Insect Samples Research Articles

Bacillus thuringiensis strains with high insecticidal activity against insect larvae of the orders Coleoptera and Lepidoptera

Abstract The concept of sustainable agricultural development is aimed at reducing the use of chemical pest control agents. Since the use of chemical pesticides poses a significant environmental threat, one of the possible solutions to overcome this problem is the study of biological approaches, in particular, the use of entomopathogenic microorganisms as biological pest control agents. Insecticidal activity of over 220 strains of Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus from the collection of the Laboratory of Spore-Forming Microorganisms of the Microbial Depository Center of the Scientific and Production Center “Armbiotechnology” and 15 strains isolated from samples of dead insects (imago) of representatives of various genera of the Coleoptera order was studied. The pathogenic effect of the strains was assessed in relation to the insect species Tenebrio molitor, Zophobas morio, Leptinotarsa decemlineata Say and Pieris brassicae, belonging to the Coleoptera and Lepidoptera orders. Among the test objects, the representative of Lepidoptera Pieris brassicae demonstrated the highest degree of sensitivity to B. thuringiensis strains up to 100%, with some Bt strains causing 60–100% mortality of Tenebrio molitor and Zophobas morio larvae of different ages. The larvae of the beetle Leptinotarsa decemlineata Say were also partially susceptible, presenting mortality levels of between 31 and 60%. The article is devoted to the study of Bt bacterial strains that have pronounced insecticidal activity against insect larvae of the orders Coleoptera and Lepidoptera.

HPLC-MS/MS and ICP-MS for Evaluation of Mycotoxins and Heavy Metals in Edible Insects and Their Defatted Cakes Resulting from Supercritical Fluid Extraction.

Edible Insects (EIs) are an alternative source of bioactive compounds such as proteins or fatty acids and micronutrients as vitamins or minerals, thus showing potential to replace traditional foodstuffs in an economical and environmentally friendly way. Nonetheless, EIs can accumulate hazardous chemicals such as mycotoxins and heavy metals. The aim of the present study is to determine mycotoxins and heavy metal content in raw insect samples and those resulting products obtained after supercritical fluid extraction (SFE). Insect samples included Acheta domesticus (cricket) meal, Tenebrio molitor (mealworm) meal, Alphitobius diaperinus (buffalo worm), and Locusta migratoria (locust). For this purpose, a QuEChERS method followed by LC-MS/MS analysis was optimized with good results for the analysis of mycotoxins, principally Aflatoxins (AFs), Ochratoxin A (OTA), and Enniatins (ENNs). In contrast, heavy metals (As, Cd, Hg, Pb) were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The results obtained revealed that Locust was positive for AFG2 at a level of 115.5 μg/kg, and mealworm was only contaminated with OTA at 58.1 μg/kg. Emerging mycotoxins (ENNA, ENNA1, ENNB, and ENNB1) were detected at lower levels < 2.2 µg/Kg. Concerning heavy metals, limits exceeding regulation were detected for Cd in the insect species studied, with levels up to 219 μg/kg, and for Pb in crickets (100.3 μg/kg). Finally, the analysis of the post-extraction solids after SFE processing revealed that heavy metals remained in the resulting SFE cakes, while mycotoxins were detected at negligible levels (up to 1.3 µg/Kg).

Identification of entomopathogenic fungi from insect pests of mango and their efficacy against mango hoppers

Surveys were made in the Sindhudurg and Ratnagiri districts of the Konkan region of Maharashtra, India during 2017-2018 for the collection of mummified insect samples from different mango orchards in order to isolate entomopathogenic fungi. The collected mummified insect samples were brought to the laboratory and incubated on PDA plates. Those samples showing growth on PDA plates were sub-cultured until pure fungus growth was obtained. They were tested for their pathogenicity in the laboratory and 14 samples were found pathogenic to mango hoppers. They were sent to NFCCI, Agarkar Research Institute, Pune for identification. Out of the 14 entomopathogenic samples identified, 7 were from Genus Fusarium, 4 were mixed cultures of Fusarium and Aspergillus, and one sample each from Genus Trichoderma, Mucor and Nigrospora. Studies showed that Fusarium species were the most common entomopathogenic fungi associated with mango pests, especially mango hoppers. Laboratory evaluation of these samples showed that amongst different isolated entomogenus fungi, &lt;i&gt;Fusarium semitectum&lt;/i&gt; sample 16(1) was more pathogenic to mango hoppers than other fungal samples. Field evaluation of this sample also proved its efficacy.

