Loxodonta africana) is listed as vulnerable, with wild populations threatened by habitat loss and poaching. Clinical pathology is used to detect and monitor disease and injury, however existing reference interval (RI) studies for this species have been performed with outdated analytical methods, small sample sizes or using only managed animals. The aim of this study was to generate hematology and clinical chemistry RIs, using samples from the free-ranging elephant population in the Kruger National Park, South Africa. Hematology RIs were derived from EDTA whole blood samples automatically analyzed (n = 23); manual PCV measured from 48 samples; and differential cell count results (n = 51) were included. Clinical chemistry RIs were generated from the results of automated analyzers on stored serum samples (n = 50). Reference intervals were generated according to American Society for Veterinary Clinical Pathology guidelines with a strict exclusion of outliers. Hematology RIs were: PCV 34–49%, RBC 2.80–3.96 × 1012/L, HGB 116–163 g/L, MCV 112–134 fL, MCH 35.5–45.2 pg, MCHC 314–364 g/L, PLT 182–386 × 109/L, WBC 7.5–15.2 × 109/L, segmented heterophils 1.5–4.0 × 109/L, band heterophils 0.0–0.2 × 109/L, total monocytes 3.6–7.6 × 109/L , lymphocytes 1.1–5.5 × 109/L, eosinophils 0.0–0.9 × 109/L, basophils 0.0–0.1 × 109/L. Clinical chemistry RIs were: albumin 41–55 g/L, ALP 30–122 U/L, AST 9–34 U/L, calcium 2.56–3.02 mmol/L, CK 85–322 U/L, GGT 7–16 U/L, globulin 30–59 g/L, magnesium 1.15–1.70 mmol/L, phosphorus 1.28–2.31 mmol/L, total protein 77–109 g/L, urea 1.2–4.6 mmol/L. Reference intervals were narrower than those reported in other studies. These RI will be helpful in the future management of injured or diseased elephants in national parks and zoological settings.The African elephant ( Loxodonta africana) is a megaherbivore, which had an extensive range across the African continent until the 1930s. Loss of habitat and poaching led to the present classification of this species as Vulnerable by the International Union for Conservation of Nature (IUCN) (Loxodonta africana africana (South African bush elephant) based on the geographical distribution (The African elephant (e (IUCN) . Accordie (IUCN) . The subribution . Elephanribution . Additioribution .Elephas maximus), mainly in Sri Lanka, Thailand and Myanmar, presumably because of the easier access to this species, as these animals are at least partially under human care ]  are one of the most valuable diagnostic tools in veterinary medicine and are used to help differentiate diseased from healthy individuals , 8. Theyman care –17. Some (ISIS)] .The objective of this study was to establish RI for hematology and selected clinical chemistry measurands for a free-ranging African elephant population. The RI were generated in accordance with the guidelines published by the American Society for Veterinary Clinical Pathology (ASVCP)  with minEthical approval specifically for this study was obtained from the University of Pretoria Faculty of Veterinary Science Research Ethics Committee and Animal Ethics Committees (certificate number REC 132-19).via a 18G needle and direct vacutainer collection from an auricular vein at first handling after the venipuncture site was swabbed with alcohol (ethanol 70%). Disposable medical gloves were worn during the blood collection. Whole blood was collected in sealed EDTA and serum vacutainers . Serum samples were left to clot for at least 30 min standing upright in a cooler box. Samples were transported cooled, until they were processed in the VWS laboratory within 6 h of collection. EDTA whole blood was analyzed with the scil Vet abc  or the Horiba ABX Micro VS60  hematology analyser. Automated hematology analysis was not routinely performed for all animals. Blood smears were made using a standard pushing (wedge) technique . Serum tubes were centrifuged at 1,300 g for 10 min, and serum aliquoted into cryotubes  and frozen at −80°C.The samples for this study originate from the free-ranging African elephant population from the KNP, South Africa. The animals were immobilized for park management purposes or other unrelated studies. Immobilization was performed by Veterinary Wildlife Service (VWS) veterinarians according to SANParks Standard Operating Procedures (SOP). Elephants were darted from a helicopter using an air-pressurized dart  propelled by a carbon dioxide powered rifle . Immobilization was induced with etorphine , azaperone , and hyaluronidase , with dose ranges based on subjective weight and age estimation by the same veterinarian (etorphine 0.003 mg/kg and azaperone 0.01 mg/kg). Once the elephant was recumbent, the ground crew approached and assisted the elephant into lateral recumbency, if required. At the end of the procedure, naltrexone  was administered intravenously at 20 times the etorphine dose (mg), and the animal observed until it had fully recovered. Sample collection proceeded according to a standardized protocol as follows: blood was taken echnique  and staiAt the time of immobilization, a physical examination was performed. Animals without injuries and free of clinical abnormalities were considered healthy. All data for the animal, including the sex, general condition, age and weight estimation, microchip number and geographical location of the immobilization site, were recorded in Excel spreadsheets. Notes were added for abnormal clinical findings or injuries, if present. Sample selection was made according to this information. All selected samples were collected between October 2014 and August 2019, meaning they were stored no longer than 5 years. This threshold was chosen as no studies could be found on stability beyond this time . SamplesData from the original hematology analyses were reviewed. Analysis was performed using EDTA whole blood after mixing at room temperature, with a Scil Vet ABC (first 25 results) and a Horiba ABX Micros ESV60 (last 11 results), using the domestic horse setting on both analyzers. These analyzers and settings have not been validated for elephant blood. Internal quality control using manufacturer-supplied quality control material was performed every day before analysis. These results from the original hematology analyses were reviewed for the present study. Firstly, the automated calculated hematocrit (HCT) was compared to a manual PCV performed at the same time. Only automated results with a HCT within 3% of the PCV were included in this study. The white blood cell count (WBC), red blood cell count (RBC) and platelet count (PLT) as measured by impedance , and the hemoglobin concentration (HGB), as measured by a cyanide-free photometric method  were considered accurate enough to be used for this study. The erythrocyte indices mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated using the following standard equations:12/L, PCV %; MCV fL, MCH pg, MCHC g/L) as well as the severity of toxicity (1+ to 4+) according to a standardized grading system described for domestic species . MorpholAnalysis was performed using the Abaxis VetScan VS2  according to the manufacturer's instructions. The Large Animal rotor  was used and included the following measurands: albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), calcium, creatine kinase (CK), gamma glutamyltransferase (GGT), globulin, magnesium, phosphorus, total protein (TP) and urea. The analytical methods for these measurands are shown in Statistical analyses were performed with MedCalc software version 19.1.7  and the Excel add-on Reference Value  Advisor version 2.1  accordinp-value of >0.2 for the Shapiro-Wilk test, not >0.05, was used to define a Gaussian distribution  or robust method . The 90% confidence intervals (CI) of the lower and upper reference limits (RL) were calculated using a bootstrap method. The ratio of the upper or lower CI to the RI was calculated by dividing the former by the latter .To examine the potential effect of storage time on clinical chemistry measurands, the number of days from storage to analysis was enumerated, and the correlation between days in storage and measurand concentration or activity calculated using Pearson's correlation coefficient, Samples were selected from free-ranging elephants, with no overt clinical abnormalities, living in the KNP in South Africa and were collected between October 2014 and August 2019. Of the original 79 selected samples, two were excluded after further detailed analysis of their capture records, as one was found deceased subsequent to a road traffic accident, and one had a snare injury. After statistical analysis and removal of 28 complete data sets with outliers, 51 samples from apparently healthy animals remained. These consisted of 42 males and 9 females. All were sub adults or adults according to the original selection datasheet, except for one male calf which was estimated to be 4 years old. Results for PCV were available for 48 of these animals. Only 23 samples from the automated hematology analysis were finally included, after applying the described screening procedures. Results thereof are shown in The CVs obtained for the clinical chemistry measurands from the repeatability study with elephant serum on the VS2 were: albumin 1.1%, ALP 3.1%, AST 4.7%, calcium 0.8%, CK 5.2%, GGT 6.6%, globulin 3.7%, magnesium 0.9%, phosphorous 1.0%, TP 1.4%, urea 3.9%. Imprecision for all measurands was considered acceptable, when compared to total allowable analytical error guidelines for veterinary species .r = 0.69, p < 0.001; CK r = 0.50, p < 0.001). In other words, older samples appeared to have a decreased AST and CK activity compared to more recent samples, which may indicate a storage effect (No significant correlation between storage time (minimum 5 months to maximum 5 years) and measurand concentration/activity was found for any measurands apart from AST and CK , published between 1977 and 1980; all these data originated from the same elephant population in eastern Africa (n = 18–23), where on later examination, infestation with the bile duct hookworm  and other parasites was discovered  and a different subspecies of African elephant (Loxodonta africana cyclotis)   report dStudies on Asian elephants are more common and involve both managed and free-ranging animals. Managed elephants often reside in tourist camps or are working elephants from the timber industry, from various locations in South East Asia, including India, Sri Lanka, Thailand and Myanmar , 17, 36.In the above mentioned studies on Asian elephants, the animals were described as clinically healthy or apparently healthy. Sample sizes varied greatly; the smallest one was performed on six individuals with longitudinal samples  with the12) and PCV (49%) described in the East African study, were higher than those determined in the current study and the minimum-maximum (1.4–6.0 × 1012/L and 18–80%) ranges much wider, in comparison to the current reference intervals (12/L) and PCV (35.1%); MCV range (106–122 fL) was slightly narrower and MCHC range (310–390 g/L) wider but comparable to the current RIs , reported in the African forest elephant (n = 5) (9 /L) from captive and immobilized African elephants (9/L) in tuskers (9/L) in free-ranging elephants in Sri Lanka, the latter being the highest reported WBC count in apparently healthy elephants  are lower than the current RIs (0.4 × 109/L)  , 39. Int× 109/L) .9/L) (9/L) (9/L)  . The lowL) (9/L) . The higelephant . The widMost of the results for clinical chemistry measurands reported in other studies of comparable geographical region were similar to the current measurements, even though analytical methods differed , 32, 43.Serum mineral concentrations in the African elephant vary by geographical location. The means of calcium, magnesium and phosphorus in the current study were higher compared to those from elephants at Sengwa Wildlife Research, Zimbabwe, but lower in comparison to elephants from Ruwenzori National Park in Uganda , 30. FirDetermination of AST and CK activity is especially important for captured animals, as increases in these enzymes are associated with muscle injury, intramuscular injection, trauma and capture stress in domestic and wild animals such as dogs, horses, some ruminants and rhinoceros –48. AnimLoxodonta africana are even lower whereas urea and globulin means were higher than the current results  from the same geographical location (KNP), using the same drug combination and clinical chemistry panel, did not consider the influence of the anesthetics relevant  without significant changes in the measurands , 62. In For future studies, the sample size should be increased, especially for the automated hematology analyses, as the current sample size was rather small and the analyzers are not validated for this species. Ideally, the same machine should be used for all samples, which in the best case scenario is validated for the species to be analyzed. Unfortunately, this was not possible for this study due to resource restrictions present in the laboratory. It is important to note that the automated hematology reference intervals are specific to the analyzers and settings used in this study. The desirable sample size for all measurands would be >120, but above 39 is considered reasonable for RI studies, which we were able to attain for blood smear analyses and WBC count as well as chemistry measurands . Larger This is the first RI study for hematology and a clinical chemistry panel relevant for the African elephant performed using appropriate statistical methods and a strict outlier elimination approach. In comparison with previously performed research on the same species and for Asian elephants, the current ranges are narrower, which will have a greater potential to identify normal and abnormal clinical pathology results in individuals of this species in the future. The established RI will function as an important tool for researchers and clinicians working with the species, provided that drugs, geographic location and nutrition are taken into consideration when interpreting results. Future studies will be needed with greater sample sizes to create RIs for different groups .The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Veterinary Science Research Ethics Committee and Animal Ethics Committee (certificate number REC 132-19), Faculty of Veterinary Science, University of Pretoria.CS contributed to conceptualization and study design, data collection, data curation, performed the data analysis, and wrote the first draft of the manuscript. EH contributed to conceptualization and study design, data collection, data curation, performed the data analysis, and acquired funding. JH contributed to data collection. MM contributed to conceptualization and design of the study, and assisted with sample provision. PB assisted with sample provision and data curation. All authors contributed to manuscript revision, and read and approved the submitted version.The 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.


