Journal of Economic Entomology

The tropical house cricket Gryllodes sigillatus is a major species used in the edible insect farming industry. Despite the rapid expansion of this sector, diagnostic tools for detecting infections in these species remain limited. The lack of validated reference genes compromises the reliability of RT-qPCR-based gene expression analyses, which are essential for the development of molecular tools for disease diagnosis and health monitoring in insect production systems. To address this gap, we evaluated the expression stability of six candidate reference genes (ACTB, EF1, GAPDH, HisH3, RPL5, and 18SrRNA) across four body parts (abdomen, head, legs, and whole body) using a combination of complementary statistical approaches, including geNorm, NormFinder, BestKeeper, the {Delta}Ct method, the R statistical environment, and the integrated RefFinder tool. Candidate genes were identified and annotated using the recently published G. sigillatus genome, through sequence comparisons with closely related insect species using BLAST and reciprocal BLAST analyses, multiple sequence alignments. All procedures complied with MIQE 2.0 guidelines to ensure methodological rigor and transparency. The results showed that ACTB, EF1, RPL5, and 18SrRNA exhibited stable and consistent expression across all analyzed tissues, whereas GAPDH and HisH3 displayed high variability and were generally unsuitable for normalization, except in head tissue where GAPDH remained stable. This study provides the first validated set of reference genes for G. sigillatus, establishing a robust foundation for accurate, reproducible, and comparable gene expression analyses. Furthermore, these findings support the development of RT-qPCR-based diagnostic tools, contributing to improved health monitoring and biosafety in insect production systems.

BACKGROUNDThe two-spotted spider mite, Tetranychus urticae Koch, is a major agricultural pest with a rapid propensity for developing acaricide resistance. Bifenazate targets mitochondrial cytochrome b (CYTB). While the G126S mutation is frequently associated with resistance, its independent role remains unclear as it often occurs with other substitutions. This study explores the molecular basis of bifenazate resistance in a Russian laboratory strain derived from a St. Petersburg greenhouse population. RESULTSDisruptive selection with increasing bifenazate concentrations generated resistant and susceptible isofemale lines. AlphaFold2 structural modeling of CYTB indicated that G126S causes a steric clash, leading to conformational destabilization, whereas other reported mutations primarily affect the ligand-binding pocket. Oxford Nanopore sequencing revealed a very low initial frequency of the G126S allele (<1%; 226/35,895 reads) in the unselected population. After one year of stepwise selection (0.00005-0.031% a.i.), the mutant allele frequency surged to 90% (7,272/8,056 reads). No other known resistance-associated mutations were found in the analyzed cytb fragment. CONCLUSIONWe report the first identification of the G126S mutation in a Russian T. urticae population and demonstrate its rapid fixation under bifenazate selection. Within this genetic background, G126S alone appears sufficient to confer high-level resistance, emphasizing the population-specific nature of resistance evolution and the critical need for local monitoring.

The pathways of non-target exposure to anticoagulant rodenticides (ARs) are poorly understood, and have yet to be examined in Ontario, Canada. The spillover of ARs into non-target rodents and high-risk landscapes has been investigated numerous times, but usually in agricultural regions as opposed to urban ones. We used snap traps to capture white-footed mice (Peromyscus leucopus) in urban wildland areas of Toronto and Vaughan, Ontario near ongoing rodenticide baiting programs. Our goal was to determine if second-generation anticoagulant (SGAR) baiting practices used by pest management professionals targeting commensal rodents may be causing rodenticide spillover into non-target rodents in urban wildland areas, which could act as a vector of ARs to predators. Only 11 out of 111 mature white-footed mice trapped near ongoing urban rodenticide operations tested positive for an anticoagulant, at five out of seven study sites. Concentrations were between 0.008-0.03 ppm, which may be sublethal for raptors. We did not detect brodifacoum, despite its detection in a recent study on Ontario raptors. Exposed individuals were caught at 0m, 5m, 20m, 40m, 70m and 100m from active rodenticide stations. They did not differ from unexposed individuals in terms of sex, age, body condition, distance to the AR source, capture date or capture site. This indicates that the pest management industrys use of rodenticides in urban and suburban settings is causing some degree of non-target spillover in Toronto and the Greater Toronto Area, and that SGAR usage should be avoided near naturalized landscapes.

