Pinus tabulaeformis and Populus euramericana reclamation were explored using high-throughput sequencing and molecular ecological network methods. The results showed that: (1) With the increase in reclamation years, the reclaimed soil properties were close to the control group, and the soil properties of Pinus tabulaeformis were closer to the control group than those of P. euramericana. (2) The dominant bacteria under the canopies of P. tabulaeformis and P. euramericana was the same. Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes, Planctomycetes, Bacteroidetes, and Cyanobacteria were the dominant bacteria in the restored soil, accounting for more than 95% of the total abundance. The average values of the Shannon diversity index, Simpson diversity index, Chao 1 richness estimator, and abundance-based coverage estimator of the bacterial community in the P. euramericana reclaimed soil were higher than those in the P. tabulaeformis reclaimed soil. The influence of reclamation years on the bacterial community of samples is greater than that of species types. (3) The results of ecological network construction showed that the total number of nodes, total number of connections, and average connectivity of the soil bacterial network under P. euramericana reclamation were greater than those under P. tabulaeformis reclamation. The bacterial molecular ecological network under P. euramericana was more abundant. (4) Among the dominant bacteria, the relative abundance of Actinobacteria was negatively correlated with soil pH, soil total nitrogen content, and the activities of urease, invertase, and alkaline phosphatase, while the relative abundance of Proteobacteria and Bacteroidetes was positively correlated with these environmental factors. The relationship between the soil bacterial community of P. tabulaeformis and P. euramericana and the environmental factors is not completely the same, and even the interaction between some environmental factors and bacteria is opposite.To compare the effects of different remediation tree species on soil bacterial communities and provide a theoretical basis for the selection of ecosystem function promotion strategies after vegetation restoration, the characteristic changes in soil bacterial communities after  In 2019, the Ministry of Natural Resources of the People’s Republic of China successively issued the Opinions on Establishing an Incentive Mechanism to Accelerate the Promotion of Ecological Restoration of Mines and the Opinions of the Ministry of Natural Resources on Exploring and Utilizing Marketization to Promote Ecological Restoration of Mines, to accelerate the promotion of ecological restoration of mines and solve outstanding problems, such as many historical arrears, many practical contradictions, and insufficient investment in ecological restoration of mines. It can be seen that promoting land reclamation and ecological restoration is an important way to repair damaged mine ecological functions and realize the sustainable development of the mining ecosystem . Soil miMedicago sativa L.  and Bromus inermis Leyss. (Smooth brome) are widely used as a community-building species for ecological restoration in northern China [Alfalfa, a high-quality perennial legume, could improve the soil’s texture and nutrients with a low degree of degradation [Currently, research on the soil bacterial community after reclamation mainly focuses on the following points: first, research on the change in soil microbial characteristics after restoration. Sun et al. studied the response of soil microorganisms to vegetation restoration in coal mining subsidence areas and microbial changes . Li Yuanrn China ,23,24. Aradation ,26. A shradation .Pinus tabulaeformis and Populus euramericana as the reclamation tree species in the different waste dumps of the Heidaigou open-pit coal mine as the study area and used high-throughput technology to obtain soil microbial information. Furthermore, we used the molecular ecological network to reveal and compare the changes and characteristics of the bacterial communities in the soil reclaimed using P. tabulaeformis and P. euramericana and compared the effects of different vegetation reclamation models on soil bacterial communities, providing a theoretical basis for the selection of vegetation reclamation.However, there are few studies on the changes in soil microorganisms in different vegetation configuration modes and different reclamation years. In the study of soil microorganisms, traditional research methods, such as various diversity indices, can only excavate the composition and abundance information of microbial communities in the surface layer; the amount of microbial information data obtained using high-throughput sequencing is rich and huge. For this reason, in this study, we selected soil with P. tabulaeformis, P. euramericana, and Platycladus orientalis (L.) Francoptmxjjkmsc. The farmland is mainly dry and dispersed. The surface is directly damaged due to open-pit mining. The method of landform reconstruction and vegetation reconstruction is adopted for restoration. The soil cover is sourced from topsoil stripped from open-cut mining, with a thickness of approximately 1.0 m. The soil is mainly loess, characterized by a relatively infertile, paucity of humus, and has not undergone any processes of maturation. The vegetation species to be restored are mainly P. tabulaeformis and P. euramericana.The study area is located in the Heidaigou open-pit coal mine in the Zhungeer Coalfield, east of Zhungeer County, Ordos City, Nei Monggol Autonomous Region, with coordinates 111°12′53″–111°20′02″ E and 39°43′11″–39°47′41″ N . The minThis sampling object was used to select the reclaimed land under different restoration years and different tree species. The north waste dump (15 years of reclamation), the east and west waste dump (12 years of reclamation), the inner waste dump (9 years of reclamation), and the Yinwan waste dump (6 years of reclamation) after the restoration of the Heidaigou open-pit coal mine were selected as the sampling sites, and the sites not affected by coal mining subsidence were selected as the control sites. The locations of the sampling sites are shown in P. tabulaeformis and P. euramericana were set up in five waste dump sites; the control site, which was not affected by mining, was set up with P. tabulaeformis and P. euramericana  was used to measure the soil pH; a Topsizer laser particle size analyzer  was used to measure soil clay, silt, and sand contents; and an Automatic Kjeldahl nitrogen meter  was used to measure soil total nitrogen. Furthermore, an ultraviolet absorption spectrophotometer  was used to measure the soil total phosphorus content, soil organic matter content, soil alkali hydrolyzable nitrogen, soil urease, soil alkaline phosphatase, and soil sucrase activity. Soil total potassium was measured using a flame spectrophotometer .® Soil DNA Kit  according to the manufacturer’s protocols. The V4–V5 area of the 16S rRNA of soil bacteria was amplified via polymerase chain reaction (PCR). The applied degenerate primers were 515F 5′-barcode-GTGCCAGCMGCCGCGG-3′ and 907R 5′-CCGTCAATTCMTTT RAGTTT-3′.The 16S rRNA genes of soil bacteria were sequenced within three days of sampling. The DNA of soil samples was extracted using the E.Z.N.AThe PCR amplification products were detected using electrophoresis, the target fragments were cut and recycled, and the fluorescence quantification was performed based on the electrophoresis detection results. The TruSeq Nano DNA LT Library Prep Kit of Illumina Company was used to prepare the sequencing library and to perform purification, quality inspection, and quantification. The qualified library was diluted by gradient and mixed according to the required sequencing amount in the corresponding proportion, and finally, the MiSeq Agent Kit V3 (600 cycles) was used as the reagent for computer sequencing. The optimal sequencing length of the target fragment was 200–450 bp.P. tabulaeformis and P. euramericana, and the OTU with the highest abundance of 75% was selected for each group to construct and analyze the molecular ecological network. The construction process of the ecological network is found in MENA . The specific analysis process is as follows: High-throughput sequencing data were obtained to determine the number of different OTUs for each sample; the relevant abundance of OTU numbers was standardized. The Spearman correlation coefficient was used to calculate the pairwise similarity of each OTU and to generate the similarity matrix. Based on the random matrix theory (RMT), the derived threshold was determined, and the adjacency matrix was constructed.Based on the reclaimed vegetation, the samples were divided into two groups: After the construction of the molecular ecological network, the topological structure and modular analysis of the network were performed. Topological analysis was used to verify whether the results of network construction meet the scale-free, small-world, and modular characteristics of molecular ecological networks, and to test the reliability of the construction results . ModularPython’s matplotlib package was used to draw a bar graph of bacterial abundance at the phylum level and a heat map of bacterial