Normalization of WISP1 circulating level and tissue expression following metabolic and bariatric surgery using rat model.

Wingless-type inducible signaling pathway protein-1 (WISP1) is a newly recognized adipokine, associated with obesity and type 2 diabetes (T2DM). This study aimed to investigate the effect of metabolic and bariatric surgery (MBS) on WISP1 circulating (serum) levels and tissue expression using rat models. We initially investigated whether WISP1 circulating levels were altered between the T2DM and normal rats. After confirmation, Sprague-Dawley (SD) rats were obtained and randomly divided as follows: Roux-en-Y gastric bypass (RYGB) group (n = 10), sleeve gastrectomy (SG) group (n = 10), SHAM group (n = 10), and normal control (NC) group (n = 10). Rats were followed for 8 weeks postoperatively. Preoperative and postoperative WISP1 circulating (serum) levels, glucose tolerance test (OGTT), insulin tolerance test (ITT), postoperative WISP1 expression (visceral adipose tissue, VAT; and skeletal muscle, SM), body weight, food intake, and fasting blood glucose levels were recorded. MBS significantly induced glucose control and weight loss. At postoperative week 8, WISP1 serum levels decreased in the MBS groups (P < 0.05); furthermore, WISP1 expression in VAT and SM significantly decreased in the RYGB and SG groups than SHAM (P < 0.05, and P < 0.05, respectively). Whereas the difference in the expression level between SG and RYGB did not amount to statistical significance (P > 0.05). MBS significantly decreased WISP1 serum levels, tissue expression in the VAT, and SM. As WISP1 is a regulator of low-grade inflammation associated with obesity and T2DM, further studies are needed to explore its relevance in MBS.

Sleep fragmentation exacerbates myocardial ischemia‒reperfusion injury by promoting copper overload in cardiomyocytes.

Sleep disorders increase the risk and mortality of heart disease, but the brain-heart interaction has not yet been fully elucidated. Cuproptosis is a copper-dependent type of cell death activated by the excessive accumulation of intracellular copper. Here, we showed that 16 weeks of sleep fragmentation (SF) resulted in elevated copper levels in the male mouse heart and exacerbated myocardial ischemia-reperfusion injury with increased myocardial cuproptosis and apoptosis. Mechanistically, we found that SF promotes sympathetic overactivity, increases the germination of myocardial sympathetic nerve terminals, and increases the level of norepinephrine in cardiac tissue, thereby inhibits VPS35 expression and leads to impaired ATP7A related copper transport and copper overload in cardiomyocytes. Copper overload further leads to exacerbated cuproptosis and apoptosis, and these effects can be rescued by excision of the sympathetic nerve or administration of copper chelating agent. Our study elucidates one of the molecular mechanisms by which sleep disorders aggravate myocardial injury and suggests possible targets for intervention.

From Sleep Deprivation to Severe COVID-19: A Comprehensive Analysis of Shared Differentially Expressed Genes and Potential Diagnostic Biomarkers.

BACKGROUND: This study aims to identify biomarkers through the analysis of genomic data, with the goal of understanding the potential immune mechanisms underpinning the association between sleep deprivation (SD) and the progression of COVID-19. METHODS: Datasets derived from the Gene Expression Omnibus (GEO) were employed, in conjunction with a differential gene expression analysis, and several machine learning methodologies, including models of Random Forest, Support Vector Machine, and Least Absolute Shrinkage and Selection Operator (LASSO) regression. The molecular underpinnings of the identified biomarkers were further elucidated through Gene Set Enrichment Analysis (GSEA) and AUCell scoring. RESULTS: In the research, 41 shared differentially expressed genes (DEGs) were identified, these were associated with the severity of COVID-19 and SD. Utilizing LASSO and SVM-RFE, nine optimal feature genes were selected, four of which demonstrated high diagnostic potential for severe COVID-19. The gene CD160, exhibiting the highest diagnostic value, was linked to CD8+ T cell exhaustion and the biological pathway of ribosome biosynthesis. CONCLUSIONS: This research suggests that biomarkers CD160, QPCT, SIGLEC17P, and SLC22A4 could serve as potential diagnostic tools for SD-related severe COVID-19. The substantial association of CD160 with both CD8+ T cell exhaustion and ribosomal biogenesis highlights its potential pivotal role in the pathogenesis and progression of COVID-19.

Start-up of pilot-scale ANAMMOX reactor for low-carbon nitrogen removal from anaerobic digestion effluent of kitchen waste.