O_LIAlthough ecological research has long focused on the effects of temperature on population growth, arthropod pests are exposed to a wide variety of environmental factors that affect their performance, such as chemical pesticides targeted against them. Moreover, these environmental factors likely do not act in isolation. Identifying the extent to which abiotic factors interact to affect pest population dynamics can strengthen current and future pest management programs. C_LIO_LIHere, we investigated the extent to which temephos, a common pesticide applied to aquatic environments for mosquito control, influences the thermal performance of juvenile survival and development rate, as well as the intrinsic population growth rate, of the invasive mosquito pest, Aedes aegypti. We implemented a response surface experimental design to measure these traits across seven temperatures and five temephos concentrations and fit temperature- and insecticide-dependent performance curves to assess impacts on the overall performance and the thermal optimum, minimum, and maximum. C_LIO_LITemephos exposure profoundly altered the thermal performance of juvenile survival by reducing survival across all temperatures, shrinking the thermal breadth, and shifting the thermal optimum to warmer temperatures. Through this, temephos also altered the thermal performance of population growth primarily by reducing its thermal breadth. C_LIO_LISynthesis and applications: Our findings demonstrate that interactions between temperature and insecticide exposure can fundamentally reshape pest population dynamics, rather than acting as independent stressors. By quantifying this interaction, we showed that temphos is most effective below the pests thermal optimum, suggesting that larvicides may yield the greatest population suppression in cooler regions or during cooler periods of the year. Incorporating such temperature-dependent efficacy into pest management strategies could improve the timing and spatial targeting of control efforts. More broadly, these results highlight the need to integrate anthropogenic stressors with climatic drivers when predicting pest risk and optimizing management under ongoing environmental change. C_LI

Iron is an essential nutrient for all types of organisms, including insects and the microbes that infect them. We predicted that insects fed an iron-supplemented diet would accumulate more iron in their hemolymph, and, because infectious microbes acquire iron from their hosts, that this extra iron would increase the severity of bacterial infections. To test this hypothesis, we studied the effects of dietary iron supplementation on infection outcomes in Manduca sexta (tobacco hornworm). Larvae were fed an artificial diet, with or without antibiotics, or the same diets supplemented with 10 mM iron. Control and iron-treated larvae were inoculated with non-pathogenic Escherichia coli or the entomopathogenic Enterococcus faecalis, and bacterial load and larval survival were measured. We found that dietary iron supplementation increased the iron content of hemolymph by approximately 20 fold; however, contrary to our prediction, this increase in iron did not result in an increase in the bacterial load of either E. coli or E. faecalis. The effect of iron supplementation on survival was more complicated. As expected, for larvae inoculated with nonpathogenic E. coli, iron supplementation had no effect. For larvae inoculated with E. faecalis, the effect of iron supplementation depended on whether antibiotics were present in the diet. Without antibiotics, iron supplementation prolonged larval survival; with antibiotics, iron supplementation decreased larval survival. The results of this study do not support the hypothesis that dietary iron supplementation increases infection severity in M. sexta. Instead, the results support the viewpoint that the relationship between dietary iron and infection outcome is complex.

