Morphological Identification Research Articles

Evaluation of NGS DNA barcoding for biosecurity diagnostic applications: case study from banana freckle incursion in Australia

AbstractMolecular diagnostics in combination with morphological identification is the method of choice for several cryptic microbial plant pathogens. For some diagnostic applications, traditional sequencing techniques can be time consuming, making them ill-suited for biosecurity incursion responses, where accurate results are needed in real time. More rapid next generation sequencing tools must be tested and compared with traditional methods to assess their utility in biosecurity applications. Here utilizing 95 samples infected with fungal pathogen Phyllosticta cavendishii, from a recent incursion in Australia, we compare species identification success using Internal Transcribed Spacer (ITS) gene barcode on conventional Sanger and Oxford Nanopore MinION sequencing platforms. For Sanger sequencing, the average pairwise identity percentage score between generated consensus sequences and P. cavendishii sequence from holotype material on NCBI database was 99.9% ± SE 0.0 whereas for MinION sequencing the average pairwise identity percentage was 99.1% ± SE 0.1. Relatively larger consensus sequences (mean 486 bp ± SE 2.4) were generated by Sanger sequencing compared to MinION sequencing (mean 435 bp ± SE 4.6). Our results confirm that both sequencing methods can reliably identify P. cavendishii. MinION sequencing, provided quicker results compared to Sanger sequencing and demonstrated diagnostic competence, with the added advantage of being portable, for front-line “point of incursion” biosecurity applications.

Genetic divergences provide evidence to accept a new Mediterranean genus Antalia (Apiaceae) and insights into allopatric divergence extended to the Pliocene

Abstract Narrow endemism is the core of plant diversity in flora of the Mediterranean Basin, which is among the world’s 36 biodiversity hotspots. The Mediterranean genus Kundmannia (Careae, Apioideae) comprises just three species, K. sicula, K. anatolica, and K. syriaca. Notably, K. sicula has a wide distribution from the western Mediterranean to Greece, while K. anatolica and K. syriaca occur only in Turkey’s Taurus Mountains, which is a phylogeographical hotspot. Initially, the aim herein was to determine the phylogenetic placement of the genus Kundmannia based on DNA sequences, but it later developed to describing a new genus, Antalia gen. nov., and revealing historical events that acted as drivers in the genetic divergence between Kundmannia and Antalia. Both ribosomal and combined chloroplast multilocus sequences were used to unravel the phylogenetic topology. Toward this aim, morphological characteristics were examined. Based on this, Antalia differs from Kundmannia by its shorter bracteoles and petals, the colour of the petals, and smaller fruits. The phylogenetic results revealed that the genus Kundmannia is nested within the tribe Careae and is not monophyletic. Antalia, which is genetically separate from Kundmannia, is described as a new genus. Detailed morphological descriptions and identification keys are provided. Causes of the genetic disjunction between Antalia and Kundmannia were carefully considered in a time-calibrated phylogenetic framework. The Mediterranean Sea has acted as a relatively effective barrier between these genera since the Messinian salinity crisis. The unwinged mericarps seem to have facilitated the work of the barrier. Historical events during and after the Pliocene and allopatric vicariance seem to have been the most important drivers of evolution between Kundmannia and Antalia and within Antalia.

Development of a multi-entry identification key for economically important fruit fly larvae (Diptera, Tephritidae, Dacinae)

Identification of fruit fly larvae is difficult due to the limited morphological characteristics present. However, this is the stage at which fruit flies are intercepted at ports of entry through horticultural imports. Molecular tools are useful but are time-consuming and expensive compared to morphological identifications. This project aims to use available information from the literature and our own research to build a multi-entry identification key for thirteen tephritid species and species groups that are of economic concern for the European Union. Third-instar larvae were obtained from different regions and hosts. Thirteen species or representatives of species groups were obtained, including Ceratitis, Dacus, Bactrocera and Zeugodacus spp. The cephalopharyngeal skeletons were dissected out, cleared in a 10% NaOH solution, dehydrated and mounted in Euparal on glass slides. Images of at least 20 larvae/species were captured using a compound microscope fitted with a camera. Measurements were taken of the mounted mandibles and the number of tubules and their position in the anterior spiracles in relation to the cephalic skeleton were noted. Differences between morphometric parameters were tested via ANOVA and verified using discriminant function analysis. A matrix was compiled including nine characters for which significant inter-specific differentiation was preliminarily detected. The key was converted into a mobile application by LucID.