The pilot-scale simultaneous denitrification and methanation (SDM)-partial nitrification (PN)-anaerobic ammonia oxidation (Anammox) system was designed to treat anaerobic digestion effluent of kitchen waste (ADE-KW). The SDM-PN was first started to avoid the inhibition of high-concentration pollutants. Subsequently, Anammox was coupled to realize autotrophic nitrogen removal. Shortcut nitrification-denitrification achieved by the SDM-PN. The NO2--N accumulation (92 %) and NH4+-N conversion (60 %) were achieved by PN, and the removal of TN and COD from the SDM-PN was 70 % and 73 %, respectively. After coupling Anammox, the TN (95 %) was removed with a TN removal rate of 0.51 kg·m-3·d-1. Microbiological analyses showed a shift from dominance by Methanothermobacter to co-dominance by Methanothermobacter, Thermomonas, and Flavobacterium in SDM during the SDM-PN. While after coupling Anammox, Candidatus kuenenia was enriched in the Anammox zone, the SDM zone shifted back to being dominated by Methanothermobacter. Overall, this study provides new ideas for the treatment of ADE-KW.

Mid-Term Diabetes Remission Outcome in Patients with BMI ≤ 30 kg/m2 Following Sleeve Gastrectomy.

BACKGROUND: Bariatric and metabolic surgery is recommended for Asian patients with type 2 diabetes mellitus (T2DM) and BMI ≥ 27.5 kg/m2. However, mid to long-term ·evidence is still lacking. METHODS: Patients' data that underwent laparoscopic sleeve gastrectomy (SG) as the primary surgery at the Affiliated Hospital of Xuzhou Medical University were analyzed. Patients with T2DM diagnosed with either fasting blood glucose (FBG) ≥ 7.0 mmol/L or glycosylated hemoglobin (HbA1c) level ≥ 7.0% and 27.5 ≤ BMI ≤ 30 kg/m2 were included. RESULTS: 24 patients (7 male and 17 female) were included in this study. With a mean follow-up duration of 4.5 ± 1.1 years, the mean percentage of total weight loss (%TWL) was 14.4 ± 6.7%. Postoperatively, nine patients (37%) still required oral anti-diabetic medications, while no patients used insulin. FBG and HbA1c levels declined to 6.3 ± 1.5 mmol/L and 6.0 ± 1.0%, respectively. Fifteen patients (63%) were with HbA1c levels < 7% and without medication requirements, five patients (21%) were with HbA1c levels < 7% with the help of oral anti-diabetic medication, and four patients (16%) were with HbA1c levels > 7% with the help of oral anti-diabetic medication. CONCLUSIONS: Our study provides further evidence that SG could result in both T2DM improvement and remission in patients with BMI ≤ 30 kg/m2. Longer follow-up duration and larger sample will be needed in the future.

Multidisciplinary approach to the diagnosis of Contracaecum magnipapillatum infections in Australian black noddies, Anous minutus (Charadriiformes: Laridae).

We provide the incidental necropsy findings associated with anisakid nematode infections of black noddy terns, Anous minutus Boie, 1844 (Charadriiformes: Laridae), from offshore islands in the southern Great Barrier Reef, Queensland, Australia. Specimens collected from the proventriculi were identified morphologically as Contracaecum magnipapillatum Chapin, 1925 (Rhabditida: Anisakidae), using light and scanning electron microscopy (SEM). The entire nuclear ribosomal DNA internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) was amplified by polymerase chain reaction (PCR) and sequenced to provide reference sequences for morphologically well-identified voucher specimens. Interestingly, after an alignment with closely related taxa using BLAST, sequences of the ITS1 and ITS2 were 100% identical to the sequences assigned to Contracaecum septentrionale Kreis, 1955, from a razorbill, Alca torda Linnaeus, 1758 (Charadriiformes: Alcidae), from Spain. These results either raise questions about the ITS as a genetic marker for some members of Contracaecum, or the identity of the specimens assigned to C. septentrionale, given that no supporting morphological data was associated with them. We highlight the need for a combined morphological and molecular approach to parasite diagnostics and the use of multiple genetic loci to resolve the molecular taxonomy of cryptic species. Morphological identifications should be taxonomically robust, transparent and precede the deposition of molecular barcodes in public repositories. The gross and histopathological findings of our investigation concur with previous reports of widespread Contracaecum infections in black noddies and support the contention that Contracaecum spp. are an unlikely primary cause of mortality.

The Prevalence of Small Intestinal Bacterial Overgrowth After Roux-en-Y Gastric Bypass (RYGB): a Systematic Review and Meta-analysis.