BCD-022 is a trastuzumab biosimilar which was shown to be equivalent to reference trastuzumab in a wide panel of physicochemical studies as well as preclinical studies in vitro and in vivo. International multicenter phase III clinical trial was conducted to comparatively assess efficacy and safety of BCD-022 and reference trastuzumab in combination with paclitaxel used as the therapy of metastatic HER2(+) breast cancer. Pharmacokinetics and immunogenicity were also studied.Patients with no previous treatment for metastatic HER2(+) breast cancer were randomly assigned 1:1 to BCD-022 or reference trastuzumab and were treated with trastuzumab + paclitaxel. Therapy continued for 6 cycles of therapy (every 3 weeks), until progression of the disease or unbearable toxicity. Primary study endpoint was overall response rate. Study goal was to prove equivalent efficacy of BCD-022 and reference trastuzumab. Equivalence margins for 95% CI for difference in overall response rates were set at .n = 3; 2.65%) and comparator . Both drug products are characterized with low occurrence rate and short life of anti-trastuzumab antibodies. Pharmacokinetics assessment after 1st and 6th study drug injection also demonstrated equivalent PK parameters by all outcome measures: AUC0–504, Сmах, Тmax, T1/2. Analysis of Ctrough did not reveal any significant inter-group differences as well.In total 225 patients were enrolled into the study, 115 in BCD-022 arm and 110 in reference trastuzumab arm. Overall response rate was 49.6% in BCD-022 arm and 43.6% in reference trastuzumab arm. Limits of 95% CI for difference of overall response rates between arms were [(− 8.05)-19.89%], thus, they lied within predetermined equivalence margins . Profile of adverse events was similar between groups (any AEs were reported in 93.81% of patients in BCD-022 arm and 94.55% of patients in reference arm). No unexpected adverse reactions were reported throughout the study. No statistically significant differences regarding antibody occurrence rate (either BAb or NAb) was found between BCD-022 . The date of registration was January 9, 2013.The trial was registered with  Based on GLOBOCAN data there will be an estimated 18.1 million new cancer cases (17.0 million excluding nonmelanoma skin cancer) and 9.6 million cancer deaths (9.5 million excluding nonmelanoma skin cancer) in 2018. Among females, breast cancer is the most commonly diagnosed cancer with over 2 million new cases and the leading cause of cancer death  . The confidence interval lies within the predefined range of clinically insignificant difference, so conclusion on equivalent efficacy of BCD-022 and reference trastuzumab was made. The equivalence of efficacy confirms biosimilarity of BCD-022 and reference trastuzumab.Comparison of other efficacy assessment parameters (secondary outcome measures) did not reveal any statistically significant differences between study arms in mITT population Fig. .Fig. 2Ep = 0.895, Yates-corrected x2 test). The difference in overall response rate between BCD-022 and reference trastuzumab arms counted 1.9, 95% CI for difference of overall response rate was [(− 12.76)-16.54%]. The limits of confidence interval lie within predefined range of clinically insignificant differences, so conclusion on biosimilarity of BCD-022 and reference trastuzumab is confirmed in PP population.Additional analysis of efficacy in PP population confirmed the results of analysis in mITT population. According to CT-scan results, the overall response rate  in PP population was 51.4% (95% CI 41.66–60.99) in BCD-022 arm and 49.5% (95% CI 39.25–59.76) in reference trastuzumab arm, respectively  patients from BCD-022 arm and in 104 (94.55%) patients from the reference trastuzumab arm. Most AEs were associated by the main disease or myelosuppressive chemotherapy (paclitaxel).One treatment discontinuation  due to AEs/SAEs were reported in comparator arm. For this patient following SAEs were registered: neutropenia grade 4, hepatic veins compression grade 4.р > 0.05). SAE were revealed in 21 patients: 8 (7.08%) patients from BCD-022 group and 13 (11.82%) patients from the comparator group (p = 0.327). According to investigators, there were 9 (4.04%) SAEs related to the study therapy: 4 (3.54%) SAEs in BCD-022 and 5 (4.55%) SAEs in comparator group. Generally, SAEs were associated with the underlying pathology, chemotherapy agents used in combination therapy or with other factors unrelated to the study therapy.Study arms had no significant difference regarding the occurrence rate of any SAEs as well as no differences in occurrence rate of SAEs related to the study therapy (р = 0.495). Two lethal outcomes (one in each arm) were registered as probably related to the study drug.  patient from BCD-022 group and 5 (4.55%) patient from the comparator group, with no significant difference revealed (.65% patip = 1.0). Grade 3–5 adverse events (CTCAE v4.03) were mostly reported for hematology disorders such as neutropenia, leukopenia, lymphocytopenia and anemia. These AEs are expected in breast cancer subjects undergoing myelosuppressive chemotherapy with paclitaxel.In general, throughout the study a total incidence of grade 3–5 adverse events was 189 cases for both arms, 94 (49.74%) and 95 (50.26%) cases were registered in BCD-022 and reference trastuzumab groups respectively .As per protocol, the screening test was performed to reveal the presence of binding antibodies (BAb) in subject’s blood, followed by the confirmatory analysis. If binding antibodies were found, the test for neutralizing antibodies (NAb) was performed. Upon assessment of binding antibodies, the neutralizing potency of anti-trastuzumab antibodies in patients’ samples was evaluated. Neutralizing activity was revealed in 3 (2.65%) patients from the study arm and 4 (3.64%) patients from the comparator arm  was found between BCD-022 and reference trastuzumab. Both drug products are characterized with low occurrence rate and short life of anti-trastuzumab antibodies.The analysis has shown that upon both single-dose and repeated-dose administration of study drug and comparator, the changes in serum concentrations of trastuzumab were similar.(0–504) was 28,969,372.5  (ng/ml)·h for BCD-022 and 28,796,527.9  (ng/ml)·h for reference trastuzumab (p > 0.05). Maximum serum concentrations of trastuzumab (Cmax) after the administration of BCD-022 and comparator were 218,720.0  and 216,710.0  ng/ml, respectively. 90% confidence interval for the ratios of AUC(0–504) in BCD-022 and reference trastuzumab groups was , and for Cmax — . Both calculated confidence intervals lie in within acceptable range (80–125%), thus the PK of study drugs is considered to be equivalent.After a single administration (1 cycle) of the study drug or the comparator, AUC(0–504) was 25,800,936.8  (ng/ml)·h for BCD-022 and 26,730,362.3  (ng/ml)·h for reference trastuzumab (p > 0.05). Maximum serum concentrations of trastuzumab (Cmax) after the 6th administration of BCD-022 and comparator were 168,735.0  and 169,220.0  ng/ml, respectively. 90% confidence interval for the ratios of AUC(0–504) in BCD-022 and reference trastuzumab groups was , and for Cmax — . Both calculated confidence intervals lie in within acceptable range (80–125%), thus the PK of study drugs is considered to be equivalent.After a 6th administration (6 cycle) of the study drug or the comparator, AUCtrough (for 6 sampling points prior to each administration) was 19,380.0 ng/ml in ВCD-022 arm and 21,572.5 ng/ml in reference trastuzumab arm, no difference was revealed between the study arms with respect to this parameter .Calculated median CAccording to the results of the study the equivalence of PK profiles of BCD-022 and reference trastuzumab is confirmed.Thus, the absence of any differences between the study arms with respect to all mentioned above parameters confirms that pharmacokinetics of BCD-022 is equivalent to that of reference trastuzumab.The development of biosimilar products is a complex, step-by-step process which involves factoring in a wide range of parameters such as physicochemical properties, functional characteristics, efficacy and safety. Even though regulatory guidelines are in place for such products, they lack the specifications and guidelines to use it in a clinical setting. As stated by Nixon et al. , the useThe study drug BCD-022 was registered in the Russian Federation under the name Herticad® in December 2015 and has been in use in the Russian clinical practice since March 2016. The initial results published by Kolyadina et al.  highlighSimilarities between the efficacy and safety data between Herticad® and the reference product, coupled with a 66% lower price in the Russian market for Herticad®, has enabled patients to have broader access to vital therapy and in turn, save additional costs in the healthcare sector. After the market entry of Herticad® the average annual treatment cost per patient with trastuzumab fell by 62%. The number of eligible patients who gained access to the treatment with trastuzumab increased from 41% in 2015 to 56% in 2017 [«Extrapolation of clinical efficacy and safety data to other indications of the reference mAb, not specifically studied during the clinical development of the biosimilar mAb, is possible based on the overall evidence of comparability provided from the comparability exercise and with adequate justification”. As for Trastuzumab biosimilar; the active substance interacts with one active site in HER2 receptors and has no different impact in the tested and non-tested therapeutic indications. In the trial BCD-022-02, the therapeutic indication is relevant in terms of efficacy and safety and the homogeneous representative population with metastatic breast cancer and HER2 overexpression, and/or amplification is sensitive for differences in all relevant aspects of efficacy and safety. As it was stated above, BCD-022  and reference Trastuzumab  are comparable regarding efficacy, safety and pharmacokinetic profiles when used in combination with paclitaxel in mBC HER2+ patients. Therefore, no additional data is required for efficacy and safety extrapolation of BCD-022  to all indications.Although Herticad® was investigated only in metastatic breast cancer it has been approved for early breast cancer, metastatic breast cancer and metastatic gastric cancer as well as the innovator trastuzumab. In Europe, it is a common practice to extrapolate biosimilar agents to indications for which it was not tested during the clinical trial. According to ЕМА «Guideline on similar biological medicinal products containing monoclonal antibodies – non-clinical and clinical issues»: With the number of biosimilars increasing, more marketed medicines are expected to reach the market over the next few years and will certainly provide a cost-effective treatment to a greater number of patients. Among the different clinical applications of biosimilar medicines, cancer treatment remains the main target area. Usage data for Trastuzumab biosimilar  in routine clinical practice for patients with HER2-positive breast cancer is currently being collected. Thus, a comprehensive pharmacovigilance study is ongoing, and the marketed biosimilar product is being constantly monitored; providing more useful information to clinicians regarding the safety and efficacy of this medicine.Results of international multicenter phase III clinical trial have demonstrated therapeutic equivalence of trastuzumab biosimilar BCD-022 and referent trastuzumab drug (NCT01764022). As it was stated above, BCD-022  and reference Trastuzumab  are comparable regarding efficacy, safety and pharmacokinetic profiles when used in combination with paclitaxel in patients with metastatic breast cancer and HER2 overexpression. This has eliminated the need to provide additional data to extrapolate the efficacy and safety of BCD-022  against all indications. Unfortunately, the economic conditions of the society and the cost of the drug has limited its accessibility to a small proportion of patients. Therefore, the introduction of Trastuzumab biosimilars into the market has provided a wider access to an alternative yet cheaper therapy to a broader network of patients.Additional file 1: Supplemental Table 1. Characteristics of the main disease in patients involved in the study (ITT population) by groups.