Exploring unified methods of killing and storing insect samples for forensic entomotoxicology using diazepam in Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae

Forensic entomologists use the maturity of necrophagous larvae to estimate the minimum post-mortem interval (PMImin), ideally taking account of effects that xenobiotics in the corpse may have on insect maturation. Forensic toxicologists may employ larvae to detect drugs in drug-related deaths when human samples are unavailable. Yet current pre-analytical practices of these two professions differ significantly, impeding the successful use of the same samples. Potential benefits of shared pre-analytical practices and opportunities for enhanced collaboration have yet to be fully explored. We employed Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae, grown in the presence of diazepam, to investigate the effects of two standard investigative practices on larvae for drug detection and for quantifying mass and length as proxies of age. Specimens were killed by either blanching or freezing and stored at -20℃ for either intermediate or long periods. Blanched larvae showed smaller changes in size and body integrity during storage, thereby producing the most reproducible estimates of PMImin. Consequently, data obtained from blanched larvae were used to evaluate the impact of diazepam on larval development. Diazepam exerted no significant effect on larval mass, and a weak effect on length. Diazepam recovery was significantly higher from blanched larvae, suggesting that freeze-killing causes drug loss. This model system demonstrates the value to forensic entomologists of the standard technique of blanching larvae, followed by storage at -20℃ for toxicological analysis. We recommend that forensic toxicologists consider blanching to kill larvae before storage at low temperatures, at least for certain drugs. This approach offers the dual benefit of high-quality specimens for both PMI estimation and drug detection.

A preliminary recording of insects on the island of Farwa Northwest of Libya

The study aimed to present the first record of insect families in Farwa Island. Farwa Island is the largest and most important island in Libya; located in Northern west part of Libya in the Mediterranean, its insect fauna is poorly studied. Short time intensive collection of insect samples was conducted in four areas (mouth of the valley, bay of containers, center of the Island, and Ras-Attalgha) in February, March and April 2021. Different methods were used for insect collection, sorting, preservation and later identification to the lowest taxonomic level and verification. 9 orders, 31 families, 40 genera and 58 insect species were collected in the study; the center of the Island had the highest numerical density and diversity of insect fauna due to its diverse vegetation compared to other areas. Diverse suitable habitats and absent insect control measures provided suitable environmental conditions for insects breeding, feeding and hiding from enemies. This study constitutes the first comprehensive survey of insect fauna on Farwa Island and adds significant value to the ongoing assessment of insect diversity in Libya. A long-term study has to be conducted to investigate detailed information about the abundance and diversity of insects and other arthropods on Farwa Island.

A Microcosm Analysis of Species-Specific Responses of Chironomidae on Heavy Metal Pollution in The Nyanza Gulf of Lake Victoria

The Chironomidae family, known as "non-biting midges" in their adult stage and "bloodworms" in their larval stage, consists of diverse dipteran insects inhabiting various global aquatic environments. Despite extensive global research, data on Chironomidae in the polluted Nyanza Gulf of Lake Victoria, Kenya, is scarce, and molecular identification methods have not been explored. This study aimed to quantify heavy metal concentrations in water, sediment, and insect samples and assess their impact on Chironomid species identified using mitochondrial DNA barcoding of the cytochrome oxidase subunit 1 (COI) gene. Analysis of Variance was used to determine if there were any statistically significant differences in heavy metal concentrations across different sample types or locations along the pollution gradient. Chironomids were collected from Nyanza Gulf, focusing on a pollution gradient. Results showed that concentrations of arsenic (As), lead (Pb), and cadmium (Cd) in insect, water, and sediment samples exceeded standard limits, while mercury (Hg) concentrations were within limits. Significant variations (p ≤ 0.05) in Pb levels were observed in water samples, and heavy metal concentrations in sediment samples varied significantly (p ≤ 0.05), with Pb showing the highest variation (p ≤ 0.0001). Insect samples exhibited significant differences (p ≤ 0.0001) in As and Hg contents. Genetic analysis identified two known species: Chironomus transvaalensis at the heavily polluted Kisumu station and Chironomus pseudothummi at the moderately polluted Kendu Bay and Homa Bay stations. Additionally, a unique Chironomus species was found on Ndere Island, a relatively clean site with restricted human activities. Sequence comparisons indicated proximity to global data but also highlighted the evolutionary significance and uniqueness of the identified species. This study demonstrated the potential use of genetic methods in determining Chironomid species diversity, community structure, and abundance in relation to heavy metal concentration. It suggests that heavy metal pollution may act as a selective pressure, driving the evolution of Chironomid species. The study recommends combining genetic approaches with other pollution sources for a comprehensive understanding of using this species in monitoring pollution

Interlaboratory Evaluation of Bretziella fagacearum Molecular Detection Assays to Guide the eDNA Monitoring of Oak Wilt Disease

ABSTRACTOak wilt disease, caused by the fungus Bretziella fagacearum, can kill mature red oaks within months of infection, severely affecting biodiversity, landscapes, and industries. The disease, originally only present in the United States, was officially reported for the first time in Canada in June 2023. The aim of this study was to suggest a standardized assay and sample processing method to optimize oak wilt detection both in infection centers and ahead of the disease front. Two previously published molecular assays, a Nested PCR and a TaqMan qPCR, were compared to detect B. fagacearum in a variety of samples in a ring trial across five laboratories. Sample types investigated included eDNA from trapped insect vectors (sorted insects and bulk content from traps), infested and healthy oak wood chips, and B. fagacearum conidia dilutions. Results demonstrated that both Nested and TaqMan assays can be used for molecular confirmation of oak wilt, and results are reproducible across different labs. There is a general agreement between both detection assays when testing true‐positive and true‐negative samples. Both methods demonstrated overall good accuracy. The TaqMan assay was more sensitive and detected lower amounts of DNA target. Both tests were 100% specific to oak wood samples, which was the best sample type to use for detection. In general, samples with high Cts were more prompted to yield false negative Nested results. Detecting oak wilt from bulk insect samples was by far more rapid than sorted sap beetles, but resulted in lower detection signals, especially with the Nested assay. The time‐period when the insect traps were set up also had considerable influence on detection results. We hope this study helps to formulate guidelines in oak wilt detection and biosurveillance management.

Insect community structure in young and productive coffee farms under leucaena shade of Jember Regency, East Java, Indonesia

The developmental phase of Robusta coffee plantations with agroforestry systems exhibits varying ecosystem characteristics (biotic and abiotic), therebyinfluencing the dynamics of insect community composition within them. One of the major coffee-producing centers in East Java is Jember Regency, where coffeecultivation commonly employs agroforestry systems with various shade trees. Understanding the abundance of insect species in agroforestry coffee plantations is important for determining insect pest control measures. This research aims to study the insect community structure in young coffee and productive coffee farms. Insect sampling was conducted in young coffee farms and productive coffee farms at the Kaliwining Experimental Station, Indonesian Coffee and Cocoa Research Institute (ICCRI), by using road sampling methods with insect nets and pitfall traps. Both types of plantations utilize Leucaena sp. var L2 as shade trees, forming agroforestry coffee plantation systems. Data analysis was performed by calculating the Shannon-Wiener diversity index (H’) to assess insectdiversity and the independent sample t-test to determine differences in insect abundance based on functional roles between the two farms. The results indicatethat the composition and abundance of insects in productive coffee farms are higher than in young coffee farms, resulting in a higher insect diversity index valuefor productive coffee farms (categorized as high), while young coffee farms fall into the moderate category. Furthermore, based on their functional roles, productive coffee farms positively influence the abundance of herbivorous, predatory, parasitoid, and detritivorous insects.

Investigating Spodoptera spp. diversity, percentage of attack, and control strategies in the West Java, Indonesia, corn cultivation

Abstract Corn, a crucial food crop in Indonesia, faces significant challenges from pests such as Spodoptera spp., impacting agricultural productivity and industrial applications. This study aimed to study the diversity and attack percentage of Spodoptera spp. on corn plants, along with the employed control techniques. The research was conducted using a survey method through field observation and farmer interviews. The insect sampling was carried out by purposive sampling, while the sampling of plants using the diagonal sampling method was done in three areas that were representative of the West Java province, Indonesia (Sumedang Regency, Cirebon Regency, and Bandung Regency). The results showed that Spodoptera frugiperda or fall armyworm has emerged as a dominant pest in all three areas. The diversity index (H′) of S. frugiperda across the Cirebon Regency, Bandung Regency, and Sumedang Regency was consistently low (&lt;1). Varying infestation rates across districts, with the Cirebon Regency showing lower rates and Bandung and Sumedang Regencies exhibiting medium-level infestation. Farmers in Bandung and Sumedang Regencies are adopting a more diverse pest management strategy, including the regular monitoring and manual collection of larvae. In addition, the survey shows a positive relationship between the knowledge, skills, and attitude of farmers, suggesting that acquiring more information and understanding agricultural techniques contribute to effective task execution and foster positive mindsets. This comprehensive investigation sheds light on the regional variations in Spodoptera spp. dynamics, providing valuable insights for sustainable pest management in the corn cultivation of the West Java Province, Indonesia.