We reviewed the literature on the prevalence of small intestinal bacterial overgrowth (SIBO) after Roux-en-Y gastric bypass (RYGB). Eight studies examining 893 patients were included. The mean age of the patients was 48.11 ± 4.89 years. The mean BMI before surgery and at the time of SIBO diagnosis was 44.57 ± 2.89 kg/m2 and 31.53 ± 2.29 kg/m2, respectively. Moreover, the results showed a 29% and 53% prevalence of SIBO at < 3-year and > 3-year follow-up after RYGB, respectively. Symptoms included abdominal pain, diarrhea, bloating, nausea, vomiting, constipation, soft stool, frequent defecation, flatulence, rumpling, dumping syndrome, and irritable bowel syndrome. SIBO is prevalent after RYGB; digestive symptoms should prompt the consideration of SIBO as a potential etiology. Antibiotic therapy has proven to be therapeutic.

Comparison of three different anastomotic methods of sleeve gastrectomy with transit bipartition using an obese rodent model.

The long-term effects and safety of single-anastomosis sleeve ileal (SASI) bypass have not been confirmed. The one anastomosis procedure carries the risk of bile reflux, and Braun anastomosis has the capacity to reduce bile reflux. This study was designed to compare the influences of bile reflux and histological changes in the esogastric sections of rats. Obese Sprague-Dawley rats underwent sleeve gastrectomy with transit bipartition (RYTB) (n = 12), SASI (n = 12), SASI bypass with Braun anastomosis (BTB) (n = 12), esojejunostomy (EJ) (n = 12), and SHAM (n = 8) surgery. During the 12-week follow-up period, weight changes, glucose improvement, and changes in serum nutrition were evaluated. Histological expression and bile acid concentration in the rats in all groups were also evaluated. No significant differences in weight loss and glucose improvements were observed in the RYTB, SASI, and BTB groups. The RYTB and BTB groups had significantly lower bile acid concentration and albumin levels than the SASI group. In addition, mucosal height in the RYTB and BTB groups was significantly lower than in the SASI group. Braun anastomosis had a significant effect on anti-reflux. BTB may be a superior primary procedure due to its potential for parallel bariatric and metabolic improvements, effective anti-reflux effects, simplified operations, and avoidance of severe malnutrition. Further clinical studies are needed to confirm these findings.

Two independent LAMP assays for rapid identification of the serpentine leafminer, Liriomyza huidobrensis (Blanchard, 1926) (Diptera: Agromyzidae) in Australia.

Liriomyza huidobrensis is a leafminer fly and significant horticultural pest. It is a quarantine listed species in many countries and is now present as an established pest in Australia. Liriomyza huidobrensis uses a broad range of host plants and has potential for spread into various horticultural systems and regions of Australia. Rapid in-field identification of the pest is critically needed to assist efforts to manage this pest. Morphological identification of the pest is effectively limited to specialist examinations of adult males. Generally, molecular methods such as qPCR and DNA barcoding for identification of Liriomyza species require costly laboratory-based hardware. Herein, we developed two independent and rapid LAMP assays targeted to independently inherited mitochondrial and nuclear genes. Both assays are highly sensitive and specific to L. huidobrensis. Positive signals can be detected within 10 min on laboratory and portable real-time amplification fluorometers. Further, we adapted these assays for use with colorimetric master mixes, to allow fluorometer free in-field diagnostics of L. huidobrensis. Our LAMP assays can be used for stand-alone testing of query specimens and are likely to be essential tools used for rapid identification and monitoring of L. huidobrensis.

The Presence of a Parasite in the Head Tissues of a Threatened Fish (Bidyanus bidyanus, Terapontidae) from South-Eastern Australia.

The silver perch, Bidyanus bidyanus (Mitchell) (Terapontidae) is a freshwater fish, endemic to the Murray-Darling river system in south-eastern Australia. Population declines have led to the fish being listed as critically endangered by the Australian Government. Knowledge about parasites and diseases of wild populations of freshwater fish are limited in Australia. During an examination of wild-caught silver perch, digenean mesocercaria were observed in the head tissues. A total of five of the 11 silver perch collected from the Wakool River, New South Wales, were infected with mesocercaria. All mesocercaria were found in the head tissues; no mesocercaria were found encysted in the eye lens. The mesocercaria were found to belong to the family Strigeidae based on the sequences of their internal transcribed spacer (ITS) region. The lack of comparable sequences of strigeid digeneans from Australian hosts precludes being able to determine if the mesocercaria found in this study are a new species or representatives of an already described species. However, genetic results confirm that this is a different species to other digeneans previously described from silver perch, thus increasing the number of digeneans reported from B. bidyanus to three species. The presence of digenean mesocercaria in the head tissues of a wild population of silver perch, as found in the present study, is of potential conservation significance. Given the critically endangered conservation status of B. bidyanus, and previous evidence of strigeid infection altering fish behaviour, ecology, and predation mortality, further research on the potential impacts of infection on wild populations is warranted.