abundance at the class level in the soil samples. The Pearson correlation between soil physical and chemical properties and enzyme activity was analyzed using SPSS 25.0. Common indices for calculating alpha diversity include the Simpson diversity index, Shannon diversity index, Chao 1 richness index, and abundance-based coverage estimator (ACE) richness index. Principal component analysis (PCA) and nonmetric multidimensional scaling (NMDS) were used to reflect the beta diversity of the bacterial communities.P. tabulaeformis and P. euramericana. There is no significant difference in the physical properties of the soil under the two types of plants. The average soil water content under P. tabulaeformis is 1.12% higher than that under P. Euramericana, while the average values of bulk density and sand content under P. tabulaeformis are 0.05 g/cm2 and 2.63% lower than that under P. euramericana. The average pH value and average cation exchange capacity (CEC) of the soil under P. tabulaeformis are higher than those of P. euramericana by 0.01 and 1.25 coml/kg, respectively, while the average total nitrogen, total phosphorus, total potassium, and soil organic matter contents are relatively low by 0.01%, 0.06 g/kg, 0.33 g/kg, and 1.89 g/kg, respectively. The activities of soil urease, sucrase, and alkaline phosphatase under P. tabulaeformis are 0.18 mg/g, 3.72 mg/g, and 0.22 mg/g lower than those under P. euramericana, respectively. After 15 years of reclamation, the soil organic matter content and urease activity were significantly different from the control group, and bulk density, soil water content, pH, cation exchange capacity, total nitrogen, total phosphorus, total potassium, and invertase activity were basically close to the control group. In short, with the increase in reclamation years, the reclaimed soil properties were close to those of the control group, and the soil properties of P. tabulaeformis were closer to those of the control group than those of P. euramericana.The soil physical and chemical properties of different reclamation tree species are shown in 2 to 2.17 g/cm2, and the proportion of soil sand particles gradually increased from 64.32% to 75.29%, while the soil water content had no obvious change. The soil pH gradually decreased from 8.29 to 8.2 with the increase in reclamation years. The cation exchange capacity, soil organic matter, total nitrogen, and total potassium content increased significantly with the increase in reclamation years, from 11.5 coml/kg, 7.09 g/kg, 0.03%, and 12 g/kg to 14.45 coml/kg, 11.73 g/kg, 0.05%, and 12.35 g/kg, respectively. The total phosphorus of the soil did not change significantly with the increase in reclamation years. The activities of urease, sucrase, and alkaline phosphatase increased significantly with the increase in reclamation years, from 0.13 mg/g, 8.44 mg/g, and 0.52 mg/g to 0.47 mg/g, 45.26 mg/g, and 0.81 mg/g, respectively.With the increase in reclamation years, the soil bulk density gradually decreased from 2.45 g/cmProteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes, Planctomycetes, Bacteroidetes, and Cyanobacteria, accounting for more than 95% of the total abundance.The bacterial community composition of each sample at the phylum level was statistically analyzed . At the Proteobacteria was the largest, ranging from 27.34% to 39.34%. In 12 a and 15 a of reclamation, the relative abundance of Proteobacteria in P. euramericana was higher than that of P. tabulaeformis. However, the relative abundance of P. tabulaeformis was lower than that of P. euramericana in 6 a and 9 a of reclamation. Proteobacteria participate in the soil nitrogen cycle process and help to restore the improvement of soil quality. The relative abundance of Actinobacteria was 16.55–36.82%, which was increasing with the increase in reclamation years. Actinobacteria was a saprophytic aerobic bacterium that preferred neutral or slightly alkaline, promoting the growth of rhizosphere bacteria, symbiotic bacteria, and endophytic bacteria. The relative abundance of Acidobacterium was 7.32–17.12%. In the samples of P. tabulaeformis, the abundance was increasing with the increase in reclamation years. This bacterium plays an important role in the degradation of plant residues. The abundance of Chloroflexi in P. tabulaeformis samples decreased with the increase in reclamation years. The nutrition mode of Chloroflexi was extremely diverse. From the perspective of vegetation type, at the phylum level, the relative abundance of Actinobacteria in the reclaimed soil of P. tabulaeformis is 0.04% higher than that in the reclaimed soil of P. euramericana. Furthermore, the relative abundance of Acidobacteria and Cyanobacteria in the reclaimed soil of P. tabulaeformis is 0.02% lower than that in the reclaimed soil of P. euramericana.The relative abundance of Proteobacteria, Actinobacteria, Acidobacteria, Gemmatimonadetes, and Cyanobacteria have a high response to the change in reclamation years. The relative abundance of Proteobacteria, Gemmatimonadetes, and Cyanobacteria decreased by 2.43%, 3.73%, and 7.61% on average each year, while the relative abundance of Actinobacteria and Acidobacteria increased by 8.31% and 3.08% on average each year.From the perspective of the changing trend of bacterial community structure, p < 0.1), while the differences in other indices are not significant (p > 0.1), but the average values of the four indices of the bacterial community in the reclaimed soil of P. tabulaeformis are higher than those in that of P. tabulaeformis.The alpha diversity index can measure the community diversity of samples. At the same time, the Simpson diversity index, Shannon diversity index, Chao 1 richness estimator index, and ACE were selected for comparative analysis of soil bacterial community diversity in different vegetation reclamation models and different reclamation years. As shown in P. tabulaeformis as the reclamation vegetation is relatively larger than that with P. euramericana as the reclamation vegetation.Beta diversity analysis can measure the similarity between different community composition samples. The PCA method based on the Euclidean distance was used to analyze the OTU of each sample, and the results are shown in P. tabulaeformis and P. euramericana are scattered, and the distance between samples with the same reclamation years is closer. This may be because the influence of reclamation years on the bacterial community of samples is greater than that of vegetation types, and this result is consistent with the result of the PCA method.Based on the phylogenetic tree, we comprehensively considered the changes in species and species abundance, calculated the weighted unifrac distance between samples, and performed an NMDS analysis. The results are shown in P. tabulaeformis group and the P. euramericana group, and the number of OTUs in each group was 725 and 818, respectively. Based on the RMT theory, the two groups of data were used to derive an adjacency matrix and to generate a network at the threshold level of 0.960. Under this threshold, the chi-square value of the P. tabulaeformis group is 60.779, and that of the P. euramericana poplar group is 69.672, which belongs to the appropriate threshold level. The analysis of the network topology  According to the analysis of the structure and diversity of the soil bacterial community, P. euramericana are greater than those under P. tabulaeformis. Based on the connectivity between nodes, it can be concluded that the relationship between bacterial communities under P. euramericana is more complex, and the relationship between different modules is also closer. Therefore, P. euramericana is more conducive to the restoration of soil bacterial diversity than P. tabulaeformis.(2) The total number of nodes, total number of connections, and average connectivity of the soil bacterial network under Actinobacteria is negatively correlated with soil pH, soil total nitrogen content, urease activities, invertase activities, and alkaline phosphatase activities, while the relative abundance of Proteobacteria, Bacteroidetes, and Armatimonadetes is positively correlated with these environmental factors. Soil total nitrogen content, soil organic matter content, and the activities of urease, invertase, and alkaline phosphatase have a greater impact on the bacterial community.(3) Among the major bacteria, the relative abundance of P. euramericana and P. tabulaeformis on soil bacterial community structure is related to the mechanisms of plant growth, which we will continue to study in our follow-up work.(4) The influence of Different restoration tree species have different effects on the soil bacterial community and the ecological function of restoration of reclaimed land. Therefore, through the experimental study of the change rule of soil microorganisms, we can timely understand the reasons for soil quality changes and select the appropriate repair tree species, which is more conducive to accelerating the selection of vegetation configuration on the reclaimed land and the speed of ecosystem restoration. However, the differences between the tree species that they derive from have not been studied in depth, and the mechanism will continue to be studied in the future.