In silico assessment of 18S rDNA metabarcoding markers for the characterization of nematode communities.

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.

Long-read datasets of Kayu Bawang (Azadirachta excelsa (Jack) Jacobs) from 3 different identified seed stands in Bengkulu (Sumatra), Indonesia and its phylogenetic relationships

Abstract* Azadirachta excelsa (Jack) Jacobs, Kayu bawang (Meliaceae) is economically valuable and widely used by the local community in Bengkulu (Sumatra) as carpentry and construction wood because of its good durability class. However, it still has ambiguous scientific multiple names, such as Azadirachta excelsa, Protium javanicum, and Dysoxylum mollissimum. Additional tools such as molecular approaches can be used to verify whether it is true or not that Kayu bawang is scientifically named as Azadirachta excelsa based on previous morphological identification. This study aimed to construct draft chloroplast genome and verify the scientific name based on molecular identification using a single rbcL gene marker. Genomic DNA was extracted from bark cambium originated from three different provenances in Bengkulu, Indonesia, namely TBT-A, TBT-K, and TBT-S. MinION from Oxford Nanopore Technologies was used to sequence the samples following manufacture protocols SQK-LSK109 yielding 481.6 Mb for TBT-A, 597.4 Mb for TBT-K, and 853.1 Mb for TBT-S, respectively. Generated data were assembled and constructed, namely 58,780 bp (14 tRNAs and 47 encoding genes) for TBT-A, 142,139 bp (4 rRNAs, 24 tRNAs, and 78 encoding genes) for TBT-K, and 84,906 bp (24 tRNAs and 53 encoding genes) for TBT-S. Based on the phylogenetic tree, Azadirachta excelsa from three identified tree stands were placed in the same group with other Azadirachta excelsa accessions.

Comparing modern identification methods for wild bees: Metabarcoding and image-based morphological taxonomic assignment.

With the decline of bee populations worldwide, studies determining current wild bee distributions and diversity are increasingly important. Wild bee identification is often completed by experienced taxonomists or by genetic analysis. The current study was designed to compare two methods of identification including: (1) morphological identification by experienced taxonomists using images of field-collected wild bees and (2) genetic analysis of composite bee legs (multiple taxa) using metabarcoding. Bees were collected from conservation grasslands in eastern Iowa in summer 2019 and identified to the lowest taxonomic unit using both methods. Sanger sequencing of individual wild bee legs was used as a positive control for metabarcoding. Morphological identification of bees using images resulted in 36 unique taxa among 22 genera, and >80% of Bombus specimens were identified to species. Metabarcoding was limited to genus-level assignments among 18 genera but resolved some morphologically similar genera. Metabarcoding did not consistently detect all genera in the composite samples, including kleptoparasitic bees. Sanger sequencing showed similar presence or absence detection results as metabarcoding but provided species-level identifications for cryptic species (i.e., Lasioglossum). Genus-specific detections were more frequent with morphological identification than metabarcoding, but certain genera such as Ceratina and Halictus were identified equally well with metabarcoding and morphology. Genera with proportionately less tissue in a composite sample were less likely to be detected using metabarcoding. Image-based methods were limited by image quality and visible morphological features, while genetic methods were limited by databases, primers, and amplification at target loci. This study shows how an image-based identification method compares with genetic techniques, and how in combination, the methods provide valuable genus- and species-level information for wild bees while preserving tissue for other analyses. These methods could be improved and transferred to a field setting to advance our understanding of wild bee distributions and to expedite conservation research.

Archaeological and molecular evidence for ancient chickens in Central Asia

The origins and dispersal of the chicken across the ancient world remains one of the most enigmatic questions regarding Eurasian domesticated animals. The lack of agreement concerning timing and centers of origin is due to issues with morphological identifications, a lack of direct dating, and poor preservation of thin, brittle bird bones. Here we show that chickens were widely raised across southern Central Asia from the fourth century BC through medieval periods, likely dispersing along the ancient Silk Road. We present archaeological and molecular evidence for the raising of chickens for egg production, based on material from 12 different archaeological sites spanning a millennium and a half. These eggshells were recovered in high abundance at all of these sites, suggesting that chickens may have been an important part of the overall diet and that chickens may have lost seasonal egg-laying