A novel diagnostic gene region for distinguishing between two pest fruit flies: Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) (Diptera: Tephritidae).

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra-primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses.

Infection levels of Gnathostomatidae (Nematoda) larvae in commercial fishes in north-eastern Australian waters and related food safety concerns.

The majority of research on the safety of marine edible fish has primarily focused on anisakid nematodes, neglecting the potential risks posed by other parasites, including those belonging to the family Gnathostomatidae. In Australia, there have been few reported cases of human infections with gnathostomatid parasites since 2011. However, due to the absence of a standardized diagnostic test in the country, it is believed that the actual number of infections is higher than reported. This study aimed to assess the occurrence and prevalence of infectious gnathostomatid parasites in selected commercial fish species in Australia. A total of 1947 marine fish from northern Australia, representing 9 families, 16 genera, and 30 species, were examined for gnathostomatid nematode infections. Overall, 12.3 % of the fish were found to be infected with at least one gnathostomatid larva. Among the species examined, the yellow-dabbled flounder (Branchypleura novaezeelandiae) exhibited the highest prevalence (83.3 %; n = 6) and the largest number of gnathostomatid larvae. The identification of the gnathostomatid larvae was confirmed as belonging to the genus Echinocephalus based on both morphological characteristics and sequence data. No significant correlation was observed between the prevalence, mean abundance, and mean intensity of infection with the length or weight of the examined fish species. Notably, several of the infected fish species are considered popular choices in the Australian market. Hence, it is imperative to raise awareness among relevant food safety authorities regarding the occurrence of these parasites. The findings from this study should be taken into consideration for the revision of current seafood safety protocols in the country.

High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap.

BACKGROUND: Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) is an internationally significant pest of grain crops and stored grain products. Wheat germ traps, routinely used in surveillance sampling of Khapra beetle provide feed-substrates used by the pest throughout its life cycle. However, Khapra beetle larvae, eggs and other traces of the pest, such as larval frass and exuviae, in wheat germ traps are difficult to sort and taxonomically identify. Additionally, high levels of polysaccharides in wheat germ can inhibit PCR based molecular detection of this pest captured in the traps. METHODS AND RESULTS: We have developed a sensitive and low-cost protocol for extracting trace levels of Khapra beetle DNA from an entire wheat germ trap. Overnight digestion of entire trap contents in 6 mL of ATL buffer, followed by a 40 min lysis step was optimal for DNA extraction. Paired with reported qPCR assays, this protocol allows the detection of a few hairs of T. granarium in a typical 2-gram wheat germ trap. CONCLUSION: This DNA extraction protocol makes it possible to perform a more rapid identification of the pest following wheat germ sample collection. The protocol has potential to improve international efforts for Khapra beetle surveillance.

Role of Circulating Tumor DNA in Colorectal Cancer.

Colorectal cancer (CRC) is a very common gastrointestinal tumor, ranking second in the global cause of cancer death. Because of the invasive nature of biopsy and cannot reflect the heterogeneity of tumor or monitor the dynamic progress of tumor, it is necessary to induce a novel noninvasive method to improve the current treatment strategies of colorectal cancer. Among all the components of liquid biopsy, circulating tumor DNA (ctDNA) may have the best future. CtDNA maintains the same genomic characteristics as those in matched tumor tissues, so it allows quantitative evaluation and analysis of mutation load in body fluid. Furthermore, because the half-life of ctDNA is from 16 min to several hours in circulation, the circulating ctDNA can be measured repeatedly within a certain period to monitor the response of CRC to treatment, the occurrence of drug resistance, and the diagnosis of recurrence.

Measurement of gastric wall thickness after laparoscopic sleeve gastrectomy: obesity comorbidities and gastric wall in Chinese patients with obesity.