Eucryptorrhynchus scrobiculatus and E. brandti are boring weevils of Ailanthus altissima (tree-of-heaven) and coexist on the same host A. altissima. In previous studies, through behavioral observation, we learned that, during the oviposition process, these weevils need to use their rostra to excavate an oviposition hole. However, the specific morphology of the rostra of the two weevils and the egg-laying mechanism during the oviposition process currently remain unknown. The morphological analysis attempts to link biological structure and function to specific environmental or behavioral characteristics, and this method has become an indispensable tool in the process of elucidating and interpreting patterns. Therefore, the morphologic characteristics of the rostra in E. scrobiculatus and E. brandti were examined and compared by scanning electron microscopy and micro-CT. This study not only plays an important role in exploring the excavating mechanism during oviposition of E. scrobiculatus and E. brandti, but also provides new insight for explaining the coexistence of two weevil species in the same host.Eucryptorrhynchus scrobiculatus and E. brandti (Coleoptera: Curculionidae: Cryptorrhychinae) are both pests of Ailanthus altissima, found in China. During ovipositing, gravid females of the two weevils need to excavate a cavity in the oviposition substrate with their rostrum, while their oviposition sites are different. (2) In this study, to explore the boring mechanism of E. scrobiculatus and E. brandti during ovipositing, the morphologic characters of the rostra of two weevils were studied in detail by scanning electron microscopy and micro-CT. (3) Their rostra appear similar, but the rostrum surface of E. scrobiculatus is rougher than that of E. brandti; their fine structures of rostrum and sensilla distribution are similar, but the sensilla twig basiconica 3 is distributed at the apex of labial palpus in E. brandti females, while not at the apex of labial palpus in E. scrobiculatus females; their rostra are hollow and their cuticle thickness is constantly changing, but the proportion of the whole rostrum tube cuticle in E. scrobiculatus is significantly larger than that of E. brandti. The above structural differences make E. scrobiculatus more conducive to oviposition in the soil and E. brandti more conducive to oviposition in the trunk of A. altissima. (4) Overall, this study not only plays an important role in exploring the excavating mechanism during the oviposition of the two weevils, but also provides new insights into the coexistence of two weevil species on the same host A. altissima.(1)  Weevils are a type of beetle belonging to the superfamily Curculionoidea. They are one of the most diverse groups in the existing biological family, with about 60,000 described species ,2.Oxyporus (Coleoptera: Staphylinidae) to fungal feeding involve modifications of the mandibles of adults and larvae; labial palpi and labrum of adults; and maxillae of larvae [Ips acuminatus (Coleoptera: Curculionidae: Scolytinae) using field emission scanning electron microscopy (FESEM) demonstrated its possible implication to act as an external carrier of pathogenic microorganism [Doubledaya bucculenta Lewis  shows an asymmetry in the mandibles of females compared to males, which helps females to dig holes between hard bamboo nodes when laying eggs [In insects, the structure and function of mouthparts and their relationship with feeding strategies have attracted increasing research attention. The morphology of insect mouthparts, as well as the adaptation characteristics of host localization, feeding, and oviposition related to mouthparts morphology, have been extensively studied. For example, the principal morphological adaptations of species of f larvae . Studiesorganism . Doubleding eggs . In weeving eggs ,7,8. Mosing eggs . Using ring eggs . The apping eggs ,9,10,11.Eucryptorrhynchus scrobiculatus Motschulsky and E. brandti Harold (Coleoptera: Curculionidae: Cryptorrhychinae) are widely distributed in China [Ailanthus altissima (Mill.) Swingle (tree-of-heaven) (Simaroubaceae) and its variety A. altissima var, cause serious damage to forestry in China at the larval stage [E. scrobiculatus larvae damage the root of A. altissima, E. brandti larvae damage the trunk of A. altissima [E. scrobiculatus and E. brandti are different. E. scrobiculatus females lay eggs in the soil near A. altissima, while E. brandti females lay eggs in the trunk of A. altissima [A. altissima [E. scrobiculatus and E. brandti, provide a detailed morphology comparison of the rostra to understand the functional role of these structures in the oviposition process, and explore the reasons for differences in the oviposition sites of the two weevils.in China ,13,14,15al stage ,17,18,19ltissima . Previoultissima . The oviltissima ,21. The ltissima . Studiesltissima ,21. HoweE. scrobiculatus and E. brandti were collected from the A. altissima forest in Xiaoxingdun village , Ningxia Hui Autonomous, China. They were reared in plastic boxes with fresh branches of A. altissima.Adults of E. scrobiculatus females and twenty specimens of E. brandti females were put into a plastic bottle filled with 70% ethanol. Each weevil was taken out of the bottle one by one, and its head (with rostrum) and body were separated with tweezers and dissecting scissors. The dissected head was put into ultrapure water for ultrasonic cleaning, and the water was changed every 5 min for a total of 15 min. The cleaned heads were dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% alcohol gradients for 15 min, which was repeated once in 100% ethanol. The mandibles, maxillae, and labium of the two weevils were dissected under a microscope , and at the same time, the samples were pasted on the sample stage according to the required shooting angle with conductive glue. The stage with the samples attached was placed in a plastic box filled with silica gel desiccant and dried at room temperature. All samples were coated with a film of gold-palladium in an E-1010 sputter ion instrument . Then, the observation and photographs were taken under an S-3400N (Hitachi) scanning electron microscope at an accelerating voltage of 0.5–30 KV.Twenty specimens of E. scrobiculatus females and five rostra of E. brandti females were put into a plastic bottle filled with 70% ethanol. The dissected rostra were fixed in Bouin solution, washed with 70% ethanol, and then dehydrated by alcohol gradient. The alcohol concentrations were 50%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (3 times), with an interval of 10–15 min. Finally, the sample was dehydrated with acetone 3 times, each for 30 min. The prepared sample was placed under a 3D X-ray microscope  for shooting and scanning. The scanning parameters were as follows: voltage 60.0 kV, current 80.0 μmA, time 0.60 s.The rostrum was separated from the body using the same method as above. Five rostra of E. scrobiculatus and 604 micro-CT slices of E. brandti were used to reconstruct rostrum tubes, respectively. These structures were trimmed, smoothed, and rendered with multiple viewers.The target structure was reconstructed using the imported data analyzed by Avizo 9.0.1. The 916 micro-CT slices of p < 0.05) were used to compare the proportion of the rostrum tube cuticle. Graphs were drawn with GraphPad Prism 9, scales and arrows on the images were marked using Photoshop 2021.The references for the classification of the sensilla were as follows ,23,24,25E. scrobiculatus and E. brandti looked similar, there were some differences. The downward curvature of the entire rostrum of E. scrobiculatus female is inconsistent. The downward curvature of the rostrum from the antennal fossa to the tip is larger than that from the antennal fossa to the head. The rostrum surface of E. scrobiculatus female is rough and has convex edges and grooves, covering a large number of setae and pores , a pair of mandibles (Md), a pair of maxillae (Mx) and a labium (Lb)  and labium (Lb) of E. brandti are not completely enclosed by mandibles; some parts are longer than the mandibles and are exposed to the outside of them. Therefore, the fine structures of the maxillae and labium of the two weevils were compared in the following study.Scanning electron microscope observation showed that although in terms of overall structure the rostra morphology of nd pores A,C,E. Thnd pores B,D,F. Thium (Lb) . From FiE. scrobiculatus and E. brandti are located in the longitudinal cleft on both sides of the labium. There are a pair of segmented movable appendages. The maxillae can be divided into five parts: cardo, stipe, galea, laciniae, and maxillary palpi. The galea and laciniae covered with a large number of setae are attached to one side of the stipe. There are some differences in the number and distribution of sensors in the stipe for the two weevils. The stipe is a large sclerite with a smoother surface connected to the end of the cardo  distributed at this position, but in E. brandti, there are three sensors  distributed, and the sensor S.tb.3 is found only at the apex of labial palpus of E. brandti females  > EBR afa (47.77 ± 0.13) > ESC bfa (45.04 ± 0.14) > EBR bfa (42.64 ± 0.07), p < 0.05  were only ventrally observed in the last segment of the maxillary palp. The sensor structure is similar to the neuronal structure of hygro-/thermo-receptors [E. scrobiculatus and E. brandti females likely play roles in monitoring the environmental conditions in the process of ovipositing.The feeding and oviposition of adult weevils are not restricted to the drilling of holes. Irregular excavations are also made, and some surface browsing is carried out, and these behaviors are inseparable from the role of the sensor. A comparison of the fine structures of maxillae and labium in two weevils revealed many sensilla distributed at the apex of the maxillae and labium, which can be divided into three types: sensilla basiconica (S.b.1–4), sensilla twig basiconica (S.tb.1–3), and digitiform sensilla (D.s). There was no difference in the number and distribution of sensilla basiconica between the two weevils at the apex of the maxillae and labium. These sensilla basiconica can be divided into two types: uniporous peg sensilla (S.b.2 and S.b.4) and porous peg sensilla (S.b.1 and S.b.3). The former may be a contact chemosensory organ, while the latter may function as an olfactory organ ,37,38. Tand GABA . In addiand GABA . The senlongatus ,43. In alongatus ,45. Yanglongatus . Therefog laying . E. scroeceptors ,47. TherE. scrobiculatus and E. brandti females were reconstructed by micro-CT. It was difficult to distinguish the inner and outer epidermis of the rostrum in micro-CT sections; therefore, we studied the general name of the inner and outer epidermis in the cuticle structure. Through the study of the rostral cuticle, it was found that the thickness of the exoskeleton from the head cavity junction to the appearance of the chewable mouthpart was constantly changing. Taking the antennal fossa as the dividing line, the proportion of cuticles after the antennal fossa was significantly greater than that before the antennal fossa. The proportion of cuticle thickness in E. scrobiculatus females in the same position was always higher than that of E. brandti females, regardless of whether it was before and after the antennal fossa or the overall rostral cuticle  was a hierarchical fiber complex