Mosquitoes in urban green spaces and cemeteries in northern Spain

BackgroundMosquitoes inhabiting urban green spaces and cemeteries in Europe represent a crucial facet of public health concern and contribute to the ecological balance. As urbanization intensifies, these areas increasingly serve as vital habitats for various mosquito species, fostering breeding grounds and increasing the risk of disease transmission.MethodsA study was conducted in the three main cities (inland, coastal, and estuarine) of the Basque Country, northern Spain, to investigate the species composition, abundance, dynamic populations, larval habitats, and host preferences of mosquitoes in urban green spaces and cemeteries. CDC traps and dipping were used to collect mosquitoes for 2 years (2019–2020).ResultsA total of 21 mosquito species were identified, with Culex pipiens s.l. being the most abundant and widespread. The three ecological forms of Cx. pipiens were found, and Cx. pipiens pipiens was the most common in both green areas and cemeteries. Morphological identification together with molecular tools identified 65 COI sequences with high homology. The highest species richness was found in the inland city, followed by the coastal city and the estuarine city. Mosquito abundance was significantly higher in green areas compared to cemeteries and in the coastal and estuarine cities compared to the inland city. The investigation of larval breeding sites highlighted the dominance of Cx. pipiens s.l., particularly in semi-artificial ponds, diverse water-holding containers (tyres and buckets) and drainage systems in green areas; in cemeteries, most of the larvae were found in flowerpots and funerary urns. Seasonal activity exhibited variable peaks in mosquito abundance in the different cities, with a notable increase in July or August. Additionally, blood meal analysis revealed that Cx. pipiens s.l. fed on several common urban avian species.ConclusionsStudies on mosquitoes are essential to understand their role in disease transmission and to design targeted and sustainable management strategies to mitigate the associated risks.Graphical

The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Molecular Identification of Endophytic Fungi Isolated from Bidara Root (Ziziphus mauritiana Lam.) Using Polymerase Chain Reaction (Pcr)

The roots of the bidara plant have the potential as antibacterial agents. The antibacterial compounds in the roots of the bidara plant originate from secondary metabolites produced by endophytic compounds known as endophytic fungi. The purpose of this research is to identify the types of endophytic fungi in bidara roots. The identification of microorganisms can be done through morphology or molecular methods. However, morphological identification alone is unable to depict the morphospecies to the phylogenetic level of a microorganism, thus molecular identification using the Polymerase Chain Reaction (PCR) method is required. This study utilized root isolates from the bidara plant with the sample code IFAZ-6, which, after sequencing, yielded a pair of base pairs measuring 558bp. The results of BLAST and phylogenetic analysis revealed that the IFAZ-6 isolate has a close relationship with the species Clonostachys rosea., which has been recorded in the database with a 100% identity level.

Evaluation of cold plasma activated water composite reagent on kiwifruit plants with bacterial canker and the external qualities of kiwifruit after spray treatment

This study aimed to investigate the efficacy of cold plasma activated water (PAW) and its complex solution (PKC) in conjunction with kasugamycin copper oxychloride wettable powder (KC) against kiwifruit bacterial canker (KBC) caused by Pseudomonas syringae pv. actinidiae (PSA). Through morphological identification and 16S rDNA sequence analysis, Pantoea agglomerans was identified as the pathogen responsible for KBC in the 'Bairui' orchard. Results indicated that PKC exhibited the highest effectiveness in controlling KBC, particularly on mildly (II) infested plants, with a remarkable reduction of 63.64% in PKC II disease index and a control efficiency of 38.64%. Additionally, the application of PKC significantly increased fruit yield, improved fruit weight, and enhanced fruit morphology, resulting in a more uniform and refined fruit shape. Analysis of fruit skin conformational parameters revealed that KBC-affected fruit exhibited reduced hardness and brittleness, increased toughness, and thinner skin. These findings are pivotal for developing effective strategies to further prevent and manage KBC outbreaks. In future studies, further investigations could focus on elucidating the mechanism of action of PAW in complex solution with copper-based pesticides and the potential mechanism of inactivation of PSA.