The burrower bug Macroscytus japonensis (Hemiptera: Cydnidae) acquires obligate symbiotic bacteria from the environment

Many plant-feeding stinkbugs belonging to the infraorder Pentatomomorpha possess a specialized symbiotic organ at the posterior end of the midgut, in which mutualistic bacterial symbionts are harbored extracellularly. In species of the superfamily Pentatomoidea, these symbionts typically are verticallytransmitted from host mothers to offspring, whereas in species of the superfamilies Coreoidea and Lygaeoidea they are acquired from the environment. In the pentatomoid family Cydnidae, vertical symbiont transmission has been reported in several species. Here, we report the first case of environmental symbiont acquisition in Cydnidae, observed in the burrower bug Macroscytus japonensis. A comprehensive survey of 72 insect samples from 23 sites across the Japanese archipelago revealed that (1) symbionts exhibit remarkably high diversity, forming six distinct phylogenetic groups within the Enterobacteriaceae of the γ-Proteobacteria, (2) most symbionts are cultivable and closely related to free-living Pantoea-allied bacteria, and (3) symbiont phylogenetic groups do not reflect the host phylogeny. Microbial inspection of eggs revealed the absence of bacteria on the egg surface. These results strongly suggest that symbionts are acquired from the environment, not vertical transmission. Rearing experiments confirmed environmental symbiont acquisition. When environmental symbiont sources were experimentally withheld, nymphs became aposymbiotic and died before molting to the second instar, indicating that nymphs environmentally acquire symbionts during the first-instar stage and that symbionts are essential for nymphal growth and survival. This study highlights Cydnidae as the only pentatomoid family that includes species that environmentally acquire symbionts and those that vertically transmit symbionts, providing an ideal platform for comparative studies of the ecological and environmental factors that influence the evolution of symbiont transmission modes.

Leptoglossus occidentalis Heidemann, 1910 (Hemiptera Coreidae) in Türkiye Its distribution, life cycle, and fungal associations

In this study, the distribution, life cycle and associations with fungi of the alien invasive species Leptoglossus occidentalis Heidemann, 1910 (Hemiptera: Coreidae) in Türkiye were investigated. During the study between 2019 and 2021, we found that the pest has spread almost all over Türkiye. We recorded the presence of the species in 35 provinces. The biological data obtained in the study were divided into ecologically similar sub-regions (Ankara-Çankırı-Kırşehir, Artvin, Isparta-Burdur, Izmir-Aydın-Manisa-Muğla-Antalya) in order to reveal the life cycle of the species. Additionally, host plant and insect samples were examined for the presence of Diplodia sapinea (Fr.) Fuckel (pine shoot blight agent) and entomopathogenic fungi species through morphological and molecular identification methods. Consequently, we found that L. occidentalis has two generations in Türkiye. Timing of the first flight was in early May-mid June and the second generation was from mid-July to late August. Although D. sapinea was detected in some of the sampled host plant tissues, it could not be isolated from L. occidentalis individuals. Accordingly, we did not find any evidence that L. occidentalis vectors D. sapinea although the insect and the fungus cooccurs in sampling areas. On the other hand, its vector potential cannot be ruled out especially during rainy periods. This potential remains as a hypothesis for future studies. Although we detected 25 fungal species isolated from L. occidentalis adults, none of them were entomopathogenic fungal species.