Methods for estimating and modeling the long-term and short-term adult flight dynamics of the conifer silk moth Dendrolimus superans (Lepidoptera: Lasiocampidae) are examined. The analysis uses light trap adult catch data collected over 21 years, from 2005 to 2025. Three models of adult flight are considered: a flight-initiation model driven by weather factors, an autoregressive model of long-term catch dynamics, and a binary model of seasonal catch. For the flight-initiation model, we propose estimating the accumulated temperature sum ST from the date when the first derivative of the remote sensing vegetation index NDVI becomes positive until the date of the first adult capture of the season. ST is shown to be sufficiently stable across all years of observation, with flight each year beginning after this temperature sum is reached. The second model demonstrates that the long-term light trap catch time series is well described by a second-order autoregressive model AR(2), in which the catch of the current year depends on catches from the two preceding years. This long-term series is compared with a previously studied larval population density series of the Siberian silk moth; both are shown to be AR(2) series with similar coefficient values, which suggesting that adult catch data may serve as a proxy for absolute larval population density. In the third model, we describe the transition from absolute-scale seasonal catch dynamics (number of adults per day) to a binary scale (0, 1), where 0 denotes days on which no adults were attracted to the trap, and 1 denotes days on which at least one individual was captured. The seasonal absolute catch series is thereby transformed into a binary series of zeros and ones, and relationships between adjacent values in such a binary series are examined. A linear relationship between the absolute and binary seasonal dynamics series is demonstrated, making it possible to estimate absolute catches from binary catch values and to analyze seasonal flight in sparse pest populations. This potentially opens new avenues for understanding how outbreak populations function at chronically low density. Author summaryForest pests can cause catastrophic damage, yet predicting their outbreaks remains challenging. During periods of low population density, standard monitoring methods become labor-intensive and uninformative, while the transition to an outbreak often occurs unexpectedly. Using a 21-year dataset of adult Siberian silk moth (Dendrolimus superans) captures from light traps, we developed an approach combining three complementary models. First, we showed that moth flight begins upon reaching a specific temperature sum, with the starting point determined by NDVI vegetation index dynamics rather than a calendar date--making the forecast more ecologically relevant. Second, long-term adult population dynamics follow a second-order autoregressive model AR(2), matching the dynamics previously observed for larval populations. This establishes light trap data as a reliable proxy for absolute population density when ground surveys are impractical. Third, we introduced a method to analyze seasonal flight using binary data (presence/absence of moths per day), which we showed is linearly related to absolute abundance. This enables studying population dynamics during periods of extremely low density, when traditional methods fail. Our approach opens new possibilities for early warning systems to detect when a population risks transitioning from a latent state to an outbreak phase.

BackgroundThe cotton bollworm Helicoverpa armigera is a major global pest controlled by genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins, including Vip3Aa. While Vip3Aa is widely deployed, the genetic basis of resistance remains poorly understood. Previous work identified disruption of a thyroglobulin-like gene (HaVipR1) as one mechanism of resistance, suggesting additional loci may be involved. ResultsUsing linkage analysis, transcriptomics, long-read sequencing, and CRISPR-Cas9 gene editing, we identify a second thyroglobulin-like gene, HaVipR2, as a novel mediator of Vip3Aa resistance. Resistance in a field-derived H. armigera line was shown to be monogenic, recessive, and autosomal, mapping to chromosome 29. Long-read sequencing revealed a [~]16 kb transposable element insertion disrupting HaVipR2, which was undetectable using standard short-read approaches. CRISPR-Cas9 knockout of HaVipR2 conferred >900-fold resistance, confirming its causal role. Comparative analyses show that HaVipR1 and HaVipR2 share conserved domain architecture, indicating that thyroglobulin-domain proteins represent a recurrent target of resistance evolution. ConclusionsOur findings establish thyroglobulin-domain proteins as a new class of Bt resistance genes in Lepidoptera and demonstrate that transposable element insertions can drive adaptive resistance while evading detection by conventional methods. These results highlight the importance of long-read sequencing and accurate genome annotation for resistance monitoring and provide new insights into the molecular basis and evolution of Vip3Aa resistance.

O_LIPhenological shifts are a major ecological consequence of climate change, yet studies often focus on single life stages meaning that the potential for carryover effects between life stages remains poorly understood. Failing to account for these effects may lead to inaccurate estimates of phenological shifts, with consequences for predicted synchrony among interacting species. This is especially relevant for temperate systems where climate warming is occurring unevenly across the year. C_LIO_LIHere, we investigated how temperature experienced the previous autumn and winter (during the pupal and egg stage) influences spring phenology in the winter moth (Operophtera brumata), a herbivorous insect with distinct life stages. Using 50 years of local climate data to create five experimental temperature regimes, we first quantified phenotypic plasticity in the duration and temporal variability of pupal and egg development. We then examined how timing of adult moth emergence affects timing of offspring hatching. C_LIO_LIWe found divergent effects of temperature on different life stages; pupal development time was shortest at intermediate temperatures while egg development time decreased linearly with increasing temperature. Furthermore, phenological shifts due to the conditions experienced by the mother were carried over to influence the phenology of her offspring. While this carryover effect was partially compensated during subsequent stages, compensation decreased under warming conditions. C_LIO_LIThese results refine our understanding of the sensitivity of the annual cycle of winter moth phenology to variation in temperature with potential implications for population dynamics and interspecific interactions. Overall, our findings highlight the need to consider the impacts of warming across multiple life stages so that carryover effects can be properly accounted for. Doing so will improve predictions of phenological shifts under future climates. C_LI

As global reptile populations continue to decline, improving reproductive success in managed populations of listed species, such as Phrynosoma cornutum (the Texas horned lizard) has become increasingly critical for species survival. One understudied area of reproductive research in reptile species is gamete collection and storage, a crucial component for maintaining genetic diversity. In Texas, semen was collected from wild P. cornutum (n = 20) in June 2025. Semen collection was performed via electroejaculation (EEJ) under alfaxalone anesthesia. Prior to semen collection, snout-vent-lengths (SVL) and weights were recorded and testes measurements were taken using a portable ultrasound. Average sperm motility and concentration across all lizards was 83.7% and 85.7 x 106 sperm/mL, respectively. While lizards with longer SVLs had higher sperm motility, weight and testis size did not affect sperm parameters. Samples were extended in INRA96 and divided for use in cold-storage longevity or cryopreservation trials. Samples under cold-storage conditions were assessed for motility daily for 10 days. Motility was not significantly reduced until 48 hours post-collection and maintained 19% motility at day 10. For cryopreservation, samples were diluted 1:1 in INRAFreeze cryopreservation media and frozen in liquid nitrogen, then immediately thawed. Average post-thaw sperm motility was 13.9%, with the highest post-thaw motility recorded at 38.2%. This is the first report of semen storage and cryopreservation in Phrynosoma and provides valuable insight into semen storage potential in reptile species.

BackgroundThe malaria transmission potential and the vulnerability of Anopheles mosquitoes to different vector control methods depend, among other factors, on the endophily, endophagy, anthropophagy and survival of each species. Local information on these bionomic parameters is generally unavailable. MethodsTo address this, we estimated species-specific values of these parameters using an augmented version of the global database of bionomics data by Massey et al. (2016). We applied inclusion and exclusion criteria to select eligible studies with relevant experimental designs that minimise bias from collection methods for parous, sac, endophagy, and endophily rates as well as for the resting duration. For the human blood index (HBI), we separated data from indoor and outdoor collections. We fitted hierarchical Bayesian models with levels based on Anopheles taxonomy to estimate these quantities. Based on the estimated bionomics, we quantified the expected vectorial capacity reduction after the introduction of a pyrethroid-pyrrole insecticide-treated net (ITN) for 57 Anopheles species. ResultsWe identified 26 eligible studies for endophagy and 61 for the parous rate, leading to a Bayesian posterior average for the Anopheles genus of 42% (95% credible interval: 18-70) and 55% (32-77) respectively. HBI values widely varied depending on the location of collection, except for some species showing strong anthropophilic behaviours. Resting duration was estimated to be 2.1 days (1.2 - 4.8) at the genus level. Few studies were available to estimate the sac and endophily rates, which prevented us from deriving precise estimates for the whole Anopheles genus. Our estimates of the vectorial capacity reduction following the introduction of a pyrrole-pyrethroid ITN ranged between 48% and 76% across species, highlighting the important differences among mosquito species in vulnerability to vector control interventions. ConclusionThis work demonstrates how data from both Anopheles species complexes and individual species can be leveraged to generate species-specific estimates of bionomic parameters, capturing the local characteristics and behaviour of malaria vectors. The dataset is readily updatable as new data become available. However, more frequent and standardised field surveys are still needed to accurately characterise local vector behaviour.