Leakage of the sleeve remains constant after laparoscopic sleeve gastrectomy (LSG). This complication may be due to a mismatch between the staple height and gastric wall thickness (GWT). Our aim was to measure the GWT in Chinese patients with obesity and investigate the relationship between GWT and gender, body mass index (BMI), body weight, and obesity-related comorbidities. The GWT of 210 resected specimens after LSG was measured using a tissue measuring device, at a compression pressure of 8 g/mm2 at three predetermined locations: antrum, midbody, and fundus. Two hundred ten patients (171 female/39 male). The gastric wall was thickest at the antrum followed by the midbody and thinnest at the fundus (3.02 mm, 2.22 mm, and 1.6 mm, respectively). Patients with gastritis and those with reflux esophagitis had thicker GWT at the antrum; male had thicker GWT at the antrum and fundus; patients with body weight > 100 kg, and those with BMI > 40 kg/m2 had thicker GWT at the fundus. Linear regression analysis revealed a significant association between GWT with body weight and BMI at the antrum and fundus; Furthermore, hypertension associated with the GWT at the fundus (P < 0.01, P < 0.01, P < 0.02, P < 0.01; and P < 0.04, respectively). This study showed that the anatomical location of the gastric wall is a major predicting factor of GWT. Furthermore, gastritis, reflux esophagitis, male gender, BMI > 40 kg/m2, body weight > 100 kg, and hypertension may increase the GWT at the antrum and fundus in Chinese patients with obesity.

Occurrence and characterisation of Eustrongylides species in Australian native birds and fish.

In Australia, nematodes belonging to the genus Eustrongylides were believed to be endemic species until the late 20th century when they were all considered to be E. excisus, invalid or inquirendae. Although these nematodes have frequently been reported in Australian fish, reptiles, and birds and cause disease or mortality among them, there has been no attempt to date to characterise them genetically. Globally, also, no one has validated or defined suitable genetic markers to distinguish between species of Eustrongylides. In this study, adult Eustrongylides from little black cormorant (Phalacrocorax sulcirostris; n = 3) and larvae from mountain galaxias (Galaxias olidus, n = 2) and a Murray cod (Maccullochella peelii, n = 1), and a Murray cod-trout cod hybrids (Maccullochella peelii x Maccullochella macquariensis, n = 1) were available for morphological examination and molecular characterisation. The adult nematodes from cormorants were identified as E. excisus. Sequences of the 18S and ITS regions were then obtained for all nematodes, which were identical among all specimens (larvae and adults) and also identical to those of E. excisus available in the GenBank. However, only one base pair difference exists between the 18S sequences of E. excisus and E. ignotus, with limited sequences available in GenBank accompanied with proper morphological data for the nematodes. With that limitation in mind, identifying our specimens as E. excisus suggests spill-over - that it is an introduced parasite species that has successfully established its life cycle among Australian native species - may have occurred. Our study is the first report of E. excisus in the little black cormorant, P. sulcirostris. Our results do not exclude the possibility of the occurrence of other species of Eustrongylides, either native or exotic, in Australia. This parasite is zoonotic and with increasing demand for fish and changing dietary preferences, such as the consumption of raw or undercooked fish, its occurrence in the flesh of the fish is concerning. This parasite is also associated with anthropogenic habitat alteration affecting the reproductive success of the infected hosts. Therefore, awareness among the relevant authorities of the presence of the parasite in Australia and its adverse impact on native animals is crucial for the success of conservation plans such as fish recovery and relocation efforts.

Regulation of Pancreatic TXNIP-Insulin Expression Levels after Bariatric Surgery Using Diabetic Rodent Model.

Purpose: The purpose of this study was to investigate the effect of bariatric surgery on pancreatic thioredoxin-interacting protein (TXNIP) and insulin expression levels. The research question is does bariatric surgery induce changes in the pancreatic TXNIP level, given that TXNIP has been proposed as a key glucose control factor? Methods: Using nondiabetic and diabetic rats, we investigated whether our streptozotocin-induced diabetic rat models exhibited changes in pancreatic TXNIP regulation. Following this confirmation, we randomly divided the diabetic rats into the following three groups: the gastric bypass group (n = 16), pair-fed group (n = 10), and sham group (n = 10). Preoperatively and 3 weeks postoperatively, all the rats underwent an oral glucose tolerance test, insulin tolerance test, and blood sampling procedures for hormonal analysis. Results: The TXNIP messenger ribonucleic acid (mRNA) and protein expression levels were significantly lower in the gastric bypass group than in the other groups. Regarding the gastric bypass group, the pancreatic mRNA expression levels of microRNA-204 (miR-204) and MafA were significantly lower and higher, respectively, than in the other groups. Furthermore, the levels of pancreatic insulin expression at the mRNA and protein levels were also significantly higher in the gastric bypass group than in the other groups. Conclusion: Bariatric surgery significantly improved glucose control and regulated the pancreatic insulin production pathways of TXNIP, miR-204, and MafA. The regulation of TXNIP, miR-204, and MafA might play an important role in the mechanism of diabetes remission following bariatric surgery.

Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar.

Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers-the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives.