarded as burn- in. Genetic distance (pairwise uncorrected p distance) was estimated from the ITS/5.8S, D1-D3 and D8-D10 LSU rDNA sequences using the p distance model and bootstrap procedure (1 × 103 replicates) in MEGA v7. All positions containing gaps and missing data were eliminated for the analyses.Analyses on all regions of rDNA were conducted separately. The forward and reverse sequences were trimmed, aligned, and visually refined using Geneious Prime v2020.0.5Ostreopsis and Coolia cell pellets were harvested in late stationary phase via centrifugation  to obtain a cell pellet of approx. 1 × 106 cells. The growth medium was decanted, and the resulting pellets were frozen at − 20 °C until further analysis. Toxin extractions were completed at the Cawthron Institute using methods described in Selwood et al.80 for screening of PLTX-like compounds, and Murray et al.98 for screening of gambierone and 44-MG respectively.80 monitors substructures of PLTX-like compounds that are generated via oxidative cleavage, using periodic acid, of vicinal diol groups present in intact toxin molecules. This yields an amino-aldehyde common to known PLTX-like compounds, used for quantification, and an amide-aldehyde that varies depending on the toxin analogue. A commercially available PLTX standard was used to generate a calibration curve and enable unambiguous identification of the oxidation products. The limit of detection (LOD) was determined as 0.5 ng/ml for the PLTX amine fragment, which equates to 0.01 pg/cell in an extract. The relative standard deviation of repeatability for LC–MS of oxidized PLTX standards was < 10% and < 8% for amino aldehyde and amide aldehyde, respectively, at 1 or 2 ng/ml, making this method suitable for monitoring trace levels of PLTX-like compounds.Briefly, Selwood et al.Coolia cell pellet was extracted twice with 90% aqueous methanol, at a ratio of 1 mL per 2 × 105 cells, and subsequently lysed via ultrasonication (10 min at 59 kHz). Cellular debris was removed by centrifugations  and the supernatant transferred to another vial. The resulting supernatants were pooled to give a final extract concentration equivalent to 1 × 105 cells/mL. The combined extracts were stored at − 20 °C for 24–48 h to precipitate insoluble matrix co-extractives, which were removed using centrifugation  prior to analysis. An aliquot of the clarified extract was transferred into a 2 mL glass autosampler vial and analysed using a modification of the LC–MS/MS method described in Murray et al.99. The compounds are identified based on retention time (2.54 and 2.58 min for gambierone and 44-MG respectively) and fragment ion ratios compared to purified reference material, on a Waters Xevo TQ-S triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC i-Class with a flowthrough needle sample manager. Data acquisition and processing was performed using MassLynx and TargetLynx software (Waters), respectively. Quantitation was achieved using a five-point, for gambierone and 44-MG (1–1000 ng/mL), linear regression calibration prepared in 90% aqueous methanol. The limit of quantification (LOQ) was determined as 0.05 pg/cell in an extract generated from a cell pellet of 1 × 106 cells.For detection and quantification of gambierones, each Supplementary Information.Supplementary Figure 1.