characterized by various arrangements embedded in heterogeneous protein matrices α-chitin (N-acetylglucosamine) nanofibers [E. brandti by scanning electron microscopy (E. scrobiculatus and E. brandti are adaptations to the oviposition position, which can avoid the failure of the rostrum during excavation.The rostral cuticle structure of  cuticle A. Therefnofibers ,49,50,51nofibers ,53,54. Icroscopy . This stE. scrobiculatus and E. brandti may be derived from the same ancestor [E. scrobiculatus and E. brandti were formed. E. scrobiculatus and E. brandti females must dig an oviposition hole with their rostra before laying eggs [A. altissima [A. altissima.Previous studies showed that ancestor . Becauseancestor . In otheing eggs ,21; the ltissima . The difltissima . On thisE. scrobiculatus and E. brandti were compared. The rostrum surface of E. scrobiculatus is rougher, the proportion of the whole rostrum tube cuticle is larger, the maxillae and labium are encased by the mandibles. These structures make E. scrobiculatus more suitable for excavating cavities in the soil. The rostrum surface of E. brandti is smoother, the proportion of the whole rostrum tube cuticle is smaller, the maxillae and labium are not completely enclosed by mandibles. These structures make E. brandti more suitable for excavating cavities on the trunk of host. In addition, the thickness change and the unique laminate structure in the rostral cuticle structure of both E. scrobiculatus and E. brandti give their rostra tubes a good rigidity and toughness, which avoids the phenomenon of rostrum fracture in the process of excavating. In summary, these features play an important role in exploring the oviposition mechanism of the weevil from the perspective of structure and function, but also provide new insights into the coexistence of two weevil species in the same host A. altissima.In this study, the differences in the fine structures of the rostra between


Camellia oleifera is an important woody grain and oil plant worldwide. However, owing to a significant decline in the number of wild pollinators globally and the associated reduction in pollination services and seed production, camellia oil is in short supply. Therefore, it is important to evaluate the pollination mechanisms and efficiency of wild pollinators in C. oleifera seed production. We explored the pollination system of C. oleifera, focusing on the flower-visiting characteristics of its candidate pollinators. We found that Apis mellifera is the best candidate pollinator, but flies and hoverflies also play important roles in the pollination system.Camellia oleifera Abel. is an important woody oil plant, and its pollination success is essential for oil production. We conducted this study to select the best pollinator candidates for C. oleifera using principal component analysis and multi-attribute decision-making. Field observations of the flower-visiting characteristics of candidate pollinators were conducted at three sites. The insect species that visited flowers did not considerably differ between regions or time periods. However, the proportion of each species recorded did vary. We recorded eleven main candidates from two orders and six families at the three sites. The pollen amount carried by Apis mellifera was significantly higher than that of other insects. However, the visit frequency and body length of Apis mellifera were smaller than those of Vespa velutina. Statistical analysis showed that A. mellifera is the best candidate pollinator; Eristaliscerealis is a good candidate pollinator; Phytomia zonata, A. cerana, and V. velutina were ordinary candidate pollinators; and four fly species, Episyrphus balteatus, and Eristalinus arvorum were classified as inefficient candidate pollinators. Our study shows that flies and hoverflies play an important role in the pollination system. Given the global decline in bee populations, the role of flies should also be considered in C. oleifera seed production. Many plants have mutualistic relationships with pollinators that ensure their reproductive success . PollinaCamellia oleifera Abel. is a woody, oil-producing plant native to China [C. oleifera is a self-incompatible plant that relies heavily on active insect pollination [C. oleifera attracts more than 50 species of pollinating insects, including bees, wasps, hoverflies, and flies [C. oleifera forests have a high mortality rate because C. oleifera nectar contains strong alkaloids and other indigestible compounds that cause posterior intestinal obstruction in bee larvae [C. oleifera pollination. However, the number of wild pollinators has been declining considerably worldwide, and the consequent reduction in pollination and seed production has caused a shortage in the camellia oil supply [C. oleifera.Insects have been identified as the world’s most important pollinators, contributing to 87% of pollination globally, making their services critical for the sustainability of natural ecosystems ,11. Cameto China . Its seeto China . C. oleilination . When itnd flies . Howevere larvae ,17. TherC. oleifera by observing the insect species that visited the flowers at different times across a range of different sites. The proportion of each insect species, flower visit duration and frequency, and other indices were quantified, and the body and flower-visiting characteristics of the insects were analysed and compared. The pollination potential of flower visitors was compared using principal component analysis and a multi-attribute decision-making model. Functional groups were determined on the basis of the scores obtained using cluster analysis.In this study, we explored the pollination system of Our research was conducted in December 2021 in Yuelu District, Tianxin District, and Wangcheng District of Changsha, Hunan Province, China .C. oleifera flowers and used a sweep net for capture. The insects captured were stored in 10 mL vials containing 75% alcohol and were then transported to the laboratory for identification by the relevant experts [To identify the visiting insects, we photographed those that sat on the  experts .C. oleifera was recorded and classified from 8:00 to 18:00 on three sunny days when the shrubs were in full bloom. The statistical method for examining the flower-visiting density was as follows. Three rows of plants were randomly selected for each sampling session at the site, and then banded sampling was carried out. The number of insects landing on 200 flowers in the row was recorded back and forth for one hour, and the data from the three rows were added together to represent the overall flower-visiting density for the site. This experiment was carried out at three sites at the same time. After obtaining the data of each site, the average flower-visiting density in different periods at the three sites was recorded as data.To select the best observation period for the flower-visiting insects, the flower-visiting density of C. oleifera during the flowering period from November to January, we conducted the following experiments in December. The temperature in the first half of the month was 6–16 °C, and it was 3–11 °C for the remainder of the month. We observed a total of 2679 insects during the study. (a) We chose five sunny days during the first half of December at the three sites. Flower-visiting insects were observed from 11:00 a.m. to 12:00 p.m., which is when they are the most active [To quantify visits by pollinators to t active . (b) We For comparative analysis of the body characteristics of the flower visitors, we used vernier callipers to measure the body length, the head width and length, and the back plate width and length of the visitor specimens collected, with the measurements repeated ten times for each species. We also photographed the main flower visitors using a stereo microscope .To explore behavioural differences among the pollinator candidates, we recorded the visiting time, namely the duration from the insect landing on the flower to the insect leaving. A total of 205 insects were recorded during this process. We also recorded the visiting frequency, namely the number of insect visits to the flowers in one minute. A total of 168 insects were recorded. Data on any foraging behaviours observed were collected [To quantify the pollen load capacity of the pollinator candidates, we added a small amount of detergent to the visitor samples stored in the 10 mL vials to remove any pollen particles carried by them . We tranp < 0.05.To explore the differences between the body and visit characteristics of the insects, the visiting time, frequency, pollen load, posture characteristics, and the canopies visited were analysed. All the analyses were carried out using SPSS, version 26 , with the statistical significance set at To screen the factors for follow-up analysis, we analysed the correlation of the insect pollen load, the body surface characteristics, the body length, proportion, flower-visiting frequency, and the flower-visiting time using SPSS, version 26, with irrelevant factors excluded .To determine the best pollinator candidate, the score for each insect was obtained using principal component analysis (PCA). The data were standardised to a mean of zero and a variance of one before conducting the PCA, examining the contribution of each feature to different principal components. According to Gutten’s lower bound principle, eigenvalues of <1 were excluded .To rank the insects as pollinator candidates, we established a score matrix according to the observations. The score for each feature was then determined using MATLAB 2019, and the score for each insect species was then obtained .To determine the pollination function group, the insects were clustered after the score for each species had been obtained. The pollination function groups were classified using SPSS, version 26, with the intergroup connection method, and the distance between the pollinator candidates was set as the Euclidean distance .C. oleifera, and the main insect species were found to be the same during this period  of the insects. V. velutina visited flowers with the highest frequency of 4.5 times per minute, whereas A. mellifera and Lucilia sericata had slightly lower frequencies. There was no difference in the mean frequencies of A. cerana and some of the visiting hoverflies, and the mean frequency was the lowest for the flies (The duration of visits varied considerably during the pollinator observations. For example, the longest visiting time for he flies .Episyrphus balteatus, which often hovered over but did not visit the flowers. Most of the bees visited to collect the nectar, but A. mellifera showed a higher enthusiasm and actively collected pollen on their pollen-carrying legs. The wasps were highly active, usually buried their heads in the anthers to forage, and made contact with the stigmas almost every time (There were significant differences in the behaviours of different insects. Flies spent more than half of their time visiting the anthers and inhabited the anther instead of the stigma. Among the hoverflies, most showed foraging behaviours and frequently made contact with the stigmas. More than half of the hoverflies came into contact with the stigma during the observation process, except ery time .p < 0.05, A. mellifera carried the largest amount, with more than 50,000 grains per individual, followed by E. cerealis, with more than 48,800 grains