Gobiomorus dormitor and G. maculatus and therefore they cannot be considered geminates. The average genetic distance of protein-coding genes between Sicydium altum and S. salvini was more than two times larger than between Atlantic and Pacific Awaous banana, suggesting different timings for their divergence across the Isthmus of Panama.We sequenced the complete mitochondrial genomes of three pairs of congeneric peripheral fishes distributed on either side of the Isthmus of Panama in order to test their status as geminate species pairs. Our phylogenetic analysis did not support a sister relationship between  The formation of the Isthmus of Panama represents one of the most dramatic geological events in Earth’s history with major climatic and biological implications. Despite its global importance, the timing of the uplift of the Isthmus of Panama remains an open question that has resulted in a thrilling scientific discussion , Gobiomorus maculatus , Sicydium altum , S. salvini , Awaous banana , and A. banana . We excised the gill arches and preserved them in a 20% dimethyl sulphoxide (DMSO), 0.5M EDTA, pH 8 solution at 4 °C until DNA extraction. All specimens were initially fixed in formalin, later transferred to 70% ethanol, and deposited and vouchered in the Neotropical Fish Collection (NFC-STRI) at the Smithsonian Tropical Research Institute in Panama (STRI).We collected specimens from six species or lineages of gobiiform fishes distributed in Atlantic and Pacific watersheds of Panama: We recovered complete mitochondrial DNA sequences as a by-product of hybrid target capture of ultraconserved elements (UCEs)  that was used as an outgroup. We extracted and individually re-aligned all 13 protein-coding genes for subsequent phylogenetic analysis. We partitioned the data by gene and by codon, and estimated the best partition scheme under the GTR + GAMMA substitution model using PartitionFinder  and G. maculatus (MF927494); S. altum (MF927496) and S. salvini ; and A. banana from Atlantic (MF927488) and Pacific (MF927489) watersheds.We obtained complete mitochondrial genome sequences with a minimum of 10× coverage for three species pairs, or lineages, of peripheral gobiiform fishes: G. dormitor to 16,536 bp in G. maculatus. All species contained 22 tRNA genes, two rRNA genes, 13 protein coding regions, as well as control region arranged in the same order. The most common start codon ATG was found in all genes except in COXI that had the start codon GTG, and in ND6 that had TAC as the start codon. The stop codon TAA terminated the genes ND2, ND4L and ATP8 in all species, whereas genes ND1 and ND5 exhibited either TAA or TAG as stop codons. Incomplete stop codons were found in some or all species for genes ATP6, COXII, COXIII, CYTB, ND3 and ND4 formed of only T or TA, which are likely completed as TAA via post-transcriptional polyadenylation.The size of the mitochondrial genomes ranged from 16,474 bp in Sicydium and Awaous showed sister relationships and can be considered as geminate species. However, G. dormitor and G. maculatus were not recovered as sister species, and consequently the hypothesis that they are geminate species was rejected , except for the COXII gene (12.74% versus 12.30%).As would be expected, given they were not recovered as sister groups, compared . DistancA. banana: ten tRNA genes were identical, with the remaining 12 ranging from 1.33 to 4.34% different. All protein-coding genes were over 2% divergent (mean = 4.75% ± 2.02), which is a common threshold for species delimitation in mitochondrial and barcoding studies   .Awaous and Sicydium geminate pairs are in the lower and upper boundaries of other geminate species pairs of peripheral fishes (e.g. D. maculatus versus D. latifrons: CYTB uncorrected p-distance = 8.6%, Galván-Quesada et al. Agonostomus monticola versus Pacific A. monticola: CYTB uncorrected p-distance = 8.3%, McMahan et al. Sicydium species inhabit fast flowing rivers and creeks between sea level and 1100 m, whereas A. banana is commonly found in estuaries and rivers only up to 300 m above sea level. Therefore, the affinity of Awaous to near-shore environments and brackish waters may have allowed them to maintain gene flow until the very last instances of marine connectivity across the Central American Seaway. Overall, these new mitogenomes constitute an important resource for future investigations on rates of molecular evolution and divergence times of geminate species, and eventually for a further understanding of the geological history and age of the Isthmus of Panama.The divergence values between


Tenebrio obscurus (Zunyi population) was sequenced and annotated. The 15,509 bp circular mitogenome contains a conserved set of 37 mitochondrial genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and an A + T-rich region. The gene order is identical to the typical insect gene arrangement. Great differences were found in A + T-rich regions between Zunyi population and Guiyang population including large deletion and substitution. Phylogenetic analysis showed that T. obscurus clustered within Tenebrionoidea and was closely related to Tenebrio molitor.Mealworms are the most common edible insect and feed additives in poultry, livestock, and aquaculture production. In the present study, the complete mitochondrial genome of dark mealworm  Tenebrio molitor), and dark or mini mealworm (Tenebrio obscurus), which is regarded by global food system experts as potentially one of the most valuable food source for humans , which will provide valuable information for molecular phylogenetics and population genetics.Mealworms are the larvae of two species of darkling beetles, the yellow mealworm (T. obscurus (JNU20160522) were provided and preserved by the cell biology laboratory of Jiangsu Normal University. The complete T. obscurus mitogenome was amplified by using a combination of universal primers  is 15,509 bp in size, which is shorter than T. obscurus mitogenome of Guiyang population. It contains a conserved set of 37 mitochondrial genes including 13 protein-coding genes (PCGs), 22 tRNA, 2 rRNA genes, and an A + T-rich region. The gene order is identical to the typical insect gene arrangement. The heavy chain codes 23 genes, including 14 tRNA and 9 PCGs, and other 14 genes are coded by the light chain. The overall base composition of the genome is as follows: A (43.1%), T (29.4%), C (17.4%), G (10.2%), with an A + T content of 72.4%. All PCGs start with ATN codon. Four PCGs end with incomplete stop codon T, while nine PCGs stop with TAA and TAG. All traditional tRNA genes ranging from 60 to 70 bp have the canonical cloverleaf secondary structure, except for tRNASer(AGN). The 16S and 12S rRNA genes of T. obscurus are 1280 bp and 780 bp in size, respectively. The A + T-rich regions show marked differences between Guiyang population (1177 bp) and Zunyi population (902 bp), which can be used as a powerful molecular marker for population investigation.The complete mitogenome of T. obscurus was most closely related to T. molitor. Our study provides additional molecular data for subsequent studies on population structure, and management strategies for this important resource insect.The ML and BI phylogenetic trees exhibited the same topology , which i


Nidirana pleuraden. Its biological function and its morphological and histochemical characteristics are unclear. In the present study, we describe the structure and ultrastructure of the postaxillary gland, and explore its main function.Owing to their incomplete adaptation to the terrestrial environment, amphibians possess complex cutaneous glandular systems. The skin glands not only regulate water loss and respiratory gas and salt exchange, but are also involved in defense against predators and microorganisms, social communication, and reproduction. These glands are distributed throughout the integument, but can accumulate in specific regions, forming visible outgrowths known as macroglands. Some macroglands are sexually dimorphic and mediate intersexual communication and reproductive success. The postaxillary gland is a sexually dimorphic macrogland in N. pleuraden. In addition to ordinary serous glands (OSG), type I and II mucous gland (I MG & II MG), a type of specialized mucous gland (SMG) is also found to constitute the postaxillary gland. The SMG is larger than other gland types, and consists of high columnar mucocytes with basal nuclei arranged radially toward a lumen. SMGs are positive to periodic acid-Schiff stain and stained blue in Masson’s trichrome stain. A discontinuous myoepithelial sheath lacking innervation encircles SMG mucocytes, and the outlets of such glands are X- or Y-shaped. Transmission electron microscopy reveals abundant secretory granules in SMG, which are biphasic, composed of an electron-opaque outer ring and a less electron-dense core. Lipid droplets, and organelles, such as rough endoplasmic reticulum and Golgi stacks, are located in the supranuclear cytoplasm of the mucocytes in SMG. Female N. pleuraden exhibits chemotaxis toward homogenate of the postaxillary gland, but male does not. On treatment with trypsin, this sexual attraction disappears.The postaxillary gland has a thinner epidermis than the dorsal region of N. pleuraden is a male-specific macrogland that consists primarily of SMGs, together with OSGs, I MGs and II MGs. Other than their extremely large size, SMGs structurally and histochemically resemble many reported specialized gland types in amphibian sexually dimorphic skin glands. Secretions of the postaxillary gland are proteinaceous sexual pheromones, which are believed to attract females at male calling intermissions.The postaxillary gland of  As a transitional type of organism which emerged from aquatic environments to inhabit the terrestrial environment during animal evolution, amphibians’ adaptation to subaerial conditions is somewhat incomplete. Their skins, which were embedded with complex and active integumentary apparatuses, play a crucial role in the survival of this taxon. Distinct from other vertebrates, numerous dermal glands are functional in the amphibian skin, which help to regulate respiratory gas, salts, and water exchange , and conIn the extant amphibians, the skin multicellular glands are simple alveolar and intradermal ones which evolved from single mucocytes and serous cells scattered in the epidermis of their bony fish ancestors . Using mPhyllomedusa leaf frogs consists of serous, mucous, and lipid glands, but with the serous ones predominate . The ordinary serous glands distributed both at the male dorsal skins and the postaxillary glands, they showed typical morphological and histochemical characteristics mentioned in other amphibians, for having relatively thick myoepithelial cells layer, eosinophilic contains and negative to AB pH 2.5 or PAS stains [Unsurprisingly, the postaxillary glands of S stains , 9, no sRana amurensis [Sylvirana latouchii [P. distincta, but the I MGs they examined were positive to the PAS staining instead of AB pH 2.5, whereas their II MGs were stained with both PAS and AB pH 2.5, implying variations in secretions of ordinary mucous glands between anuran species.Like the topological distribution of OSGs, two kinds of ordinary mucous glands were identified from dorsal skins and the postaxillary glands. The subdivision of type I mucous glands and type II mucous glands was mainly based on the morphologies of their constituent secretory cells  and histochemical responses of the luminal or intracytoplasmic secretions (intensely positive to AB pH 2.5 in I MGs and positive to PAS in II MGs). Our observations on the two distinct ordinary mucous gland types correspond to the previous reports that demonstrated I MGs and II MGs exist in the skin of murensis , and theatouchii . Antoniaatouchii  also fouN. pleuraden consisted of derived mucous glands primarily under our LM, SEM and TEM observations, was analogous to most discovered SDSGs, except the femoral glands in Mantellidae , [Cycloramphus , ), the illy SSGs, ), and thpresent, ). High cpresent, , 17, 23.present, . Noticeapresent, , 10, 23,R. iberica show no response to PAS [Hypsiboas punctatus, and more frequently, in serous glands [Histochemically, SMGs in the postaxillary glands reacted positively to PAS stain, like most other SMGs in different species, with one exception that the SMGs of e to PAS . It was e to PAS . Similars glands , 23. It s glands , which as glands , 56, 57 s glands  rather ts glands , 9. AlthN. pleuraden stand for one glandular type in different stages of maturation or asynchronous secretory phases caused by local fluctuation, considering the SMGs’ specific topological affinity to the postaxillary glands.According