The coleoid cephalopods (octopus, cuttlefish, and squid) are emerging model organisms for neuroscience, development, and evolutionary biology. Determining their sex early in life is critical for population management and controlled experiments. Here, we present a protocol to non-invasively determine the sex of multiple cephalopod species as young as 3 hours post-hatching using a skin swab and quantitative PCR (qPCR). We describe steps for designing qPCR primers, swabbing live animals, extracting DNA, running the qPCR, and analyzing the results. For complete details on the use and execution of this protocol, please refer to Rubino et al.1 HighlightsO_LISwab live cephalopods as early as 3 hours post-hatching C_LIO_LIExtract DNA from cephalopod skin swabs C_LIO_LIPerform qPCR-based sex determination C_LIO_LIDesign and validate qPCR primers for new species C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/715692v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@3aa68dorg.highwire.dtl.DTLVardef@8c7e61org.highwire.dtl.DTLVardef@1bd45d9org.highwire.dtl.DTLVardef@134cc4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Accurate species identification is crucial to assess the medical and veterinary relevance of a mosquito specimen, but it requires high experience of the observers and well-equipped laboratories. This study aimed to evaluate whether low-cost imaging in combination with geometric wing morphometrics can provide accurate identification of invasive, morphologically similar Aedes species. The right wings of 670 female specimens covering 184 Ae. aegypti, 156 Ae. albopictus, 166 Ae. j. japonicus and 164 Ae. koreicus, were removed, mounted and photographed with a professional stereomicroscope (Olympus SZ61, Olympus, Tokyo, Japan) and a macro lens (Apexel-24XMH, Apexel, Shenzhen, China) attached to a smartphone. The coordinates of 18 landmarks on the vein crosses were digitalized by a single observer for each image. In addition, the landmarks of 20 specimens per species and imaging device were digitalized by six different observers to assess the degree of the observer error. The superimposed shape variables were used to compare the species classification accuracy of linear discriminant analysis (LDA), support vector machine (SVM), Random Forest (RF), and XGBoost. In the single-observer landmark data, the LDA achieved the best classification results with a mean accuracy of 95 % for landmarks from microscope images and 92 % for those obtained from smartphone images. In the multi-observer landmark data, LDA consistently performed worse than the other three classifiers, and the reduction in the accuracy was more pronounced for smartphone images than for microscope images. This pattern was associated with a higher degree of observer error for smartphone images, as confirmed by a landmark-wise comparison across all landmarks. Geometric wing morphometrics provides a reliable method to distinguish the most common invasive Aedes species in Europe. Thereby, the image quality obtained by smartphones equipped with a macro lens is sufficient and represents a cost-effective alternative to professional microscopes. However, due to the greater degree of observer variation for smartphone images, landmark coordinates for such images should ideally be collected by a single observer.

Invasive round goby Neogobius melanostomus have advanced eastward through the state of New York and provinces of Ontario and Quebec over the past two decades and are approaching Lake Champlain, one of the largest lakes in North America. This manuscript describes international efforts to monitor round goby populations during 2021-2025 on (a) the southern approach to Lake Champlain via the Hudson River and Champlain Canal, and (b) the northern approach to Lake Champlain via the Saint Lawrence River and Richelieu River. Monitoring utilized environmental DNA (eDNA), backpack electrofishing, beach seining, benthic trawling, and viral hemorrhagic septicemia virus (VHSV) testing. In the Champlain Canal, round goby were captured as far north as the downstream side of the C1 dam (97 kilometers [km] from Lake Champlain) while eDNA detections occurred as far north as the upstream side of the C2 dam (90 km from Lake Champlain). In the Richelieu River, round goby were captured as far south as Saint-Marc-sur-Richelieu (82 km from Lake Champlain) while the southern-most eDNA detections occurred near the Canadian side of the international border (4 km from Lake Champlain). Water temperature influenced habitat usage of round goby in the Champlain Canal, with catch rates in near-shore areas declining at < 10 {degrees}C. All VHSV test results were non-detections at the mouth of the Richelieu River, while one positive and two inconclusive results occurred along the Champlain Canal. Together, these data have informed multiple mitigation measures and have implications for management of aquatic invasive species across North America.