Phytoplasma Associated with White-backed Planthopper on Rice Plants in Sidrap Regency, South Sulawesi

South Sulawesi is one of the largest rice production centers in Indonesia. Several important diseases of rice plants, such as those caused by viruses and phytoplasmas, can be transmitted by insect vectors, especially leafhoppers and stem plant. Symptoms of diseases caused by viruses and phytoplasmas are quite diverse but visually similar and difficult to distinguish. This study aims to analyze the presence of phytoplasma associated with white-backed planthopper which are commonly found in rice plantations. The research method used is by conducting surveys and explorations of insect samples in six villages in Sidrap District. White-back planthoppers found on rice plantations showing symptoms of yellowing and stunted leaves were sampled for further analysis, including total DNA isolation of insects, standard PCR amplification for insect and Nested-PCR for phytoplasma identification, gene sequencing for both amplicons, and nucleotide analysis using BLAST method and Mega X program. The PCR with CO1 primer successfully amplified a 700 bp amplicon from insects, whereas nested-PCR using fP1/rP7 primers followed by m23SR/R16F2n amplified phytoplasma supposedly around 1800 bp and 1250 bp of 16S RNA gene, respectively. The DNA sequencing analysis results indicate that the insect samples were identified as 83% Sogatella vibix species based on homology percentage analysis using BLAST and Mega X Program. As for the phytoplasma, it leans more towards the 16SrI group or Candidatus phytoplasma asteris (Aster yellows phytoplasma) with a homology percentage of 99%.

Curating BOLD records via Bayesian phylogenetic assignments enables harmonization of regional subgeneric classifications and cryptic species detection within the genus Tachina (Diptera: Tachinidae)

Abstract DNA barcoding is a popular tool for species identification of insect samples, with important applications for plant protection. However, Barcode of Life Database queries related to tachinid samples can produce ambiguous results due to contradictory species assignments. Therefore, we applied Bayesian phylogenetic inference to 811 Tachina Meigen, 1803 COI-5P sequences containing all contradictory species assignments of Tachina Barcode Index Numbers. Based on phylogenetic assignment, the assemble species by automatic partitioning (ASAP) analysis, the MonoPhy test and analyses of genetic divergence, we identified 805 sequences representing species of the Tachina genus, whereas six sequences were incorrectly assigned to Tachina. The dataset contained 21 described species and at least seven unidentified species. Additionally, we found that the described species Tachina marklini Zetterstedt, 1838, includes two cryptic species based on ASAP partitioning of sequences, phylogenetic inference and geographical metadata. Furthermore, we harmonized conflicting subgeneric classifications of the Palearctic and Nearctic by employing different subgenera assignments from both partitions. We aimed at monophyletic subgenera partitions. We largely confirmed the validity of the Palearctic Tachina subgenera and Nowickia as a subgenus of Tachina. However, Palearctic Nowickia species formerly allocated to the outdated genus Fabriciella appeared to constitute one monophyletic group with the subgenus Rhachogaster. We conclude that this set of bioinformatics tools is suitable for curating COI-5P-based DNA barcode libraries. Although, curation is limited due to possible species introgression, horizontal gene transfer, incomplete lineage sorting and human errors the resulting dataset enabled the revision of subgenera concepts and the unambiguous cryptic species detection.

Comparative Toxicological Analyses of Traditional Matrices and Blow Fly Larvae in Four Cases of Highly Decomposed Human Cadavers.

In forensic investigation, determining the time and cause of death becomes challenging, especially in cases where the remains are found in advanced decomposition, rendering traditional toxicological samples unavailable or unreliable. Entomotoxicology, an emerging methodology within forensic science, leverages insect specimens collected from cadavers as alternative toxicological samples. Several laboratory and field research studies have highlighted the efficacy in detecting various drugs, toxins, and elements absorbed by insects feeding on cadaveric tissues, even at low concentrations. However, correlation studies between drug concentrations in conventional matrices and insects remain controversial due to unknown factors influencing drug metabolism and larval feeding activity. This paper presents four real cases in which human cadavers were discovered in advanced stages of decomposition, and toxicological analyses were performed on both insect samples and available matrices. The results presented complement the scant literature currently available on the application of entomotoxicology in real cases, providing insights into the correlation between larvae and human specimen results. Furthermore, guidelines to collect and preserve entomological evidence at the crime scene and during the autopsy for use in entomotoxicological analyses are provided. This advancement holds promise in aiding forensic investigations, particularly in cases where traditional methods cannot be applied or require supporting data for further validation.