per individual. There were no significant differences for Phytomia zonata, A. cerana, and V. velutina. There were also significant differences in the proportion of normal pollen and pseudopollen carried by the different insects. The ratio of pseudopollen to the normal pollen carried by most insects was approximately 0.1, and the ratio of flies was generally large, among which L. sericata had the highest ratio of up to 0.43.There were significant differences in the powder-carrying capacity of the pollinator candidates (V. velutina was the largest species, with its body length reaching 20.42 mm. Bees and hoverflies were similar in appearance, but the bees were larger in size (p < 0.05, Among the 11 main pollinator candidates, E. cerealis, A. mellifera, and V. velutina for further analysis. E. cerealis is densely tomentose, especially on the back. A. mellifera has dense yellow villi on the body and dense hairs on the feet. V. velutina has dense villi on the body surface, especially on the chest. The flies are mainly covered with relatively hard and smooth bristles and short black hair. Among the hoverflies, P. zonata has dense villi on the body and feet, and the pollen grains could be seen on the specimens. E. arvorum is fluffy on the back, while E. balteatus is fluffy on both sides of the body (There were also many differences in the body surface characteristics among these insects. Considering the proportion of the species, the visiting time, frequency, and the pollen load, we selected the body .p < 0.01, A. mellifera was much higher than that of the other insects, reaching 0.91 (Using correlation analysis, we selected five factors with high correlation from the six previously recorded factors for follow-up analysis (ing 0.91 .A. mellifera is the best pollinator candidate; E. cerealis is a ‘good’ pollinator candidate; P. zonata, A. cerana, and V. velutina are ‘ordinary’ pollinator candidates; four fly species, E. balteatus, and E. arvorum were classified as ‘inefficient’ pollinator candidates (Two kinds of clustering results were obtained according to different methods. The two results showed that ndidates .C. oleifera pollination [C. oleifera nectar results in a reduction in the bee numbers, other pollinators, such as flies and hoverflies, become the dominant pollinators and compensate for the absence of the bees [Considering the body and flower-visiting characteristics of the candidate pollinators, bees might be the best candidate pollinators. However, flies and hoverflies also play an important role in lination ,27. Whenlination ,29. For the bees . This dythe bees . This shthe bees .In line with a previous study , we founA. mellifera as the best C. oleifera candidate pollinator and found that flies and hoverflies also play crucial roles in the pollination system. PCA and the MADM model showed similar results for the pollinator potential of flower visitors. However, the results of both analyses differed from those based on taxonomic affinities. This suggests that the classification of flower visitors into different functional groups should consider the contribution of different flower visitors to pollination while disregarding the taxonomic affinities [C. oleifera has multiple visitor functional groups, including flies, hoverflies, bees, and wasps. However, only a subset of these visitors serves as effective pollinators [We used statistical methods to select finities ,38. Likelinators . Most stlinators .E. cerealis is a ‘good’ candidate pollinator; P. zonata, A. cerana, and V. velutina are ‘ordinary’ candidate pollinators; and the remaining flower visitors are ‘inefficient’ candidate pollinators for C. oleifera. This division highlights the relative importance of diverse pollinators [C. oleifera has pseudopollen, which is ineffective for pollination [C. oleifera anthers [Additionally, we determined that linators . In thislination . Therefo anthers . This in


The COVID-19 pandemic resulted in an overload of health services and healthcare professionals. The result is a setback in health promotion and prevention, delays in diagnosis, and deaths from other diseases that are currently receiving inadequate attention. This article illustrates the risk of this negligence. This study aimed to report a case of coinfection of disseminated cryptococcosis and BK virus in a patient without a previous diagnosis of human immunodeficiency virus infection and COVID-19 negative in the context of the COVID-19 pandemic. Despite receiving antifungal therapy, the patient died. This fatal case is a warning regarding delay of diagnosis and neglect of other serious illnesses owing to the current pandemic, including fungal diseases and neglected diagnoses.  Coronavirus disease 2019 (COVID-19), declared as a pandemic by the World Health Organization (WHO) in January 2020, has already killed more than 4 million people globally  , 4.Wright et al. in 2020   referreCryptococcus, especially by the species C. neoformans [ Its main manifestation is neurocryptococcosis, meningitis caused by fungi of the genus formans  . This sA male patient, 37 years old, homosexual, English teacher, was referred to the Specialized Care Service of Infectious Diseases at Dr. Miguel Riet Correa University Hospital, Rio Grande, Brazil on September 8, 2020. He reported fever, enlarged cervical nodes, asthenia, adynamia, and anorexia for 30 days, and moderate-intensity headache that had worsened in the last 10 days. He had several medical visits to external professionals in the preceding 30 days with the same complaints. He had received only symptomatic treatment (dipyrone 500 mg q 6 h) and only investigated for SARS-CoV-2 by reverse transcription-polymerase chain reaction and serological tests. It should be mentioned that the results of both tests had been negative.On physical examination at Dr. Miguel Riet Correa University Hospital, an ulcerated lesion (2×1 cm) was noted on the face, specifically on the foreheadand close to the scalp with irregular borders and fibrinous background that had evolved over 10 days . Other fAntibodies to HIV were present and other serologic tests were negative. The computed tomography of the brain and the chest radiograph showed no abnormalities. The values of hematocrit, hemoglobin,leukocyte count, segmented neutrophils, and platelets were 36%, 12.7 g/dL, 10,340, 82%, and 367,000, respectively,with preserved renal and liver function. Since the patient had a history of a scratch by a domestic cat with sporotrichosis hyperendemic in the region [ Cryptococcus species  revealed yeastswith morphology characteristic of  species . MoreoveCryptococcus species, confirmed by the growth ofC. neoformans on culture, proving the diagnosis of disseminated cryptococcosis. The isolate showed no resistance in vitro to Amb or Flu by microdilution technique . Microscopy examination of blood culture and CSF  showed eOver the next 24 h, his course evolved with disorientation, left deviation of the mouth and palpebral ptosis with heart rate, respiratory rate, and systemic blood pressure of 117, 22, and 90/ 60 mm Hg,respectively. Finally, he was transferred to the Intensive Care Unit. On September 12, the patient was vomiting and consequently aspirated gastric contents. He had anisocoria with left mydriasis and sensorium lowering that progressed to a comatose staterated 3 on the Glasgow scale. A new CT scan of the brain was requested, revealing generalized edema, diffuse ablation of cerebral sulci and fissures, and slightly hyperdense duralsinuses, possibly related to cerebral venous thrombosis. Nodular thickening of the skin and subcutaneous tissue in the right frontal region was seen, corresponding to the area of theskin lesion. The patient required orotracheal intubation and treatment was started with cefepime 1 g/8h IV and clindamycin 600 mg/8h IV for aspiration pneumonia. It should be mentioned that aspiration material was not collected for analysis.The patient died within 48 h after admission to the Intensive Care Unit, on September 13. The HIV viral load tests and CD4+ count were requested but their results wereinconclusive. No other test was performed due to his rapid deterioration  . Figure This study presents a case of disseminated cryptococcosis in a patient with HIV infection, which had evolved for more than 30 days before the diagnosis of this viral-fungal co-infection. In the context of the COVID-19 pandemic, his full diagnostic investigation was neglected, resulting in severe disease progressing to death. The patient lacked a severe headache (headache is common in neurocryptococcosis) and did not have a prior diagnosis of HIV infection. A skin lesion with a high load of fungal propagules was the first important evidence of a disseminated presentation of cryptococcosis. These cutaneous manifestations occur in only ~5% of cases and are generally associated with low T CD4+ cell counts  , 10. WDespite the absence of data on viral load and CD4+ counts, the evolution of the condition suggests a high degree of immunosuppression, a hypothesis supported by the skin lesion with high fungal burden, and by the detection of BK virus DNA in the cerebrospinal fluid of the patient  , 14.The BK virus (BKV) is a pathogen of the family of polyomaviruses, and its infection is quite common with a prevalence of up to 90% antibodies in adults. It is also found in the whole blood sample of about 30% of HIV patients  , 14. TCryptococcus species and BKV affecting the central nervous system. We found only three reports of co-infection ofC. neoformans and BKV, all in transplanted patients with BKV causing kidney injury  victims of COVID-19, due to errors or diagnostic failures  .Tili et al. in 2020 affirmed that the pandemic culminates due to some medical negligence and inequalities that delay or even paralyze the health services and care for other acute and chronic conditions  . The toCryptococcosis is on the list of neglected diseases of WHO, even though it is among the five most lethal diseases  . Late dThe reported case is another victim of this severe opportunistic mycosis, probably due to its late diagnosis, with a limited prior investigation restricted to COVID-19. Even with treatment, the lethality rate in cryptococcosis is within the range of 20 and 50%  . TherefThe authors are grateful to the Health Science Post-graduation program, College of Medicine from the Federal University of Rio Grande, Rio Grande, Brazil, Coordination for the Improvement of Higher Education Personnel, Brazil, and the Capes PrInT–FURG Program .This project does not have specific funding for its conduction.J. L. B., V. R. P., L. S. M., M. O. X., and V. R. A. performed the sampling and conducted the diagnosis and identification tests. J. L. B. and R. P. B. wrote the first draft of the manuscript. J. L. B., V. R. P., L. S. M., M. L. R., and R. P. B. contributed to the conduction of the study and data preparation. R. P. B. and M. L. R. contributed to care and monitoring of the patient and researching the clinical information. D. A. S. and M. O. X. managed the project, analyzed the data, and finalized the manuscript. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.The authors declare that there was no conflict of interest in this study.No financial interests have been declared related to the material of this manuscript. This project was approved by the Health Research Ethics Committee of the Universidade Federal do Rio Grande (number: 234/2018).