IntroductionHuman land-use intensification and the resulting habitat loss are primary drivers of insect pollinator declines. Habitat restoration offers a promising approach to counteract these declines, yet landscape-level evaluations of bee responses to restoration and management remain limited. We conducted a two-year, landscape-scale study in Wisconsin, USA, to assess how different intensities of tallgrass prairie restoration and management affect bumble bees (Bombus spp.). ObjectivesThis study aimed to determine whether (1) bumble bee abundance and diversity increase with assisted restoration, and (2) outcomes differ between low-(seeded only) and moderate-intensity (seeded and managed with prescribed fire) interventions. MethodsUsing catch-and-release surveys, we measured bumble bee abundance and diversity at 32 sites representing a gradient in restoration intervention: no intervention (unassisted recovery), low intervention, and moderate intervention. ResultsBumble bee abundance and diversity were higher at assisted restoration sites (low and moderate intervention) than at unassisted sites. Although both tended to be greater at moderate than low intervention intensities, these differences were not statistically significant. Bumble bee community composition also differed across intervention intensity, driven by shifts in dominant species (e.g., B. impatiens and B. griseocollis). Rarer taxa, including endangered and vulnerable species, occurred only at assisted restoration sites, with the largest populations at moderate intervention sites. Across all sites, bumble bee responses were strongly and positively associated with floral abundance, but not with semi-natural habitat in the surrounding landscape. ConclusionOur findings demonstrate that assisted grassland restoration can effectively increase bumble bee abundance and diversity, supporting its value as a conservation practice for pollinators. Implications for Practice: (1) Grassland restorations targeting plant communities can successfully support nontarget pollinators across a range of management intensities and landscape contexts. Adding seeds of pollinator-preferred plants could improve restorations with low floral abundance and diversity. (2) Management of existing restorations is important to maintain abundant floral resources and diverse pollinator communities. Because sites varied widely in prescribed fire use, our findings likely represent a conservative estimate of its benefits, and higher intervention intensity (e.g., repeated seeding, regular fire, mechanical or chemical shrub and invasive plants control) may further enhance outcomes for bumble bees.

Net radiation (Rn) can be estimated using models that apply the Brunt equation for the incoming longwave radiation and air temperature (Tair) for the outgoing longwave radiation under reference conditions. This study aimed to estimate Rn using two previously regionally calibrated Brunt model, thereby eliminating the need site-specific calibration, and to assess whether Tair can be used as a substitute for canopy temperature (Tc) under well-watered crop conditions. Measurements were conducted in sesame and cotton fields during the first year and in a cotton field during the second year. Canopy temperature was measured during the second year, and the calculations were performed at hourly and daily time scales. Regardless of the method used to estimate sky emissivity or whether Tc or Tair was used, errors were greater at hourly time scale. The overall RMSE, MAE, Bias and KGE values at the daily time scales were 11.88, 9.13, 2.53, and 0.91, in the first year, and 13.45, 10.56, 0.10 and 0.74, in the second year, respectively. When using both regionally calibrated Brunt model, Rn simulation performance was superior to that of the Allen/FAO method. The comparison between Rn estimated using Tair and Tc, indicated statistical differences. Nevertheless, linear regression and error metrics showed that these differences were modest, especially at daily time scale. Thus, for practical purposes both regionally calibrated Brunt equations can be used to calculate clear-sky emissivity and improve Rn estimations, and Tair can be used as a substitute for Tc at the daily time scale under well-watered conditions.