to the structural, ultrastructural and histochemical traits in SMGs, they could be easily distinguished from OSGs and OMGs. Even though there were evidences on OMGs transforming into SMGs after testosterone injection in plethodontids , we do nN. pleuraden prefers the homogenate of male postaxillary glands. However, male frog is neither attracted nor repelled by the homogenate of the postaxillary gland. Taken together, these demonstrate the secretions of the postaxillary gland effect as sex attractant rather than aggregation or territorial pheromone [Compared with distilled water, female heromone . Thinninheromone . Female heromone , 43, we heromone , 60, 61.R. dalmatina, R. iberica and R. italic), the SDSGs are diffused in the male dorsal skins rather than segregate in macroglandular structures [Rana species, but when we take a glance at a broader scenario, it becomes less suitable since anurans who communicate with acoustic signals also possess SDSGs have been documented several times [N. pleuraden [Many SDSGs have been proved to involve in pheromone productions in amphibians, regardless of urodelean species , 41, 62 al times , 46, 61,leuraden .Leiopelma hamiltoni, a primitive anuran lineage, calling was not observed, but chemicals played a fundamental role in its social communications [N. pleuraden is a relatively advanced species of Neobatrachia [It is widely accepted that chemical cues are the salamander counterpart of auditory vocalization in frogs and toads , thus a ications . Becauseatrachia , and thePseudophryne bibronii, male chemosignals combined with acoustic signals improve the ability of females to find nest sites [N. pleuraden. The frog is nocturnal and hides in very dense hydrophytes during the breeding season  is much larger than other glands, its high columnar mucocytes have basal nuclei, arranging radially toward an obvious central lumen. What inside the lumen and cytoplasm of the mucocytes is eosinophilic, positive to periodic acid-Schiff stain, and stained blue with Masson's trichrome. A discontinuous myoepithelial sheath without direct nerve supply is observed to surround mucocytes of SMG, as well as the X- or Y-shaped glandular outlets distributing at the superficial epidermis. Secretory granules in SMG appear to be biphasic, reveal as a less electron-dense core encircled by an electron-dense outer ring. In addition to numerous secretory granules, lipid droplets, and organelles, such as rough endoplasmic reticulum, Golgi stacks are also detected in the mucocytes. The homogenate of the postaxillary gland attracts female N. pleuraden, but has no attracting or repelling effect to males. And if introduces male advertisement call, or trypsin treatment, the sexual appeal vanishes. Taken together, we think the postaxillary gland plays a key role in the specie’s reproduction: its secretions are proteinaceous sexual pheromone, which facilitates females to locate males when they cease calling. Moreover, this study provides new evidences to the field of anuran chemical communication, which is on early stages of development with limited cases. Knowledge on the topological distribution, structural diversity and histochemical variance of sexually dimorphic skin glands in an enlarged phylogenetic scenario, likewise the accurate identification of the glandular secretions would help to elucidate how prevalent anurans communicate via chemosignals, and the interaction among multiple sensory modules in this group.The present study examines the structure, histochemical characteristic and bio-function of the postaxillary gland in N. pleuraden were collected at the Caohai National Nature Reserve  in a night of late June, 2017. For behaviour tests, in total 15 male and 40 female N. pleuraden were captured at the reserve in nights during June 24th to July 3th, 2018. There were 5 sets of ethological trials conducting sequentially. Animals were collected and maintained separately in lidded plastic boxes (15 × 10 × 6 cm3) with moist tissue paper inside on experimental days’ eves, and released back to the field (except for the stimuli providers) after a certain set of trials have been accomplished the next night. Considering this sampling-experimenting-releasing arrangement, the possibility that identical individual was introduced to more than one set of trials could not be ruled out.For the structural and ultrastructural examinations, two male N. pleuraden used in this study were sexually mature as we located the male frogs via their advertisement calls and the females were larger than 55 mm in snout-vent length with swollen abdomens (an indication of gravidity). Seven males (two in 2017 & five in 2018) were euthanized by bath immersions in overdosed (3 g/L) tricaine methanesulfonate (MS-222) solution for 20 min and then washed by distilled water before skin or gland excisions.All the Animal collection and experiment permissions were issued by the Management Office of the Caohai Nature Reserve (CHNR2017062101 & CHNR2017062301) for this study and all experimental procedures involving animal attendances strictly followed the regulation formulated by the Animal Care and Use Committee of Chengdu Institute of Biology, CAS.2) and the postaxillary glands (about 1 cm2) were stripped from the two euthanized male N. pleuraden, and the widths, heights and thicknesses of the postaxillary glands were measured. Samples for light microscopy (LM) were fixed in Bouin’s fluid for 24 h, after which they were rinsed in running water for 30 min and immersed in 70% ethanol for another 30 min for discoloration. Dehydration was achieved in ascending ethanol series . Then the samples were cleared in xylene, embedded in paraffin and sectioned transversely at 6 μm using a rotary microtome . Sections were stained with: (1) hematoxylin-eosin (HE) for general cytology and histology; (2) combined Alcian blue (pH 2.5) and periodic acid-Schiff (AB-PAS) for acid mucopolysaccharides, sialic acids, neutral carbohydrates and glycoproteins; (3) Masson's trichrome method for collagen fibers and myofibers. The staining procedures followed the instructions in corresponding stain kits . Stained sections were sealed on microslides with resinene, examined and photographed using a Nikon E200 microscope equipped with an industrial digital camera . The thicknesses of the epidermal layers in the dorsal skin regions and the postaxillary glands, as well as the maximum heights of simple alveolar glands and inner secretory cells were measured using ImageView (www.microdemo.com/download/imageview). Values represent mean ± standard error consistently. In dorsal skin and the postaxillary gland sections, we referred different simple alveolar gland types as ordinary serous glands (OSG), type I mucous glands (I MG), type II mucous glands (II MG) and specialized mucous glands (SMG) according to widely accepted preexisting terminologies [Dorsal skin patches about 16–25 mm6–25 mm2 nologies , 51.2) were fixed in 3% glutaraldehyde , then rinsed in 0.1 M Sorensen phosphate buffer (pH 7.2) for 3 times (15 min each time) and postfixed for 1 h in 1% osmium tetroxide in the same buffer. The samples were again rinsed and dehydrated through a graded series of ethanol . After the immersion in isoamyl acetate to displace ethanol, the samples were mounted on aluminium stubs, dried in a critical point apparatus , coated with gold palladium in a sputtering device , and observed in a scanning electron microscope  at 15 kV.For scanning electron microscopy (SEM), the postaxillary glands samples , operating at 60 kV and photographed using an ES1000W Erlanshen CCD Gatan digital camera . Spliced figures of structural and ultrastructural observations’ results were prepared using Adobe Photoshop CS6 (www.adobe.com/Photoshop).For transmission electron microscopy (TEM), glands samples were fixed, postfixed and dehydrated following the same procedure in SEM sample preparation. After dehydration, the samples were cleared in propylene oxide and embedded in epoxy resin. Semithin (1 μm) and ultrathin (70 nm) sections were cut using a ultramicrotome  and diamond knives . Semithin sections were stained with 1% toluidine blue in 1% sodium carbonate and examined under a light microscope for preliminary gland localization. Ultrathin sections were mounted on uncoated 200 mesh copper grids and electron-dense stained with uranyl acetate (25 mg/ml), followed by lead citrate (2 mg/ml). Grids were viewed using a transmission electron microscope  distilled water and homogenized using a glass tissue homogenizer. Cold distilled water was added to the homogenate to make the final volume 10 ml. After a 30 s shaking, the homogenate was divided into 10 aliquots (1 ml each), which were stored at 4 °C and used on the night of the preparing day. Each aliquot contained the equivalent of one fifth of one postaxillary gland or a tenth of a removed dorsal skin patch. When used as chemical stimulus, one aliquot was injected into a sponge block (1 cm2), and then the sponge block was attached to one end of the experimental tank (below water level). Sponge blocks injected with distilled water, postaxillary gland aliquot mixed with trypsin solution  dissolved in 0.9% sodium chloride to bring the concentration to 25 g/L, added 1 ml into the aliquot and gently shook for 5 min at ambient temperature) and distilled water mixed with trypsin solution were also placed in the tank as optional stimuli for tested frogs.To perform animal preference tests, we prepared both chemical and auditory stimuli. As for chemotaxis detections, the two bilateral postaxillary glands (weight about 0.14 g) or a dorsal skin patch of the same weight were excised from a euthanized male www.praat.org).The advertisement calls of male frogs were recorded using a solid state recorder , and the connected microphone  was pointed to calling frogs in close vicinity. A representative recording consisting of six calls  in which the call duration, the fundamental frequency and the dominant frequency were 0.70 ± 0.01 s, 539.04 ± 16.83 Hz and 1874.75 ± 19.93 Hz, respectively, was loop played to tested subjects via two portable speakers  wired to two laptops and positioned equidistantly to the center of the experimental tank at the opposite ends. The playbacks were set to present non-simultaneously through the two speakers, with 20 s delay in one speaker. Before the introduction of tested animal in behavioral trials that involved acoustical stimuli, calls from the two speakers were adjusted to 54 dBC SPL . The visualization of frog advertisement calls  at both ends were referred as the selected zones . The experimental tank was self-made: a 1 m polyvinyl chloride rain gutter with rectangular cross section (18 cm wide) was sealed at both ends using matched plastic lids. Markers were painted on the edge of the tank with black marker pen to subdivide it into 3 areas: from the center point to extend 10 cm bi-directionally was assigned as the waiting zone, whereas the two areas  To test whether the postaxillary gland secrete sexual attractants: female chosen between the homogenate of postaxillary glands and distilled water; (2) To test whether the postaxillary gland’s secretions are intrasexual attraction or repellent: male chosen between the homogenate of postaxillary glands and distilled water; (3) To test whether male dorsal skin secrete female attractants: female chosen between the homogenates of male dorsal skin and the distilled water; (4) To test how auditory signals interact with chemosignals: female chosen between the homogenate of postaxillary glands and distilled water, when male advertisement calls were presented in both ends of the experimental tank; (5) To reveal nature of the potential sexual attractants released by the postaxillary gland: female chosen between the homogenate of postaxillary glands added trypsin solution and distilled water added trypsin solution. Trials were conducted between 20:00 and 02:00 when frogs are active. The yard of a famer house  near the Caohai Nature Reserve was rented to execute behavioral tests, so the environmental factors were similar to the animals’ natural breeding sites. The five series of trials were initiated on June 25th, June 27th, June 29th, July 1st and July 4th, respectively. The mean air, water temperature and humidity were measured as 20.0 ± 0.3 °C (18.5–21.8 °C), 20.2 ± 0.2 °C (18.8–21.1 °C) and 75.6 ± 1.1% (70.9–82.8%) respectively.n = 10 for each position) were also compared following the above procedure.Recorded behavioral videos were analyzed using the embedded player of IPClient. Timing began at the point when a tested frog left the waiting zone, and the total time that a frog spent on staying at each stimulus side was calculated (use the midline of the experiment tank as side switch sign). Two series of duration time data obtained from one set of preference trials were compared. First, normality of the distribution was examined using Shapiro-Wilk test. If the data were normally distributed, homogeneity of variances was checked through Levene’s test. Pooled t-test was used for variances-equal data, otherwise the t’-test was applied. When normality test failed, the Wilcoxon rank-sum test was introduced. The epidermis’ thicknesses of the dorsal skins and the postaxillary glands (n = 30 for each gland type) were analyzed using one-way ANOVA followed with the Bonferroni-adjusted least significant difference (Bonferroni-adjusted LSD) test, after the variables had been confirmed to possess normal distributions and equal variances. The same analyzing procedure was brought to compare the heights of the 4 types of gland .Heights of the secretory cells in the three kinds of mucous glands  of 0.05 and performed in SAS 9.3 .Additional file 1. Basic information on Nidirana pleuraden and ethological experiment equipment. Supplementary figures show the regression of the postaxillary gland out the breeding season of N. pleuraden, the animals’ breeding sites, waveform and spectrogram of the male advertisement call, and set-up of the animal preference test.


Bos grunniens) is a yak breed with strong adaptation to the high-elevation, cold and anoxic environments. Its complete mitochondrial genome is 16,323 bp long with an asymmetric base composition, and harbors the 37 typical mitochondrial genes and one noncoding control region. The PCGs are initiated with the typical ATA or ATG codons, and are terminated with TAA, TAG or the incomplete stop codon T––. Phylogenetic analysis suggests that Tianjun yak is most closely related to the polled yak.Tianjun yak ( Bos grunniens) is a local yak breed from Tianjun County, Haixi Mongolian-Tibetan Autonomous Prefecture, Qinghai Province, China, and has a strong adaptation to the high-altitude, cold and anoxic environments. To facilitate its genetic assays, its complete mitochondrial genome was assembled from high-throughput Illumina sequencing data in the present study. The annotated sequence has been deposited into GenBank under the accession number MN163006.Tianjun yak . A voucher specimen is held in the Key Laboratory of Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences . The genomic DNA coded as NO.20190824, which was extracted from Tianjun yak, is stored at −80 °C (ultra deep-freeze refrigerator) in the sample storage room of our department. Genomic DNA isolation were carried out with the QIAamp DNA Blood Mini Kit . Library construction and high-throughput sequencing with the Illumina HiSeq X™ Ten Sequencing System  were conducted by Annoroad Gene Technology . In all, 3.02 Gb of raw data were retrieved, and were used for mitochondrial genome assembly using MITObim v1.9  or ATG (the 10 others) codon. Three types of stop codons are employed, i.e. TAG (ND2), TAA  and the incomplete stop codon T . The 22 tRNAs range in size from 60 (AGNtRNA-Ser) to 75 bp (UURtRNA-Leu). The two rRNAs are 957 bp (12S rRNA) and 1571 bp (16S rRNA) in size, respectively. The control region is 893 bp long, and is located between tRNA-Pro and tRNA-Phe.The mitochondrial genome of Tianjun yak is structurally similar to those of previously published ones of Bison: Bison bison (GU946976)  (Froese et al. A Bayesian phylogenetic tree was reconstructed using MrBayes v3.1.1 (Ronquist and Huelsenbeck


Foods and beverages were classified into food categories, ranked, and compared by weighted proportional frequency and energy contribution for food security groups by age. Significant differences between household child food security groups were determined using the Rao-Scott modified chi-square statistic. The weighted proportional frequency of beverages  and their energy was significantly higher among food insecure compared with food secure while the reverse was true for water frequency among 12–17 years. Beverage and mixed dish frequency were higher among food insecure compared with food secure 6–11 years while the reverse was true for frequency and energy from snacks. Frequency-differentiated intake patterns for beverages and snacks by food security across age groups may inform dietary recommendations, population-specific dietary assessment tools, interventions, and policy for food insecure children.Food insecurity is associated with nutritional risk in children. This study identified and compared the most frequently consumed foods, beverages, and food groups and their contributions to energy intake among U.S. children and adolescents  by food security status. Dietary intake from the day-1, 24-h dietary recall, and household child food security status were analyzed in the 2007–2014 National Health and Nutrition Examination Survey ( The U.S. Dietary Guidelines for Americans Advisory Committee identified many children and adolescents as having low intakes of fruits, vegetables, whole grains, and dairy concomitant with excessive intakes of sodium, saturated fats, added sugars, and refined grains . Such diAdolescents and children in food insecure households, with “limited or uncertain availability of nutritionally adequate and safe foods or limited or uncertain ability to acquire acceptable foods in socially acceptable ways” , may be However, little is known about the specific eating patterns and food and beverage exposure patterns among U.S. children and adolescents with regard to food security status. Eating patterns, including frequency and amount of foods and beverages consumed, snacking and meal skipping, time of eating occasions and other eating behaviors, influence energy intake and contribute to dietary quality . ResearcNHANES is a nationally representative, cross-sectional survey of the National Center for Health Statistics (NCHS) and Centers for Disease Control and Prevention ,15. The n = 8,123, All participants of this secondary analysis completed the dietary component of What We Eat in America (WWEIA)/NHANES 2007–2008, 2009–2010, 2011–2012, and 2013–2014. Children were 6–17 years  where n = the sample size, i = each participant, Ri = the number of reports of individual food codes for the ith individual [n∑i = 1(Riwi) where wi = sample weight for the ith individual was used to determine the weighted proportion of foods to the total foods reported, or the contribution of each food category reported to the total food category reported, given as: n∑i = 1(Riwi)/n∑i = 1(Tiwi) (100) where Ti = total number of reports of all food codes for the ith individual. The weighted proportion of reported energy was similarly calculated with substitution of energy for frequency and total energy for total number of reports. Foods were ranked by weighted frequency and contribution to energy individually, by food sub-category and broad category (selected data shown in tables). The Rao-Scott modified chi-square determined significant differences among food secure, low, and very low food secure groups (p < 0.05/3 or p < 0.02 using a Bonferroni adjustment for multiple comparisons to mitigate the probability of Type 1 error) and among food secure and insecure groups (p < 0.05). The results of significant differences among broad food groups were used to focus presentation of the results and discussion. All analyses were completed in SAS version 9.4 using SAS survey procedures with adjustment for survey design elements, non-response, and interview weights to allow inference to U.S. population.The data of food secure and food insecure children, including low and very low food secure categories were stratified by ages: 6–11 and 12–17 years because of similar diets within age ranges, food security reporting, known differences in food security by age in the same household and the NHANES methodology of self-reported dietary recall by age groups. Despite small participant dividual . The weip ≤ 0.0004, p = 0.001).Overall, ~90% of U.S. children and adolescents were food secure and 10% food insecure, with the smallest proportion being very low food secure (1–2%). Household poverty-income-ratio and race/ethnicity differed among 6–11 and 12–17 years by food security status  reported by frequency was statistically significantly greater for food insecure compared with secure children 6–11 years  and energy contribution (p = 0.02) was lower among food insecure compared with secure children of similar age (14.4% vs. 16.1% and 18.3% vs. 21.0%). Additional significant differences resulted among food secure, low and very low food secure groups (p = 0.02) for “snacks and sweets” .The weighted proportion of the broad category “beverages” . A greater contribution of energy from “beverages” (p = 0.03) was also determined for food insecure compared with secure (13.0% vs. 11.2%). Alternatively, significantly more frequent intake of “water” was observed among food secure (p = 0.004), compared with insecure and low and very low food secure groups .Compared with food secure adolescents 12–17 years (12.7%) “beverages” as a broad category were more statistically significantly frequently consumed by low (15.5%) and very low food secure (14.4%) and also combined food insecure groups  reports among food secure adolescents while accounting for 4.5% and 3.5% of low and very low food secure reports. “Bottled water”, however, was less frequently consumed among food secure and low food secure groups  compared with very low (5.2%) food secure reports. “Tap water” and “unsweetened bottled water” were the most frequently consumed items.Both the frequency and amount of food and beverage intake are important behavioral exposures characterizing dietary intake. Frequency data permits consideration of the most commonly consumed foods while amount shows the “dose”. In this analysis, U.S. children and adolescents had similar frequency of consumption of food categories regardless of food security with the exception of beverage and snack categories. Frequency alone is often overlooked as a component of dietary patterns among children and only two studies are known among adults ,24. TradFrequency differences in “beverages” and “snacks and sweets” by food security status supports a previous summary of the literature for children 6–11 years for higher added sugar intake , and are“Soft drinks” were the highest ranking items in the “beverages” category among older food insecure children with caffeinated soft drinks ranking prominently. Older, prevalently low-income children may be working and contributing to family income  and usinLess frequency and energy contributions of “snacks and sweets” among food insecure compared with secure may represent a relatively more healthful dietary pattern among food insecure 6–11-year-old children. “Snacks and sweets” may be viewed as non-essential foods where budget may be conserved and include high-energy, high-sodium foods ,35. As sFrequently consumed foods and beverages among children and adolescents may be used to inform opportunities to promote available and familiar foods that are sources of the nutrients or dietary components that are lacking ,27, and While applications of stealth nutrition may help, the overall poor dietary intake of U.S. children regardless of food security status demand more dramatic changes to improve dietary selection. Primary care contact or public health education among youth provide an ideal environment for education and discussion of dietary habits and suggestions for substitution of soft drinks, for example, with water or low-fat dairy and promotion of fruits and vegetables. Dietary recommendations for food groups and categories may be further translated to specify frequently consumed foods comprising groups recommended for increase or decrease. Federal nutrition assistance programs such as Supplemental Nutrition Assistance Program; Special Supplemental Nutrition Assistance Program for Women, Infants, and Children; and the National School Lunch and Breakfast Programs may similarly apply knowledge of the frequently consumed foods to tailor education and menu components to the 59% of 2016 U.S. food insecure households participating . The NatDietary intake and reporting are reliant on memory and prone to error . Much leAmong children and adolescents 6–17 years old, similar foods ranked among those frequently consumed. However, frequency-differentiated intake patterns exist for beverages and snack foods by food security across age groups. The main findings reported in this paper may inform dietary recommendations, development of population-specific dietary assessment tools, interventions, menus, and the composition of food packages, and food policy for food insecure children, and adolescents.


The device achieves an areal capacitance of 196.5 mF cm-2 with high cycling stability. The flexible properties of PVA, the conductivity of CNT, and the pseudo-capacitance of PANI contribute to the superior performance. Present work develops a facile and effective way for preparing flexible electrode materials.The flexible and rechargeable energy storage device with excellent performance is highly desired due to the demands of portable and wearable devices. Herein, by integrating the bendability and stretchability of Polyvinyl alcohol (PVA), pseudocapacitance of Polyaniline (PANI), and the charge transport ability of carbon nanotubes (CNTs), PVA/CNT/PANI flexible film was fabricated as supercapacitor electrodes with excellent electrochemical performance and flexibility. Full-solid supercapacitor is prepared based on PVA/HIn present work, we fabricated PVA/CNT/PANI flexible film as supercapacitor electrodes with excellent electrochemical performance and flexibility. A facile and economic strategy was developed to prepare PVA/CNT/PANI flexible film as supercapacitor electrodes.−2 with high cycling stability.The device achieves an areal capacitance of 196.5 mF cmThe flexibility of PVA, the conductivity of CNT, and the pseudo-capacitance of PANI are contributed to the superior performance.The portable and wearable electronic devices are receiving great attention in recent years , 2, whicCarbon materials, conductive polymers, and metal oxides or their composites are most generally used electrode materials of supercapacitors –19. CarbFor pseudocapacitive materials, conductive polymers have attracted great attention due to their large theoretical capacitance, better capacity retention, low toxicity, and eco-friend –26. The −2. There is still 71.4% capacitance retention rate after 5000 cycles, which exhibited excellent cycling stability. Present work provided high universality to develop high strain polymeric materials with excellent electrochemical properties.In present work, PVA/CNT/PANI flexible film was prepared as supercapacitor electrodes. CNTs are used as a charge transfer pathway to enhance the conductivity of polymer and double-layer capacitance. PANI polymer provides pseudocapacitance, and the PVA matrix provides bendable and stretchable ability. Flexible solid symmetric supercapacitor was assembled by PVA/CNT/PANI film as active electrodes, and achieves capacitance of 196.5 mF cmCNT was supplied from Aladdin Reagent Co., Ltd. PVA, ammonium persulfate (APS), aniline (ANI), ethyl alcohol, hydrochloric acid, sulfuric acid (98%), and nitric acid (96%) were purchased from Aladdin.3 and H2SO4 in volume ratio of 1:3 under sonication. Then, it was heated to 90 °C for 120 min. The acidulated CNT was washed with deionized water and dried at 80 °C for 6 h. PVA/CNT film was fabricated using the following sententious process.The dispersion of CNTs in the solution is very poor, and there will be an obvious agglomeration phenomenon. Using strong acid oxidation and ultrasonic treatment can improve the dispersion of CNTs in solution. Typically, 500 mg CNT was added into the 40-ml mixed solution of HNOAs shown in Fig. 2SO4 gel was used as electrolyte. A 10-ml aqueous solution including 1 g PVA was prepared under vigorous stirring, which is followed by addition of 0.56 ml of H2SO4. The acquired PVA/H2SO4 gel is agreed to cool. The as-prepared PVA/CNT/PANI films were applied on both sides of PVA/H2SO4 gel to complete assembly of a typical supercapacitor with carbon cloth as the current collector.The PVA/HThe acquired samples and devices are investigated by field emission scanning electron microscopy , Raman spectra and Fourier transform infrared (FTIR) spectra . The electrochemical impedance spectroscopy (EIS), galvanostatic charge-discharge (GCD), and cyclic voltammetry (CV) were characterized by CHI660E electrochemical workstation (Shanghai Chen-hua instrument co. LTD).As shown in Fig. After covering PANI on PVA/CNT film, different surface morphology could be observed with complicated microstructure as shown in Fig. −1 correspond to disordered carbon and ordered graphitic sp2 carbon, respectively. For PVA, the obvious peak is much less, and the peak around 2847 cm−1 is attributed to -CH2 [−1 is originated from vibration of the C-H bond, and two peaks at 1411 and 1582 cm−1 could be contributed by C-C [−1 deviated from the peak position of PVA, which may be due to the overlap of PANI on PVA. Figure −1 results from C-N bond. A series of peaks in the range of 1430-1530 cm−1 is due to C=C stretching vibration [−1. These results indicate the successful preparation of desired composite films. The different polymerization time induces different film morphology and loading amount of PANI, so the electrochemical performance of devices based on these films are further investigated.Figure  to -CH2 . For PVAd by C-C . The pea−1 is 1016.8mF cm−2, and that of 6 h and 12 h are 906 mF cm−2 and 881.3 mF cm−2, respectively. The results indicate that the 9-h sample shows the best performance which is consistent with the observation of SEM that the cracks among flowers provide enough space for wetting between active materials and electrolyte, and thus could improve the reaction and storage ability of electrons.The electrochemical properties of the PVA/CNT/PANI samples were evaluated by the three-electrode test. The composite films prepared by cutting in situ polymerization sample were used as a working electrode, and the CV curves were measured. Figure −2) at a scanning speed of 5 mV s−1. For the flexible PVA/CNT/PANI symmetrical supercapacitor, the capacitance performance is also tested by GCD in Fig. The working status of PVA/CNT/PANI films as electrodes in a flexible supercapacitor is further investigated and a full-solid supercapacitor is fabricated as described in the experimental section using a 9-h PVA/CNT/PANI film. The device has a typical sandwich structure. As seen in Fig. −2 and after 5000 cycles, the capacitance retention rate reached 71.4%, showing good cyclic stability. The flexible properties of PVA, the conductivity of CNT, and the pseudo-capacitance of PANI contribute to the superior performance. Present work provided a simple but efficient method for the preparation of flexible electrode materials.In summary, PVA/CNT/PANI film was prepared by a facile method. Due to the synergistic effect of PVA, CNT, and PANI, the obtained films have good flexibility, bendability, and electrochemical properties. The area capacitance reached 196.5mF cm


Indonesia has high mosquito diversity, with circulating malaria and arboviruses. Human landing catches (HLC) are ethically questionable where arboviral transmission occurs. The host decoy trap (HDT) is an exposure-free alternative outdoor sampling device. To determine HDT efficacy for local culicids, and to characterize local mosquito fauna, the trapping efficacy of the HDT was compared to that of HLCs in one peri-urban (Lakkang) and one rural (Pucak) village in Sulawesi, Indonesia.Anopheles per night (n = 22 ± 9) than the HDT (n = 3 ± 1), while the HDT collected a significantly greater nightly average of Culex mosquitoes (n = 110 ± 42), than the outdoor HLC (n = 15.1 ± 6.0). In Pucak, there was no significant difference in Anopheles collected between trap types; however, the HDT collected significantly more Culex mosquitoes than the outdoor HLC nightly average (n = 53 ± 11 vs 14 ± 3). Significantly higher proportions of blood-fed mosquitoes were found in outdoor HLC (n = 15 ± 2%) compared to HDT (n = 2 ± 0%). More blood-fed culicines were collected with outdoor HLC compared to the HDT, while Anopheles blood-fed proportions did not differ. For the HDT, 52.6%, 36.8% and 10.5% of identified blood meals were on cow, human, and dog, respectively. Identified blood meals for outdoor HLCs were 91.9% human, 6.3% cow, and 0.9% each dog and cat. Mosquitoes from Pucak were tested for arboviruses, with one Culex pool and one Armigeres pool positive for flavivirus, and one Anopheles pool positive for alphavirus.In Lakkang the outdoor HLCs collected significantly more Anopheles specimens. Although the HDT can attract a range of different Asian mosquito genera and species, it remains to be optimized for Anopheles in Asia. The high proportion of human blood meals in mosquitoes collected by outdoor HLCs raises concerns on the potential exposure risk to collectors using this methodology and highlights the importance of continuing to optimize a host-mimic trap such as the HDT.The HDT collected the highest abundance of culicine specimens. Outdoor HLCs collected the highest abundance of  Anopheles diversity, with 21 previously confirmed malaria vector species and species complexes [Aedes aegypti and Aedes albopictus, the primary vectors of several important arboviruses, including dengue and chikungunya virus [Aedes species are present in Indonesia and many are capable of transmitting the causative agents of rift valley fever and filariasis [Culex are ubiquitous throughout much of Indonesia, with many identified as the vectors of filarial parasites, as well as a number of lesser known arboviruses [The Indonesian archipelago, located along the equator, is made up of over 17,000 islands , 3. Thisomplexes –8. Also ya virus –12. Numelariasis –20. Furtoviruses , 21–27.The Republic of Indonesia has the fourth largest population in the world and half of that population live in malaria endemic regions , 29. Disversus those conducting HLCs, as well as the positive impacts of prophylaxis [Although HLCs are the sampling method most indicative of vectors biting hosts, they have come under scrutiny due to ethical concerns around exposing collectors to infectious bites . The WHOphylaxis , 36, thephylaxis , 38.Anopheles coluzzii, a typically indoor-feeding vector, at a ratio of nearly ten to one compared to outdoor human landing catches [Anopheles gambiae (sensu lato) species complex, this tool has not been evaluated as a sampling method in Asia, where it could provide malaria and arboviral endemic regions with a human exposure-free sampling device for entomological investigations [The host decoy trap (HDT) represents an exposure-free and passive sampling device suitable for outdoor use that attempts to artificially recreate the stimuli that mosquitoes use when host-seeking and feeding . By mimi catches . It was  catches . More re catches . The stu catches . Howeverigations , 40.To the best of our knowledge, this study is the first evaluation of the human decoy trap outside of Africa, representing the first field test of the behavioural principles employed in the design of the HDT on Asian (Indonesian) mosquito fauna. Due to its unique geological history and stable year-round climate, Sulawesi, the largest island of Wallacea, has particularly elevated levels of species richness and endemism –43 and w2 and has a population of approximately 1200 inhabitants, primarily engaged in rice farming and fishing. Typical dwellings here are based on an open concept two-story wood and plaster construction with metal roof and open-air access to outdoors much of the day and evening. Lakkang is considered to be malaria receptive. Dengue is diagnosed by platelet count and/or symptoms due to lack of suitable infrastructure .Located in Tallo, Makassar Regency of South Sulawesi, Lakkang Village is 3.9 km from Makassar city center. However, this peri-urban village is surrounded by rivers, making the isolated location distinct from its large neighboring city Fig. a–c. The 2 with a population of approximately 500 inhabitants and the main local economic activity is corn and rice farming. Here, the majority of housing consists of a one-room wooden construction on stilts, with permanent openings to the outside (open windows and doors and open-structured floor panels). Human excursions into the surrounding forested area represent points of contact between wildlife and humans. Pucak is considered to be malaria receptive. There are no data for dengue cases in Pucak.Pucak Village is situated in Tompu Bulu, Maros Regency, South Sulawesi Province Fig. a, b, d. www.kestrelinstruments.com).In both Lakkang and Pucak, collections were performed between April and May 2017, during the rainy season, for 2 weeks, on 4 consecutive nights each week . The HDT comprised a black cylinder, the surface of which was maintained at a temperature of 30–40 °C via hot water and wrapped in an adhesive plastic sheet . Mosquitoes are attracted to the host odors from the tent and induced to land by the visual and thermal properties of the HDT; they land and are trapped on the adhesive surface. At the end of every collection period, the adhesive sheet was removed, and trapped mosquitoes were extracted using glue board solvent , with only very infrequent loss of body parts. Collections with HDT were performed at the outdoor collection sites only. HDT collections were done in 4 h shifts (18:00–00:00 h), with one collector inside the HDT tent per night. The single collector inside the HDT tent would rotate randomly on consecutive nights to remove collector bias.HDTs were produced by Biogents AG  based on previously described research  and prepCollection village, site, date, collector identity, hour, genus, sex, and abdominal status were recorded for each mosquito collected by the HLC method. For each mosquito collected using the HDT, all the above data were recorded except for hour of collection. Mosquito morphological identifications were based on multiple taxonomic keys based on genera , 45. ForAnopheles mosquitoes (n = 392) were sequenced at the mitochondrial DNA cytochrome c oxidase subunit 1 (cox1) locus for species identification. DNA was extracted from individual specimens using a modified version of the simple alkaline method by Rudbeck & Dissing [2O was added and the tubes inverted 10 times. DNA was suspended in a final volume of 1000 µl containing 0.016 M NaOH and 0.029 M Tris-HCl, pH 8.0. The cox1 gene was amplified using LCO and HCO primers [2, 200 µM dNTPs, 5 pmol of each primer, 1 unit of Taq DNA polymerase, and 1 µl of genomic DNA as prepared above. Amplification was performed in 25 µl volumes in 96-well PCR plates  in a Mastercycler Nexus thermocycler . Thermocycling conditions for cox1 were as in Lobo et al. [All  Dissing . Individ primers , 48. Eaco et al. . PCR proo et al.  and St. www.dnastar.com) was used to align cox1 sequences. Using single nucleotide polymorphisms (SNPs), contigs were divided into sub-contigs. Consensus sequences were manually inspected for insertions, deletions, and repeat regions to ensure these sequence differences did not inflate divergence and decrease identity scores. cox1 sequences were assembled into final species identities using 95% identity, as mitochondrial lineages are more apt to diverge within a species [cox1 contigs to the databases [Starting with a minimum match of 95%, the Seqman Pro Assembler  and H2714 (5′-CTC CAT AGG GTC TTC TCG TCT T-3′)  amplification of the mitochondrial  bp; see ); as wel bp; see ). PCR co bp; see  and St L bp; see .n = 665 out of 672) from Pucak were tested for arboviruses. Individual specimens were pooled in groups of up to 30 based on genus to maximize efficiency: Aedes, Anopheles, Armigeres and Culex. A standard TRIzol RNA extraction as described by the manufacturer (Invitrogen) was used to isolate viral RNA. Extracted RNA was stored at − 20 °C until further testing. After RNA extraction, a reverse-transcriptase PCR was performed following the protocol for SuperScript III one-step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) for all specimens using previously described primers: Flavivirus F (5′-TAC AAC ATG ATG GGA AAG AGA GAG AA-3′) and Flavivirus R (5′-GTG TCC CAG CCG GCG GTG TCA TCA GC-3′); Alphavirus F (5′-(CT)AG AGC (AGT)TT TTC GCA (CT)(GC)T (AG)GC (ACT) (AT)-3′) and Alphavirus R (5′-ACA T(AG)A AN(GT) GNG TNG T(AG)T C(AG)A ANC C(AGT)A (CT)CC-3′); and Bunyavirus F (5’-CTG CTA ACA CCA GCA GTA CTT TTG AC-3′) and Bunyavirus R (5′-TGG AGG GTA AGA CCA TCG TCA GGA ACT G-3′) [Heads and thoraxes of mosquitoes (CT G-3′) –57.MASS [post-hoc Tukey comparisons between collection methods  and so was not included as a factor in subsequent GLMs. HLC collectors working in pairs created a larger plume of CO2 and other olfactory cues, likely impacting mosquito numbers considerably [Anopheles specimens was too low to examine biting activity in either Lakkang or Pucak individually.Statistical analyses were completed in R version 3.5.2 . Catch aMASS ) with nemultcomp ). When siderably . TherefoA total of 2292 mosquitoes were collected in the study. Lakkang collections produced 1620 specimens, and Pucak 672. Across both study sites, the HDT collected 1361 mosquitoes, while the outdoor HLC collected 864 and the indoor HLC collected 67. Overall mean nightly catches were 85.1 ± 19.68 for the HDT, 27.3 ± 6.40 for the outdoor HLCs and 2.9 ± 0.88 for the indoor HLCs.cox1 sequences of 382 Anopheles mosquito specimens were aligned into nine sequence groups with a stringency of greater than 95% identity within each group , An. nitidus/An. letifer, and An. nigerrimus/An. letifer, respectively (Table The Anopheles mosquitoes (n = 373) were collected. Anopheles barbirostris made up the majority of the collection , with smaller proportions of An. epiroticus/An. sundaicus (s.l.) , An. epiroticus , An. nitidus/An. letifer , An. nigerrimus/An. letifer , and < 1 of both An. peditaeniatus  and An. vagus . Three specimens were morphologically misidentified as Anopheles and consequently molecularly verified as Cx. bitaeniorhynchus and Ae. vexans. Outdoor HLC collected all seven species of Anopheles mosquitoes in Lakkang as well as the single Ae. vexans specimen. Indoor HLC and HDT collected three of these species of anophelines: An. barbirostris, An. epiroticus/An. sundaicus (s.l.), and An. epiroticus.In Lakkang, 7 species of Anopheles mosquitoes (n = 6) were collected, An. barbirostris (n = 5) and An. vagus (n = 1). Both of these species were collected using outdoor HLC. Two morphologically identified Anopheles specimens were molecularly identified as Cx. bitaeniorhynchus. These Culex specimens were collected with HDT and indoor HLC.In Pucak, two species of Anopheles were collected by the outdoor HLC, while the HDT collected a greater abundance of Culex mosquitoes. For Anopheles, the outdoor HLCs collected significantly more mosquitoes per night  = 23.275, P < 0.0001) than the HDT (3.0 ± 1.3), representing a seven-fold difference  = 8.1756, P = 0.004) .Mosquito abundance in Lakkang differed by trap type; more Culex collections compared to the HLC. For Anopheles, the HDT collected a nightly mean of 0.1 ± 0.1 mosquitoes and there was no significant difference in this compared to outdoor HLC, which caught a mean of 0.6 ± 0.3 per night  = 2.9109, P = 0.088)  = 17.137, P < 0.0001) .Mosquito abundance in Pucak also differed by trap type; the HDT again collected a greater abundance of Anopheles specimens were collected with outdoor HLCs, 85.7% , while outdoor HLCs and indoor HLCs collected 19.8% and 1.9%, respectively  and Pucak (99.5%), with 2.6% Anopheles, 2.7% Aedes, and 0.6% Mansonia in Lakkang and 0.2% Anopheles and 0.2% Armigeris in Pucak  but accounted for only 3.1% of the catch in Pucak  compared to Pucak HDT (99.5%). Indoor HLCs did not collect any Armigeris or Mansonia mosquitoes. There were similar proportions of Anopheles (20.9%) and Culex (72.1%) in Lakkang, but indoor HLC had a higher proportion of Culex (83.3%) compared to Anopheles and Aedes in Pucak  . Whereas, Anopheles blood-fed proportions between the HDT (1/25) and outdoor HLC (18/178) were not significantly different .Outdoor HLCs collected a significantly higher proportion of blood-fed mosquitoes (66/433) compared to the HDT (23/1360), 0.15 ± 0.02 and 0.02 ± 0.0 (n = 163) were tested for blood meal by PCR to identify the host source (Table n = 19), 52.6%, 36.8% and 10.5% of identified blood meals were on cows, humans and dogs respectively. For mosquitoes collected with the HDT, a single Aedes and a single Anopheles specimen fed on cows, while the only blood-fed Mansonia mosquito fed on humans . Identified blood meals for outdoor HLCs (n = 111) were 91.9% human, 6.3% cow, and 0.9% each dog and cat. Aedes mosquitoes collected with outdoor HLC had fed on humans and cows , as had Anopheles mosquitoes . Culex mosquitoes collected with outdoor HLC fed on humans, cows, dogs, and cats  respectively (Table Mansonia mosquitoes collected with outdoor HLC only fed on humans (n = 5). Of the Anopheles species that were molecularly identified, 39 had identified blood meals. Anopheles barbirostris (29/39) blood-fed on humans and cows (n = 26 and n = 3), respectively. Anopheles epiroticus/An. sundaicus (s.l.) (5/39), An. epiroticus (3/39), An. nitidus/An. letifer (1/39) and An. peditaeniatus (1/39) all blood-fed on humans.Engorged females n = 16 were tesCulex pool positive for flavivirus, one Anopheles pool positive for alphavirus, and one Armigeres pool positive for flavivirus.Mosquitoes from Pucak tested for arboviruses indicated one Anopheles peaked between 18:00–19:00 h with smaller subsequent peaks between 20:00–21:00 h and 22:00–23:00 h  in Lakkang were 27.5 °C and 80.8% RH, and in Pucak were 26.4 °C and 85.6% RH . As the HDT was effective at sampling culicines (both in comparison to HLC within this study and relative to previous results), it therefore seems likely that the difference in catch stems from fundamental differences between Indonesian and African anopheline behavior. This challenges the notion that ‘universal’ tools for sampling or controlling diverse secondary malaria vectors will be effective, implying instead that tailored solutions, adapted to the specific behavior and ecology of local vectors, will be needed. Such tools will require empirical study of these traits in less well understood vector species to provide the basis for locally effective innovations.The difference in collection performance may be due to the specific behavioral traits of local anopheline species. Based on our results, host-seeking behaviors in Indonesian anophelines are presumably sufficiently different to those of the Gambiae Complex species to lead to differential responses to the host-associated cues used in the HDT. Inclusion and configuration of these cues in trap design has been based on both laboratory