Adapting to changing methodology in a long‐term experiment

AbstractLong‐term experiments are critical for understanding ecological processes, but their management comes with unique challenges. As time passes, projects may encounter unavoidable changes due to external factors, like availability of materials, affecting aspects of their research methodology. At the Kellogg Biological Station Long‐Term Ecological Research Site, one of the many National Science Foundation‐funded long‐term research stations, a three‐decade project recently experienced a supply‐chain‐induced change in insect sampling methodology in their lady beetle observation study. Since 1989, lady beetles (Coleoptera: Coccinellidae) have been sampled weekly over the growing season using yellow sticky cards. In 2021, the original sticky traps were discontinued by the manufacturer and replaced with a similar, but not identical trap. We conducted a 3‐year study while the new traps were phased in to examine how the trap change would impact the observed biodiversity patterns at the site. We examined community metrics and individual taxa captures to examine within‐year and between‐year differences in performance between the card types. Overall, we noted several small but statistically detectable differences in capture patterns between the two trap types. After accounting for other sources of variation, we observed a difference in Shannon diversity of insects captured on the two card types, but not richness or abundance, for the overall insect community. Yet, these differences were dwarfed by the magnitude of difference observed between years within card types. For individual taxa, similar patterns held: between trap differences could be detected statistically, but the number of differences in capture rate between trap types was less than the number of differences observed for the same trap, between years. Thus, we conclude that while subtle changes in methodology could impact data produced in long‐term experiments; in this case, the magnitude of this change is smaller than other factors such as time and plant treatment. However, if sustained changes in the capture rates of focal taxa are observed, future data users may use our observations to specifically quantify and correct for these shifting patterns related to the protocol change.

Insect Species Diversity in Bangek River Area Lubuk Minturun Padang

The study employed the insect net method to sample insects from the Bangek River. Primary and secondary data were collected for analysis. The research was conducted at Bangek River, Lubuk Minturun District, Padang City, West Sumatra Province. Insect sampling involved a single event using an insect net within the administrative area of Lubuk Minturun. A total of 35 insects were captured from the Bangek River, comprising 5 species from the Lepidoptera order, 28 from the Odonata order, and 1 species each from the Hemiptera and Orthoptera orders.

Beyond the surface: Microplastic pollution its hidden impact on insects and agriculture

This study comprehensively investigates the impact of microplastic pollution on soil and plant health, focusing on the test organism S. frugiperda and maize crops. Various plastics were integrated into the soil at different concentrations, with continuous monitoring of soil parameters revealing nuanced effects on soil physicochemical properties. Leaf area consumed by S. frugiperda caterpillars was meticulously quantified, elucidating the influence of plastic treatments on plant susceptibility to herbivory. Fourier Transformed Infrared (FTIR) analysis provided insights into the presence of hydrocarbon-based polymers, while Environmental Scanning Electron Microscopy (ESEM) analysis highlighted the pervasive distribution of microplastics within plant samples. Rigorous methodologies, including controlled extraction and chemical digestion, identified microplastics in plant and insect samples, emphasizing their potential transmission from soil to plants and insects. Advanced analytical techniques such as FTIR and ESEM characterized microplastics' polymer types and properties. The study contributes insights into the nuanced relationship between microplastics and insects, exploring their hormetic effects on insect populations and potential synergies with additives and environmental contaminants. Findings reveal microplastic concentrations in plant samples ranging from 10 μm to 100 μm, with prevalent polymers being Polyethylene (PE) and Polypropylene (PP). Detailed soil analysis underscores microplastics' adverse effects on soil physical, chemical properties, and nutrient status, informing strategies for mitigating plastic pollution and safeguarding environmental and agricultural sustainability through evidence-based policymaking and interdisciplinary collaboration.

Unlocking precision: Advancing rapid field molecular identification of Tuta absoluta across its life cycle using locked nucleic acid strategies

Rapid and accurate insect identification is crucial for effective plant quarantine procedures. This study introduced a reverse transcription recombinase-assisted amplification-lateral flow strip (RT RAA-LFS), specifically designed for on-site detection of the globally quarantined pest, Tuta absoluta, within a 25-minute timeframe. A pivotal strategy involved integrating three locked nucleic acid (LNA) modifications into a species-specific probe, enabling RT RAA to selectively and efficiently amplify the unique haplotype of T. absoluta's mitochondrial cytochrome c oxidase subunit I (COI) gene. After determining the amplification product dilution factor and the volumes of primers, magnesium acetate, and nucleic acid lysate in the amplification system, the finalized RT RAA-LFS system is capable of direct visual detection, complemented by handheld fluorescence detection for quantum dots (QDs) fluorescence on the lateral flow strip. This technique successfully identifies various developmental stages of T. absoluta in crude nucleic acid lysates without the need for genomic DNA extraction. Moreover, it detected the target gene in the genomic solution, reaching concentrations as low as 1.6 pg or 51 copies per 50-µL reaction system, aligning with qPCR results. Field validation with 50 insect samples supports the correlation between RT RAA-LFS quantum dots' fluorescence intensity and LFS color intensity, affirming the system's reliability in estimating the target gene copy number (R=0.584, p < 0.01). This validation emphasizes the practical efficacy of the RT RAA-LFS system for T. absoluta detection, highlighting its potential as a rapid on-site tool in agricultural pest management.