Miracinonyx trumani is an extinct species that roamed the Pleistocene prairies 13 000 years ago. Although M. trumani is more closely related to the cougar (Puma concolor) than to the living cheetah (Acinonyx jubatus), it is believed that both A. jubatus and M. trumani possess a highly specialized skeleton for fast-running, including limbs adapted for speed at the expense of restricting the ability of prey grappling. However, forelimb dexterity of M. trumani has not been yet investigated. Here, we quantify the 3D-shape of the humerus distal epiphysis as a proxy for elbow-joint morphology in a sample of living cats to determine whether the extinct M. trumani was specialized to kill open-country prey using predatory behaviour based on fast running across the prairies and steppe terrains of the North American Pleistocene. We show that M. trumani had an elbow morphology intermediate to that of P. concolor and A. jubatus, suggesting that M. trumani had a less specialized pursuit predatory behaviour than A. jubatus. We propose that M. trumani probably deployed a unique predatory behaviour without modern analogues. Our results bring into question the degree of ecomorphological convergence between M. trumani and its Old World vicar A. jubatus.The North American cheetah-like cat  These meshes were repaired with the software Geomagic Essentials . The CT-scanned specimens were processed using 3D Slicer .a), and the second row (no. 2) runs through the groove that separates these structures (a). The digitization was carried out using the software Avizo v.9 (Fisher Scientific). The raw 3D coordinates were uploaded into R environment [gpagen from Geomorph package [One of us (AM-S) digitized two semilandmark rows to characterize the main morphological features of the distal articular surface. The first row (no. 1) follows the boundary of the articular surface surrounding the trochlea and capitulum a, and thructures a. The diironment  and procironment  sliding ironment  with fun package . Next, w package .Figure A. jubatus, P. concolor and M. trumani to focus on the morphological differences between these two living species and to assess for similarities between the elbow topology of M. trumani and either A. jubatus or P. concolor. Then, we repeated the Procrustes superimposition and PCA described above with this subsample, and we computed a K-means [K = 2) to classify M. trumani within one of these two living species because other classification methods, such as linear discriminant analyses, should be avoided when the number of variables is very high [Subsequently, we selected only the specimens of  K-means  analysisery high .M. trumani. Topological deviations between these meshes were measured using Geomagic essentials software . We performed a morphing procedure using the surface model of the elbow joint of the specimen P. concolor (no. 409) as a starting shape to the average shape of P. concolor, A. jubatus and to the elbow of M. trumani. As the three models start from the same standard model (i.e. same number of mesh triangles and surface areas), the differences obtained in the topological deviation analysis will only be attributable to those shape changes applied to the three models.We also generated the average shape of the two resulting groups and we compared them with the shape of the distal articular surface of . 3b shows the bivariate plot depicted from the first two eigenvectors (PCs) obtained from a PCA of elbow shape. The first PC mainly separates out the Puma–Acinonyx lineage (b) according to their more squared and shorter  articular surface, as well as their shallow trochlear groove (c). By contrast, the second PC mainly separates A. jubatus from P. concolor (c), as the former has a more proximally expanded capitulum and a more vertical trochlear crest (c). The elbow of M. trumani clustered between the range of elbow shape variation of P. concolor and A. jubatus (b) but it was classified with P. concolor by K-means analysis .Figure 1 lineage b accordir groove c. By conconcolor c, as thear crest c. The el jubatus b but it P. concolor, A. jubatus and M. trumani (a) shows that the trochlea crest of P. concolor is projected distally to a greater extent than A. jubatus. The capitulum of A. jubatus is highly curved, while P. concolor has the least curvature in this region. Both the trochlea crest and the capitulum curvature of M. trumani are somewhat intermediate of those of P. concolor and A. jubatus, but they are more similar to that of the former.Our topological analysis among the elbows of  trumani a shows tM. trumani and both living taxa are shown separately in figure 2b. The capitulum and trochlea groove are more expanded in A. jubatus than in M. trumani, but the trochlea crest is less expanded in A. jubatus. The most distal part of the capitulum of A. jubatus is similar to that of M. trumani. The trochlea groove of the two models is almost identical. The elbow of P. concolor is much more similar to that of M. trumani, but with negative values close to zero in their topological deviations. Both the trochlea crest and the most lateral part of the capitulum at the proximal edge are similar in both taxa. The most distal part of the capitulum of P. concolor exhibits negative values relative to the topology of M. trumani, suggesting that P. concolor possesses a capitulum crest much flatter than M. trumani.The quantifications of the topological deviations between . 4A. jubatus possess limbs adapted for speed at the expense of restricting the joint motion to the parasagittal plane. This restriction entails the loss of the ability to supinate the forearm, which is essential for prey grappling [figure 1a, M. trumani plots between the range of elbow shape variation of its closest living relative (P. concolor) and its Old World counterpart (A. jubatus), suggesting that there is no modern analogue for the elbow morphology of M. trumani.Extreme pursuit predators such as rappling –16. As sK-means analysis indicates that the elbow of M. trumani clusters with that of P. concolor and not with that of A. jubatus. This positioning of the M. trumani elbow from Natural Trap Cave does not necessarily reflect a specialized Puma-like predatory behaviour. Rather, it shows the retention of the ability to supinate the forelimb to grapple prey, unlike the condition seen in A. jubatus [M. trumani to supinate the forelimb compared to that of A. jubatus also explains the retention of fully retractable claws, unlike A. jubatus, which are essential to immobilize relatively large prey prior to delivering a killing bite [M. trumani exhibited a brachial index  that is closer to that of A. jubatus than to P. concolor , the jaguar (Panthera onca) or the tiger (Panthera tigris), suggests that M. trumani was a more cursorial form, but it retained fully retractable claws and some degree of supination at the elbow, which reinforces the idea that there is no modern analogue for the predatory behaviour exhibited by M. trumani. However, it is worth noting that the brachial index for the living lion (Panthera leo), which is an ambush predator that inhabits open habitats [M. trumani .M. trumani, they cast doubts on the ‘anachronist’ hypothesis proposed to explain the excessive speeds of modern pronghorns as a result of a coevolutionary relationship with the extinct M. trumani. On the other hand, the excessive speeds of the modern pronghorn could be explained as an evolutionary by-product of its adaptation for decreasing transport costs in response to the spread of grassy habitats [Therefore, although our results are focused on a single specimen of habitats .The data are provided in the electronic supplementary material , which c


In Pakistan, public sector investment has significantly increased in higher education over the last two decades. This study, therefore, aims to estimate the private economic returns of Ph.D. faculty members over their non-PhD counterparts in Pakistani universities. We use questionnaire-based survey data of 784 respondents comprising Ph.D. and non-Ph.D. faculty members. In the first step, earning function is estimated for the entire group. In the next step, the lifetime private economic returns are calculated with the help of the simulation process, which yields the net present values (NPV) of the lifetime earnings for the two subgroups. Our findings show that the lifetime private economic returns of a Ph.D. degree are higher than a non-Ph.D. degree. In the case of a domestic Ph.D. degree, the average lifetime economic returns of the Ph.D. are 46.5% higher than those of non-Ph.D. faculty members. In contrast, foreign-country Ph.D. degree holders earn 29.8% extra than non-Ph.D. faculty members. Therefore, the net lifetime returns of foreign Ph.D. holders are 16.7% points less than the domestic degree holders because the cost of doing a Ph.D. degree from a foreign university is higher. Our sensitivity analysis reveals that changing the retirement age from 60 to 55 and 65 does not affect these results. However, the difference between the net returns decreases if the retirement age is 55 and increases in the case of 65. Similarly, increasing the completion time of a Ph.D. also affects the net lifetime private economic return negatively. The research on education as a form of investment and investigation of the role of human capital started in the 1960s. The interest in this area was enhanced by the observation that investment in human capital positively affects output, especially the rise in earnings per worker Schultz . The theThe literature on returns to higher education indicates that schooling positively impacts an individual’s lifetime earnings  the impact of one extra year of completing the Ph.D. degree, (b) the impact of changes in retirement age and (c) the effects of varying interest rates.The prior studies have examined the private rate of returns at different levels of