Hunting habits die hard: Conserved prey preferences in army ants across two distant neotropical rainforests

AbstractArmy ants are widely recognized as keystone species in neotropical rainforests due to their role as important arthropod predators. Their large‐scale raids involve thousands of workers scouring the forest floor in pursuit of prey, primarily capturing other invertebrates. Up to 20 species of army ants coexist in a rainforest, and dietary niche differentiation has been proposed as a mechanism to alleviate competition among them. Based on only a handful of study sites, however, our understanding of the precise dietary preferences and the extent of niche differentiation remains notably limited. In this study, we aimed to expand our knowledge of army ant communities by resolving an Ecuadorian predation network consisting of 244 prey species and 13 army ant species representing the five known neotropical army ant genera: Cheliomyrmex, Eciton, Labidus, Neivamyrmex, and Nomamyrmex. We collected 2156 prey items from 180 army ant raids/emigrations, and of these, we identified 1945 prey items to the family level, 1313 to the genus level, and 664 to the species level based on morphological identifications and DNA barcodes. Prey consisted primarily of other ants (1843 prey items; 153 ant species), to the largest part ant brood (N = 1726). Hence, most army ant species chiefly plundered the nests of other ants, while the three swarm raiding species, that is, Lab. praedator, Lab. spininodis, and Ec. burchellii, exhibited a relatively high proportion of non‐ant invertebrate prey in their diet. The predation network showed a high degree of specialization (H2′ = 0.65), characterized by little dietary niche overlap among sympatric species. We compared the Ecuadorian network with one previously studied in Costa Rica and found that, despite the large geographic distance, prey preferences remained remarkably similar. We discovered species‐specific preferences for captured ant genera and species, despite some species turnover in both army ants and prey. Additionally, army ants also exhibited consistent spatiotemporal raiding preferences across study sites. In conclusion, predation preferences within army ant communities exhibited consistency in multiple niche dimensions across two distant neotropical rainforests, suggesting a notable level of predictability within army ant predation networks.

Concordance between conventional cytology and cell block of suspected malignant pleural effusion: A record-based study in a tertiary care center of eastern India

Background: Cytological study of pleural fluid helps establishing the underlying cause of pleural effusion. It is considered the most important tool in diagnosing malignancy in effusions. However, accurate identification of cellular morphology can be a problem in conventional cytology. Cell block preparation from pleural effusion aids in this diagnosis due to the preservation of tissue architecture, and multiple sections can be obtained for further studies. Aims and Objectives: The objectives of this study were to compare the diagnostic results of conventional cytology and cell block studies of suspected malignant pleural effusions and find out the diagnostic agreement or concordance between the two methods. Materials and Methods: A record-based study was conducted at the Department of Pathology on conventional cytology and cell block study reports of 150 suspected malignant pleural effusion cases over 35 months. The diagnoses were expressed in five categories – non-diagnostic, negative for malignancy, atypia of undetermined significance, suspicious for malignancy, and malignant. χ2 test was used to identify the percentage of malignancy reported. Agreement of both methods was assessed through Kappa statistics. Results: About 4.67% of cases were reported as malignant by conventional cytology, whereas cell block study diagnosed malignancy in 19.33% of cases. Relative diagnostic agreement between the two methods was 0.73 and concordance by Cohen’s Kappa was 0.34, which signifies fair concordance. Conclusion: Cell block study, though having a fair concordance, is much superior to conventional cytology smears in detecting malignant cells and diagnosing a pleural effusion as malignant.

Design, synthesis, and applications of nucleic acid-specific benzoxazole-N,N-dialkylphenylamines derivatives for nucleolus imaging in the cells

Considered the brain of the nucleus, alterations in the structure and function of the nucleolus are linked to numerous cellular functions and, consequently, contribute to several diseases. The identification of nucleolar morphology and activity via novel biomarkers presents new avenues for the development of therapeutic approaches for a variety of human diseases, including cancer, neurodegeneration, and aging. Therefore, specific detection of the nucleolus with fluorescence probes is of critical importance for clinical applications. In the present study, a series of benzoxazole-N,N-dialkylphenylamines derivative compounds were designed and synthesized based on the benzothiazole-based fluorescence probe Thioflavin T (ThT). Among the compounds, BX-3 and BX-16, which carry electron-withdrawing substituents in the benzoxazole ring, were observed to have higher fluorescence emission at wavelengths of 470 and 465 nM, respectively. The general morphology and divisions of the cells were examined under inverted and light microscopes, respectively, and the fluorescence potentials of selected compounds were determined using immunofluorescence microscopy. The fluorescence intensity of molecules and 3D interactive surface plot images of cells were analyzed using ImageJ software. Cell imaging analyses showed that BX-6 and BX-13, like ThT, specifically stain the cell nucleolus. Moreover, molecular docking studies showed that the compounds could identify the RNA-rich nucleolus by binding with high affinity to the guanine region in the RNA structure. The results suggest that the compounds may be an initial route in developing specific biosensor compounds for nucleolus imaging.