behavioral assays and subsequent field-based proof of concept studies  that havAn. vagus, An. nitidus, An. peditaeniatus and the Barbirostris group. In Thailand, An. epiroticus displayed exophagic and zoophilic preferences and the majority of these species were collected in buffalo traps compared to HLC [An. nigerrimus and An. vagus. Additionally, the human blood index calculated for An. barbirostris was 0.27, indicating lower human blood meal preference compared to other vectors in the region [St. Laurent et al.  identifid to HLC . A studyd to HLC  did not e region . FurtherCulex, Mansonia and Armigeres, collecting significantly more Culex mosquitoes and similar amounts of other genera. Existing sampling methods for these groups have been tested and found to be effective in the Southeast Asian region, including CDC light traps [The HDT, as configured in this study, appears to be effective for collecting all other mosquito genera, ht traps  and tentht traps . These tDespite their importance in transmitting filariasis , 68, JapAnopheles vectors that feed on humans, the method is still limited in how closely it can mirror outdoor HLCs. Although the risks of utilizing willing, trained volunteers providing informed consent to perform HLCs are widely accepted due to the otherwise impossible to approximate epidemiological risk factors, research demonstrating configurations of the HDT that more closely mirror HLC collections could potentially eliminate this risk. Although this study failed to demonstrate the ability for the HDT to mimic HLCs, it does demonstrate some of the risk involved; outdoor HLCs collected more blood-fed mosquitoes than HDTs in both Lakkang and Pucak, the majority of this difference is based almost entirely on human blood-fed mosquitoes, which comprised 91.9% of outdoor HLC blood-fed mosquitoes. Since the HDT protects collectors from mosquito bites and only uses their odors to attract mosquitoes, a possible reason for the majority of human blood meals  were mosquitoes that fed on HLC collectors. This discrepancy could also be due to the different mosquito compositions collected. Further studies, involving matching DNA from blood-fed mosquitoes to collectors could establish the strength of the relationship between HLCs and collector exposure through comparison with HDT blood-fed rates. Given HLCs accounted for a disproportionately high number of blood-fed mosquitoes, their use in entomological studies when risk of arboviral transmission is present should be avoided where possible.Inarguably, outdoor HLCs are optimized for human host-seeking anophelines, as supported by the results of this study and previous literature . AlthougCulex and one Armigeres while one Anopheles pool tested positive for alphavirus, highlighting the potential risk to collectors. While there are no reports of any arboviruses in circulation in human populations in Pucak, local diagnostic capacity may limit accurate detection and reporting of these. Viral transmission may be occurring since viruses are present in the mosquito vectors.In this study, two pools of mosquitoes tested positive for flavivirus, one Anopheles species in a region without previous collection. Of the nine molecularly identified consensus sequences, six could be identified to species: An. barbirostris; An. epiroticus; An. peditaeniatus; An. vagus; Cx. bitaeniorhynchus; and Ae. vexans. Of the four molecularly identified Anopheles, three are confirmed malaria vectors in Indonesia: An. barbirostris; An. epiroticus; and An. vagus [Anopheles peditaeniatus has not been confirmed as a malaria vector in Indonesia; however, it has been shown positive for Plasmodium falciparum by ELISA in Sri Lanka [Anopheles but molecularly confirmed to be Cx. bitaeniorhynchus and Ae. vexans. Culex bitaeniorhynchus is a vector of Wuchereria bancrofti, Murray Valley encephalitis virus, Japanese encephalitis virus, and Batai virus [Ae. vexans is capable of transmitting Eastern equine encephalitis virus, Western equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, and Japanese encephalitis [Based on the HLC results, this study was able to characterize n. vagus , 77–80. ri Lanka  and Thairi Lanka . The preai virus –86, whilphalitis –89. ThisAn. nitidus; however, the BOLD database search identified these Anopheles specimens as An. letifer. The discrepancy in the BLASTn and BOLD database sequence results coupled with the presence of cox1 sequences for An. letifer in BLASTn and An. nitidus in BOLD, and lack of associated sequences in databases could indicate an addition to the Hyrcanus or Umbrosus Group. However, it is far more likely that distributions and phylogenetic relationships between species in these two groups may vary due to the wide geographical distribution of sequences available in databases, complicating interpretation. For example, the An. letifer sequences in GenBank are from Singapore [An. nitidus sequences in BOLD are from Thailand. Similar results were seen for group 5. Therefore, additional research that implements nuclear and mtDNA sequencing within Indonesia is necessary to accurately identify species that are malaria and arboviral vectors and to create baseline information for nucleotide databases for Sulawesi specifically.While molecular tools can disambiguate vector identities, they must be based on comprehensive libraries of reference genetic material. In our study, three consensus sequences had conflicting BLASTn and BOLD database results: group 2, 4, and 5. Group 2 belongs to the Sundaicus Complex, a main malaria vector in Indonesia . Group 4ingapore , while tAnopheles vector populations were composed of up to seven species, consisting of predominantly An. barbirostris. Seven different Anopheles species were identified in Lakkang, whereas only two Anopheles species were found in Pucak. The diversity in mosquito vectors in two 1 km2 villages further illustrates the biodiversity of the Indonesian archipelago and its ability to host a variety of vectors. Appropriate innovations in surveillance and control, especially in regions of high biodiversity with heterogenous vector ecology and behavior, are undoubtedly a challenge [Local hallenge , 91.Aedes, Anopheles and Mansonia were found in higher proportions in Lakkang, while Culex and Armigeres were found in higher proportions in Pucak. These differences are attributed to the presence of available habitats preferred by species of these genera. Lakkang is surrounded on all sides by the Tallo River and the main economic activity is rice farming. Due to its riverine location and predominance of rice paddy land cover, sunlit water bodies are readily available, making the surrounding area an excellent habitat for many Anopheles species, including the three main species found in this study, namely An. barbirostris, An. epiroticus and An. sundaicus (s.l.) [Culex larvae [Culex preference for rain-fed rice fields [Culex specimens relative to Anopheles specimens at these study sites. However, this could also be a result of zoophilic Anopheles being present at the sites that were not targeted by the trapping regimen in this study. Lastly, previous studies have shown an increase in the host-seeking behaviors of An. gambiae (s.s.) and an increase in Mansonia mosquito populations during periods of rising relative humidity [Culex larvae could explain the elevated numbers of Culex mosquitoes collected in Pucak, however future studies would need to further investigate this relationship.Genera composition varied between Lakkang and Pucak. s (s.l.) –94. Wherx larvae . More gee fields . The richumidity , 98. TheIndoor and outdoor mosquito biting profiles were compared with HLC. Indoor HLCs collected low proportions of all mosquito genera in both villages compared to outdoor HLCs, despite the predominantly open housing structures found in both study villages. Given there are limited physical barriers to impede mosquito ingress into dwellings, these low numbers indicate mosquito biting is preferentially occurring outdoors, suggesting implementation of outdoor interventions may be most effective in both Lakkang and Pucak, Indonesia, if disease transmission is confirmed in this susceptible region.Anopheles mosquitoes peaked in activity between 18:00–19:00 h. This corroborates previous reports of An. barbirostris, the predominate molecularly identified vector in this study, that indicate An. barbirostris feeds outdoors in the early evening [An. barbirostris are dependent on geographical location in Indonesia [Culex mosquitoes in Lakkang or Pucak. In both villages, Culex activity increased during the second half of the night peaking at 22:00–23:00 h in Lakkang and continuing to rise up to the end of collections at 00:00 h in Pucak. Although collections in this study represent a fourth of the daily timeline, it aligns with other field studies from India and Thailand that have reported peaks in Cx. quinquefasciatus flight activity around 22:00 h [Cx. pipiens was found to increase until 22:00 h [Culex biting activity documentation in Indonesia stresses the importance of continued recording of mosquito biting times so accurate local transmission risks can be determined.Biting activity is an important factor for determining the behaviors and possible transmission times for vector-borne diseases.  evening . Howeverndonesia . There h 22:00 h , 101. In 22:00 h . A limitIdentifying and evaluating species compositions and behaviors in Indonesia will require continued interest and investment from researchers. The region’s high biodiversity and archipelagic geography make efforts to comprehensively survey the islands either prohibitively expensive or piecemeal and individualized to localities. Furthermore, behavior of local vector fauna differs markedly to the relatively well-studied African vectors. Combining longitudinal data sets for trap types and comparison studies will yield better understanding for ethical trapping and effective intervention practices as the world continues to pursue the elimination of malarial and arboviral transmission.Although the HDT collected the highest density of mosquitoes in this study, this was entirely due to culicine specimens, which were not evaluated at the species level. Outdoor HLCs collected the highest abundance of anopheline specimens. Therefore, although the stimuli presented in the HDT can be employed to sample a range of different Asian mosquito genera and species, it remains to be optimized for specific host-seeking behaviors in Asia. The high proportion of human blood meals from mosquitoes collected by outdoor HLCs reinforces what is known about the exposure risk to collectors for the methodology and the importance of continuing to optimize a host-mimic trap such as the HDT. Further study involving matching DNA from blood-fed mosquitoes to collectors could establish the strength of the relationship between HLCs and collector exposure, especially through comparison with mosquito blood-meals of a host-mimic trap, such as an optimized HDT. The HDT could also be evaluated as a potential outdoor intervention strategy if the high capture rates can be demonstrated to impact overall mosquito populations within a locality.