Commensalibacter nepenthis sp. nov. and Commensalibacter oyaizuii sp. nov., from fluid in a Nepenthes pitcher cup and a butterfly (Junonia lemonias) in Thailand.

Two novel Gram-negative, aerobic, rod-shaped, non-motile bacteria, strains TBRC 10068T and TBRC 16381T, were isolated from a fluid sample from a close-pitcher cup (Nepenthes gracilis) and an insect sample (Junonia lemonias), respectively. Comparing the 16S rRNA gene sequences with those found in EzBioCloud's publicly available databases revealed that the two strains exhibited a close genetic relationship with Commensalibacter intestini A911T; the calculated sequence similarities were 98.56 and 97.70 %, respectively. The average nucleotide identity and digital DNA-DNA hybridization values of the two Commensalibacter strains, as well as those of their closely related type strains, were found to be lower than the species demarcation threshold of 95 and 70 %, respectively. The phylogenomic analysis of strains TBRC 10068T and TBRC 16381T showed that they belong to the genus Commensalibacter. However, they formed distinct lineages separate from all other strains of Commensalibacter by use of 81 bacterial core genes. In addition, the comparative genomic analysis revealed that the core orthologues of strains TBRC 10068T and TBRC 16381T, compared to the closely related type strains of Commensalibacter species, had distinct genetic profiles. Strain TBRC 10068T contained 163 unique genes, whereas strain TBRC 16381T contained 83. The three Commensalibacter species possessed Q-9 as the primary isoprenoid quinone homologue. The results of a polyphasic taxonomic investigation indicated that strains TBRC 10068T and TBRC 16381T represent two separate new species within the genus Commensalibacter. The species were designated as Commensalibacter nepenthis sp. nov. with the type strain TBRC 10068T (=KCTC 92798T) and Commensalibacter oyaizuii sp. nov. with the type strain TBRC 16381T (=KCTC 92799T).

Identifying archived insect bulk samples using DNA metabarcoding: A case study using the long‐term Rothamsted Insect Survey

AbstractInsect populations are declining in many parts of the world, but a lack of long‐term monitoring data is impeding our ability to understand and mitigate the causes of insect biodiversity loss. Whilst high‐throughput sequencing (HTS) approaches, such as DNA metabarcoding, have the potential to revolutionize insect biomonitoring through rapid scalable identification, it is unclear to what extent HTS can be applied to long‐term stored insect samples. Archived insect samples could inform forecasting and provide valuable information regarding past changes to biodiversity. Here, we assess the efficacy of DNA metabarcoding to identify archived samples from the longest passive monitoring scheme in the United Kingdom: the Rothamsted Insect Survey (RIS). With a focus on aphids as the target taxa of a national network of suction‐traps, we analyze a 16‐year time‐series of stored samples (2003–2018) using DNA metabarcoding from one of the RIS suction traps as an exemplar. We achieved this by using a non‐destructive DNA extraction protocol, ensuring the integrity of archival samples for further studies. We compared the identities of aphids determined by both metabarcoding (as inferred amplicon sequence variants [ASVs]) and morphological identification and found that metabarcoding detected most genera with varying success (mean &gt; 76%). When comparing the two methods objectively (i.e., including taxa not detected morphologically), however, congruence decreased (51%). We show that minimum sequence copy thresholds can minimize metabarcoding false positives, but at the expense of introducing false negatives, highlighting the need for careful data curation. Detectability of taxa identified morphologically and similarity between the two methods did not significantly vary over time, demonstrating the viability of metabarcoding for screening archival samples. We discuss the advantages and challenges of metabarcoding for insect biomonitoring, particularly from archival samples, including improvements to sample handling, processing, and archiving. We highlight the wider potential of HTS approaches for stored samples from insect monitoring schemes, unlocking the immense potential of global historical time series.