Patagonian partnerships: the extinct Dusicyon avus and its interaction with prehistoric human communities.

The southern Mendoza province, located in the northern region of Patagonia, was inhabited by hunter-gatherer groups until historic times. Previous archaeological studies have reported canid remains among faunal assemblages, which were assumed to be part of the human diet. However, the taxonomic identification and significance of these canids within human groups have raised questions. In this study, we used ancient DNA analysis, morphological examination and stable isotope analysis (δ13Ccol and δ15N) to re-evaluate the taxonomic assignment of a canid discovered at the Late Holocene burial site of Cañada Seca. Previous morphological identifications suggested that it belonged to the genus Lycalopex, but our results conclusively demonstrate that the individual belongs to the extinct fox species Dusicyon avus. This finding expands Dusicyon avus' known geographical distribution to Patagonia's northern extremity. Furthermore, statistical predictions based on genetic divergence undermine the hypothesis that hybridization between Canis and Dusicyon, facilitated by the introduction of domestic dogs, played a role in the extinction of Dusicyon species. On the other hand, our findings indicate that a Dusicyon avus individual shared a similar diet and was probably buried alongside humans, suggesting a close relationship between the two species during their lives and deaths.

Untangling the cephalopod market: authentication of seafood products in Greece with DNA-barcoding

Cephalopod products are very popular in Greece and are considered a delicacy. However, heavy processing and marketing practices interfere with visual inspection and impede morphological identification, allowing species substitution. Mislabeling of seafood products remains a worldwide issue despite existing labeling regulations at local and European level. For the detection of fraudulent products, a variety of identification methods have been developed, however DNA barcoding remains the most favored. This study aims to investigate the cephalopod species sold in the country's seafood market and assess their mislabeling rates. Two mitochondrial genes, the cytochrome c oxidase subunit I (COI) and the 16S ribosomal RNA (16S), were selected for the analyses. A total of 156 samples were collected from fishmongers, markets, and restaurants across four cities. Identification was successful in 93.58% of the samples and 59 discrepancies (40.41%) between the label and the identified species were recorded. However, in some cases, substitution might have been unintentional, caused by negligence, lack of detailed information on the label, and the overall low awareness of the legislation by retailers. High mislabeling rates were estimated, especially when compared against the average global substitution rate in seafood products but align with those reported by studies on cephalopod products. This study is the first to investigate mislabeling rates in cephalopod commodities in Greece. With the aim of a transparent seafood trade, our results highlight the need for monitoring of all seafood products available in the country's market.

Isolation and Identification of Competitive Fungi on Medium for Black Wood Ear Mushroom in Korea and In Vitro Selection of Potential Biocontrol Agents

Black wood ear mushroom (<i>Auricularia auricula-judae</i>) is one of the most economically important mushrooms in China, Japan, and Korea. The cultivation of wood ear mushrooms on artificial substrates is more efficient in terms of time and cost compared with their natural growth on trees. However, if the substrate cultivation is infected by fast-growing fungi, the relatively slow-growing ear mushroom will be outcompeted, leading to economic losses. In this study, we investigated the competitive fungal isolates from substrates infected with fast-growing fungi for the cultivation of ear mushrooms in Jangheung and Sunchon, Korea. We collected 54 isolates and identified them by sequencing their internal transcribed spacer region with morphological identification. Among the isolates, the dominant isolates were <i>Trichoderma</i> spp. (92.6%), <i>Penicillium</i> spp. (5.6%), and <i>Talaromyces</i> sp. (1.8%). To find an appropriate eco-friendly biocontrol agent, we used five <i>Streptomyces</i> spp. and Benomyl, as controls against <i>Trichoderma</i> spp. and <i>Penicillium</i> spp. Among the six <i>Streptomyces</i> spp., <i>Streptomyces</i> sp. JC203-3 effectively controlled the fungi <i>Trichoderma</i> spp. and <i>Penicillium</i> spp., which pose a significant problem for the substrates of black wood ear mushrooms. This result indicated that this <i>Streptomyces</i> sp. JC203-3 can be used as biocontrol agents to protect against <i>Trichoderma</i> and <i>Penicillium</i> spp.