education, including primary, secondary, matric, intermediate, etc. Moreover, previous studies focused on the impact of one extra year of schooling on private returns. This research calculates the lifetime private economic returns of Ph.D. over non-Ph.D. faculty members working in the higher education sector in Pakistan.To estimate the returns to educational investment at the higher education level in Pakistan to find the lifetime economic returns of Ph.D. over non-Ph.D. faculty members in the education sector of Pakistan.Compare the lifetime economic returns of Ph.D. faculty members who have Ph.D. degrees from domestic and foreign universities.The study focuses on these objectives:The rest of the paper is organized as follows. Section 2 presents the results of the studies that have already been conducted. Section 3 explains the data set, sample size, and the estimation technique used in our analysis. The preliminary results are analyzed in the first part of section 4. The second part has an empirical analysis that presents the difference in the rate of returns of Ph.D. over non-Ph.D. faculty members in Pakistan. Finally, the summary and discussion of the estimated results are exhibited in section 5.This section covers a brief review of the prior literature on the private economic returns of educational investment in different levels of education. Becker  considerMeanwhile, there is a tradeoff between schooling years and the working life span, so the lifetime earnings will decrease with one extra year of schooling. Yu et al.  examine Psacharopoulos  analyzesBlundell et al.  examine Ganyaupfu  estimateIqbal and Bashir (2010) investigate the private return rate at different education levels, which shows that returns of Ph.D. degrees would be increased by increasing the retirement age. The rate of return at the higher education level is estimated using the internal rate of return (IRR). The study results were based on the four levels of education, including Professional Bachelor, Master, M.Phil, and Ph.D., which shows that the rate of return for Master and M.Phil is higher than for the Bachelor and Ph.D. level. The direct and opportunity costs are the critical reasons for the low rate of return of a Professional Bachelor’s degree. Afzal et al.  examine Education level determines the difference in private economic returns of individuals. Numerous factors impact the private economic returns of Ph.D. over non-Ph.D. faculty, including the study area, experience, level of education, degree country, gender, and the nature of work or job. As per the literature, the level of education and experience are the major factors contributing to the growth of private economic returns See Fig. . But thej amount at time T* = tj where j is the number of years of schooling.As per the literature, education costs are forgone earnings and direct costs, but investment in education pays higher returns. If these private economic returns are higher than foregone earnings and direct costs, investment in higher education is worthwhile. Suppose that education starts paying Yj) and the annual wage increment (ΔYj).For simplicity, it is assumed that education costs constantly increase at a certain level. Moreover, the cost of education will be different by the level of education with the J years of schooling. The study’s objective is to estimate the private economic returns of Ph.D. over non-Ph.D. faculty of Pakistan using the augmented human capital earning function. Initially, the earning function was based on years of schooling and professional experience, but the current study focuses on the level of education instead of the years of schooling that positively correlated with earnings. An individual’s lifetime earnings are based on the starting wages (Y1) & 21 (J2) years of education. The starting wages of an individual with a Ph.D. (J2) and non-Ph.D. (J1) degrees are Y2 and Y1, and the constant annual increments are (∆Y2) & (∆Y1) till the retirement age (T), respectively. The initial point of Yj and (∆Yj) depends on the level of education. The lifetime earnings of an individual are given by the present value of expected lifetime earnings associated with education (PVE), given starting wage (Yj), and constant increment (∆Yj).The study estimates the rate of return of Ph.D. and non-Ph.D. faculty with 18 , Area of Study (AOS), Marital Status (MS), Funded vs. non-Funded (FON), Country of Degree (COD), Job Switch (JS), Employment Sector like a public or private, and Level of Study (S). The dependent variable of Eq. . Our objective is to estimate the private rate of return of Ph.D. over non-Ph.D. faculty with the help of earning function. Moreover, the rate of return will be calculated by the NPV method, where the coefficient values of regression analysis help calculate the private return. In this study, “T” denotes retirement age (60 Years), while the starting working age for non-Ph.D. and Ph.D. faculty members are assumed to be 25 and 29 years, respectively. Therefore, the values of the coefficients are first estimated from Eq. . Then, tThe study data were collected through a questionnaire survey, and the survey respondents were Ph.D. and non-Ph.D. faculty members of Pakistani universities. The survey respondents belonged to different cities in four provinces of Pakistan, including Islamabad, Rawalpindi, Lahore, Multan, Rahim Yar Khan, Bahawalpur, Faisalabad, Karachi, Jamshoro, Larkana, Quetta, Mardan, and Peshawar. The data were collected using a stratified random sampling technique where the total population is divided into subgroups known as strata, such as Ph.D. and non-Ph.D. faculty members, male and female, and public & private sector universities. The survey respondents were those faculty members who completed their terminal degrees from 2001 to 2020.^2 is the critical value of the normal distribution at α/2 , e^2 is the margin of error of 5%, P is the Sample proportion 0.5, and N is Population Size. The total population of the Ph.D. and non-Ph.D. faculty members in different Pakistani universities are 47,346, containing 14,614 Ph.D. and 32,782 non-Ph.D. faculty members  is the difference between the present value of cash inflows and cash outflows over time discounted by some rate. Therefore, the method incorporates the cost of acquiring education and lifetime earnings. As discussed earlier, the study calculates the NPV of a Ph.D. and non-Ph.D. lifetime earnings for faculty members in Pakistan. If NPV is positive (NPV > 0), the return on educational investment is positive. If NPV < 0, the return on education investment is negative. Finally, there will be no loss or profit if NPV = 0. The NPV is calculated using Eq. .7\documeAs discussed earlier, the objective is to calculate the net present value of lifetime earnings of Ph.D. over non-Ph.D. faculty members. So, Eq.  defines The undiscounted expected lifetime earnings of a Ph.D. faculty member are 67.8 and 70.4 million Pakistani rupees (PKR) that hold degrees from Pakistani and foreign universities, respectively. The Ph.D. degree cost is divided into two parts such as explicit (Direct Costs) and implicit costs. The explicit cost is PKR 6,73,327, and the implicit cost is PKR. 2.72 million. The total degree cost in Pakistan is almost PKR. 3.4 million, and most Ph.D. scholars earn, on average, PKR. 5,09,410 during the degree (Interim earnings). Therefore, after deducting interim earnings from the total cost, the total net cost of the degree in Pakistan is PKR. 2.89 million.The total cost of a foreign Ph.D. degree is PKR. 7.51 million comprises explicit and implicit costs of PKR. 4.8 and 2.72 million, respectively. On the other hand, a foreign Ph.D. degree holder earns PKR. 1.12 million during the degree. Therefore, the total net cost is PKR. 6.38 million after deducting interim earnings from the total cost.The human capital earning function is applied to calculate an individual’s lifetime private economic returns. Therefore, regression analysis (using earning function) and Net Present Value (NPV) methods are used. In step one, the earning function method is used for different education levels, experience, sectors of employment, and other control variables. In the second step, lifetime private economic returns of Ph.D. over non-Ph.D. faculty are calculated through the NPV method. In the first step, the forgone earnings were not in consideration, so in the second step, total cost (forgone earnings and direct expenditures) was subtracted from Present lifetime earnings.Most studies have used the Internal rate of return (IRR) method, but the IRR method will not achieve the required results (As per study objectives) of this study, such as  has been used to detect the multiclonality problem. The value of VIF is 2.79, which is less than 10, so the multicollinearity problem is not established. Furthermore, the Wu-Hausman test is used to detect the problem of endogeneity, which showed the For calculating NPV, an average 7% interest rate on Pakistan Investment Bonds (PIBs) is used. The data on interest rates is accessible on the Census and Economic Information Center (CEIC) and Economic Survey of Pakistan 2021 , the NPV of a Ph.D. degree is Rs 6.85 million, which is 46.5% higher than the earnings of the non-PhD faculty.Compared to this, the foreign Ph.D. degree holder earns 29.8% higher returns than a non-Ph.D. faculty. This means that the NPV of a Ph.D. degree is higher for those who have completed Ph.D. degrees from domestic universities. This is due to the significantly higher cost of financing a Ph.D. degree from abroad. The results of Bashir and Iqbal  study alTable The earnings of male Ph.D. faculty are 3% higher than the combined results Table  of both Table Figure The cumulative net present value of lifetime earnings of non-Ph.D. faculty is higher than Ph.D. till the age of 32 and 38 years depending upon the source of the Ph.D. degree  sectors.