Comparative Evaluation of Analytical Techniques for Quantifying and Characterizing Polyethylene Microplastics in Farmland Soil Samples

The presence of microplastics in soil has become a significant global concern, posing a threat to environmental, agricultural, and human health. However, the use of various detection methods has led to significant discrepancies in the statistics reported for the abundance of soil microplastics across the available literature, resulting in poor comparability across the available literature. Studies have shown that plastic film residue is among the main primary sources of microplastics in farmland soils. Therefore, we manufactured and selected polyethylene microplastics (PE-MPs) from plastic film and combined them with soil samples after density separation treatment, forming a spiked test soil sample, which was subjected to density separation and extraction using a ZnCl2 solution. The experiment used visual inspection by stereo microscopy, micro-Fourier transform infrared spectroscopy, micro-Raman spectrometry, laser direct infrared imaging, and pyrolysis-gas chromatography–mass spectroscopy for the quantitative analysis of spiked soil. This study systematically assessed and compared these five common soil microplastic detection methods. The results revealed different quantitative detection accuracies across various particle size ranges. Micro-Fourier transform infrared spectroscopy showed a broader applicable particle size range (20–5000 μm) and higher detection rate (91%), while the detection range (>50 μm) and rate (70%) of visual inspection through stereo microscopy were lower. For the identification of microplastic morphology, laser direct infrared imaging performed better, whereas micro-Fourier transform infrared spectroscopy had the advantage in determining the types of polymers present over micro-Raman spectrometry. The advantages, disadvantages, and detection rates of the above five detection methods were clarified during the detection process. Considering these findings and similar studies, we suggest three combinations of the evaluated methods to optimize the detection of PE-MPs.

Improved identification of fracture total factor characteristics and cross-scale flow channels by interpreting initial production data of horizontal wells

Fracture identification after hydraulic fracturing of tight gas reservoirs is crucial to fracturing evaluation, productivity analysis, and production plan. Owing to fracture branching, bending, extending, and reversing in reservoirs, a simplified straight fracture cannot fully describe fracture characteristics. The novelty of this study is that the coupling mechanism between the complex fracture network characteristics and well production performances is figured out, thus achieving the identification of fracture morphology in fractured reservoirs. In this study, a combined model including Laplace transform, boundary element method, Stehfest numerical inversion, and fractal theory presents transient well performances. The key contributions to productivities at different production stages are dynamically identified via a sensitivity analysis. The results clearly indicate that the fracture total length is the key factor determining productivities due to no fracture interference at initial production stage. Furthermore, the logarithm of productivity linearly depends on the logarithm of production time and the productivity contribution rate equals the ratio of fracture length to total length. Thus, an updated inversion method is proposed by interpreting initial production data based on a tree-shaped fracture network associated with fracture total factor and cross-scale characteristics. The inversion results well match with their targets, with an error of no more than 5 %, ensuring the accuracy and validity of the proposed method. This innovative approach with low requirements on field data is convenient to yield an accurate prediction of fracture morphology, thereby providing a feasible strategy for promoting sustainable production of clean energy in unconventional gas reservoirs.

Population genetic structure of bottlenose and whitespotted wedgefishes from the Southwest Indian Ocean using a dual marker approach

Wedgefishes (Rhinidae) are threatened by unsustainable fishing globally, and especially in the Southwest Indian Ocean (SWIO), due to their high-value fins in the shark trade. The whitespotted wedgefish Rhynchobatus djiddensis and the bottlenose wedgefish R. australiae are both classified as Critically Endangered on the IUCN Red List of Threatened Species, yet a lack of species-specific knowledge and taxonomic uncertainty still exists within this genus. Genetic approaches aid in taxonomic classification and identifying distinct populations for targeted conservation. Morphological specimen identification of samples (n = 189) collected across the SWIO was confirmed based on the cytochrome oxidase c subunit I (COI) and/or nicotinamide adenine dehydrogenase subunit 2 (ND2) gene regions. The genetic diversity and population structure within and between species and sampling locations were investigated using a dual marker approach: (1) 2 concatenated mitochondrial gene regions, namely COI and the control region (n = 117), and (2) 9 nuclear microsatellite markers (n = 146). The overall genetic diversity was moderate, with an indication that different evolutionary forces are at play on a mitochondrial versus nuclear level. The 2 species were delineated based on both marker types, and for R. djiddensis, the sampling locations of South Africa and Mozambique were genetically homogeneous. For R. australiae, significant differentiation was found between sampling locations, with Madagascar and Tanzania being genetically the most similar. This information provides critical insights into the distribution range and population structure of the whitespotted wedgefish species complex that can support the sustainable management of wedgefishes.