With discoveries of brain and spinal cord mechanisms that control gait, and disrupt gait coordination after disease or injury, and that respond to motor training for those with neurological disease or injury, there is greater ability to construct more efficacious gait coordination training paradigms. Therefore, it is critical in these contemporary times, to use the most precise, sensitive, homogeneous , and comprehensive measures available to assess gait coordination, dyscoordination, and changes in response to treatment. Gait coordination is defined as the simultaneous performance of the spatial and temporal components of gait. While kinematic gait measures are considered the gold standard, the equipment and analysis cost and time preclude their use in most clinics. At the same time, observational gait coordination scales can be considered. Two independent groups identified the Gait Assessment and Intervention Tool (G.A.I.T.) as the most suitable scale for both research and clinical practice, compared to other observational gait scales, since it has been proven to be valid, reliable, sensitive to change, homogeneous, and comprehensive. The G.A.I.T. has shown strong reliability, validity, and sensitive precision for those with stroke or multiple sclerosis (MS). The G.A.I.T. has been translated into four languages ), and is in use in at least 10 countries. As a contribution to the field, and in view of the evidence for continued usefulness and international use for the G.A.I.T. measure, we have provided this update, as well as an open access copy of the measure for use in clinical practice and research, as well as directions for administering the G.A.I.T. There have been important discoveries of brain and spinal cord mechanisms controlling gait coordination e.g., ,4,5,6,7),5,6,74,5Based on the available discoveries, there is a greater ability to construct more efficacious gait coordination training paradigms for those with neurological injury or disease. With new interventions targeted at improving the neural drive of gait coordination, it is critical in these contemporary times to use the most precise, sensitive, homogeneous , and coGait coordination is defined well by Krasovsky and Levin , page 21“Locomotor coordination is a context-dependent property of the motor system, having both spatial and temporal components. Spatial coordination is the relationship between the position of different body segments or joints, whereas temporal coordination is the relative timing between segment or joint positions throughout the task. These components are never mutually exclusive……”Therefore, an example of a gait coordination measure is ‘knee flexion angle at toe-off’. In this example, the spatial component is knee flexion angle and the temporal component is the gait event time of toe-off. Without both the spatial and temporal components simultaneously being performed, there is no measure of coordination. The technology-based gold standard of gait coordination measurement and force production includes gait kinematics and gait kinetics, customarily measured using motion capture and force plate systems in well-equipped gait research laboratories . StudiesIn clinical practice and in some gait research settings, there is no access to such technology. Furthermore, the time constraints of clinical practice make it impractical to utilize this instrumented technology in its current form due to the labor-intensive process of collecting and analyzing the data. However, observational gait coordination measures can be used in that case. The ideal observational gait coordination measure would be constructed according to the following characteristics:Homogenous, that is, all items measuring gait coordination/dyscoordination, according to the above definition of coordination, and no items assessing compensatory strategies.As comprehensive as possible, that is, measuring as many coordination joint movements and other gait components as possible.Numerical scoring scheme overall, and per item, with score numbers within an item assigned across levels of dyscoordination.Good psychometrics .Good sensitivity in measuring the recovery of gait coordination, both endogenous and in response to treatment.Practical, with a reasonable scoring time and the use of equipment available in most clinical settings.Two separate research groups conducted a review of observational gait measures ,17. Theyp = 0.0001; 95% confidence interval (CI)= 0.95–0.99), and inter-rater reliability , including between an experienced and an inexperienced clinician  [p = 0.045, 95% CI= 0.023–2.18)) [The G.A.I.T. psychometrics were originally tested in stroke survivors. The G.A.I.T. was constructed with items measuring the spatial coordinated movement components of gait (movement excursion), that occur at specified temporal events of stance and swing phases of the gait cycle. Within each item of the measure, deviation from normal coordination is scored at multiple levels of dyscoordination. Psychometrics were studied and found to be good, according to intra-rater reliability (intraclass correlation (ICC) = 0.98; 6–0.999) . The G.A3–2.18)) .Very recently, two independent groups have estimated the minimal clinically important difference (MCID) for the G.A.I.T. in stroke survivors ,30. In aRecently, the G.A.I.T. psychometrics were studied for those with multiple sclerosis (MS). Construct validity was tested . The resIn view of the evidence for the continued usefulness and international use for the G.A.I.T. measure, we have provided this update, as well as an open access copy of the measure for use in clinical practice and research . We are


Apis cerana, is an important pollinator of crops and natural phytocoenosis in Asia. Natural barriers in the form of forests, mountains, rivers and seas have created the prerequisites for the formation of subspecies and ecotypes of the A. cerana. We analyzed the genetic structure of A. cerana samples from the Russian Far East, Vietnam and South Korea. High haplotype diversity was found in all samples of A. cerana based on analysis of the tRNAIeu-COII locus sequences. The found polymorphisms can be used as genetic markers to differentiate subspecies of A. cerana.The oriental honey bee, Apis cerana samples from the Russian Far East, South Korea and Vietnam. An analysis of the polymorphism of seven microsatellite loci and an assessment of the haplotype diversity of the mtDNA tRNAleu-COII locus were performed. A fragment of about 431 bp in tRNAleu-COII was sequenced. The analysis showed the presence of 14 haplotypes, while the predominant haplotype was Japan1. Microsatellite data revealed two differentiated clusters. The first cluster contained tropical climate A. cerana samples from Vietnam, and the second cluster combined temperate climate A. cerana samples from the Russian Far East and South Korea.In this article, we present the results of the genetic analysis of  Apis cerana F.) plays an important role in local ecosystems and agriculture in Asia. The natural range of A. cerana has embraced almost all Asian countries, from Indonesia to 47°54′ N latitude in Khabarovsky Krai of Russia , hatRNAleu-COII haplotypes were detected in our study . The majority (21 sequences) of our 46 A. cerana sequences of the tRNAleu-COII locus belonged to the Japan1 haplotype  and the Korea14 haplotype (four samples from FE). Previously, Tan et al. (2007) [A. cerana samples on Hainan island belonged to the haplotype Japan1. These 69 haplotypes differed from the Japan1 haplotype by 1 to 12 substitutions. The greatest differences (12 substitutions) were found with haplotype H3 (DQ381965.1). The H3 haplotype also included two samples from our study, which formed a separate branch on the phylogenetic tree  and determine the genetic structure of the study samples. Analysis of the structure output data in Structure Harvester showed that the total sample consists of two main clusters . The first cluster was represented by SK and FE, and the second cluster included sample Vn (excluding one colony). A. c. indica selected in Vietnam from A. cerana colonies selected in South Korea and the Far East. Thus, microsatellite data analysis showed that samples from Vietnam may indeed belong to the A. c. indica.Despite the small sample size, there was a division into two clusters. We observed differentiation of the A. cerana populations from the Far East, South Korea and Vietnam. In total, 14 tRNAleu-COII haplotypes were identified during the analysis of 46 sequences with a fragment size of 431 bp. As in previous studies [tRNAleu-COII locus, the following problem was identified: the authors of the studies used different fragments of this locus, which is due to the sample preparation and capabilities of the sequencer used. Accordingly, the longer the fragment, the more likely it is to identify new haplotypes.The aim of this work was to establish whether there are genetic differences between geographically distant of  studies ,11,17, JA. c. indica based on morphometric methods [tRNAleu-COII haplotype diversity) confirmed that it may belong to this subspecies. Thus, this set of microsatellite loci can potentially be used to differentiate A. c. indica. However, this requires further research with an increase in the number of samples and expansion of the geography of sampling. The genetic structure of samples from the Far East and South Korea was uniform. Using a selected set of microsatellite loci, we were unable to identify differences between them. Analysis of the polymorphism of microsatellite loci showed that the studied samples form two clusters. One of the clusters was formed by samples from South Korea and the Far East, and the other was formed by a sample from Vietnam. Most of the samples from Vietnam (11 out of 14) were previously identified as  methods . Our datA. cerana phenotypically divided into four subspecies/morphoclusters: A. c. cerana , A. c. indica (in southeast Asia and southern India) and A. c. himalaya  and A. c. japonica (Japan). Radloff et al. (2010) [A. cerana populations across its full geographical range and showed that they are subdivided into six morphoclusters. Similar to studies of A. mellifera subspecies [A. cerana subspecies are few and are mainly based on the analysis of polymorphisms of mtDNA genes [A. cerana populations from Vietnam belong to morphocluster IV (previously named A. c. indica and A. c. javana). Whereas populations from South Korea and the Far East belong to morphocluster I . Thus, we see that there is still no single approach to the classification of A. cerana subspecies and further studies at the genetic level are required. The accurate identification of the A. cerana subspecies will allow for the preservation of the unique gene pool of the local subspecies.According to Ruttner 1988), 88, A. cef et al. 10 [A. ceNA genes ,21. Acco