Some Anopheles species that act as malaria vectors are members of species complexes, a concept whereby sibling species cannot be differentiated solely on the basis of morphological characters. Therefore, species complexes represent a major problem in malaria vector control, because within an Anopheles complex, vectors cannot be differentiated from non-vector species, unless molecular techniques are used to identify them. The Anopheles tessellatus species complex is an important potential vector in South, East, and Southeast Asia, including certain regions of Indonesia. However, no in-depth studies have been conducted on this species complex in that country. Therefore, this study investigated the taxonomic status of An. tessellatus from diverse populations across five Indonesian islands (Sumatra, Java, West Nusa Tenggara, East Nusa Tenggara, and Sulawesi) and identified interpopulation genetic variation based on molecular data of the ITS2, COI, and COII genes. Phylogenetic relationships were constructed using the Maximum Likelihood method. Haplotype and network analysis were also conducted. The results indicate that An. tessellatus constitutes a monophyletic group comprising three well-defined lineages that exhibit clear intraspecific genetic differentiation. Cluster 1 corresponds to the population of Sumatra, Cluster 2 represents population from Sulawesi, and Cluster 3 encompasses populations from Java, West Nusa Tenggara, and East Nusa Tenggara. These findings demonstrate high haplotype diversity and low nucleotide diversity within the species. Populations from West Sumatra, Manado, Tojo Una - Una, and North Morowali (Sulawesi) have the potential for speciation with a genetic distance of 0.61 - 0.94% for COI, between 0.81 - 0.95% for ITS2, and between 0.62 - 0.71% for COII. These findings underscore the need for further integrative studies to obtain a more comprehensive understanding of the An. tessellatus complex in Indonesia and its role in malaria transmission.

In wildlife forensic practice, species identification and estimation of the Minimum Number of Individuals (MNI) for highly processed specimens have long relied on weight-based conversion methods, which may result in underestimation of the number of individuals involved in a case. Focusing on confiscated casque products of the helmeted hornbill (Rhinoplax vigil), this study combines macroscopic morphological examination with mitochondrial DNA barcoding (16S rRNA, COI, and Cytb) to explore a more robust approach for individual quantification. The results demonstrate that the conventional "weight-based" approach overlooks critical biological information contained in anatomical structures and cannot accurately reflect the actual number of individuals involved. Based on this, we propose an anatomy-based criterion centered on the principle of structural uniqueness: specimens retaining biologically unique beak or casque structures should be directly assigned to a single individual, whereas weight-based estimation should only be applied when original anatomical features are entirely absent. In addition, considering material loss during processing, we propose approximately 85 g as a reference threshold for estimating the number of individuals in heavily processed solid casque products. This approach improves the scientific rigor and accuracy of forensic identification and provides reliable technical support for the conviction, sentencing, and law enforcement of wildlife trafficking cases involving helmeted hornbill and other endangered species.

This study aimed to evaluate DARO Systems detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) against serum and oral fluid surveillance methods within a controlled study consisting of one PRRSV infected seeder pig and 46 naive nursery pigs. Findings showed DARO Systems comprehensive herd-level surveillance approach detected PRRSV earlier than traditional testing methods.

Mosquitoes in urban environments are considered unpleasant by citizens, and they can represent serious health risks as disease vectors. Invasive Aedes species are continuously spreading in Europe and have been increasingly reported from urban settlements. Some of these mosquito species have been associated with small artificial habitats. Therefore, their presence may also be expected in small plastic garden ponds, which are numerous in urban settings across many parts of Europe. Here, we aimed to determine whether urban waters host a high occurrence of mosquitoes and to identify the potential abiotic, biotic and landscape drivers of mosquito presence and abundance in urban ponds. We sampled 53 urban ponds in the city of Budapest (Hungary) of both natural and anthropogenic origin, and 40 garden ponds, which cover both highly urbanised areas and those with high green index in the suburbs. We collected data on macroinvertebrate communities, pond management, physico-chemical parameters, and pond characteristics (mainly morphology and vegetation cover) measured in situ. Two different mosquito detection techniques were used: dip-net sampling and eDNA. While only one invasive species (Aedes koreicus) was detected, occurring in a single pond, several other species were present, including potential malaria vectors increasingly reported from urban environments: the Anopheles maculipennis complex, An. claviger and An. plumbeus. Fish presence was negatively associated with both the presence and abundance of mosquitoes, regardless of pond type. Contrary to expectations, urbanisation did not play a major role in explaining mosquito presence or abundance. These results highlight the importance of local pond characteristics, and particularly the role of fish presence, in regulating mosquito populations in ponds in the urban landscape, although the broader ecological effects of fish on pond communities should also be considered.

Venom is an important functional trait that helps predatory animals capture prey. Centipede predatory venoms are complex cocktails of multiple proteins, such as neurotoxins (scoloptoxins), cytotoxins, {beta}-pore-forming toxins, and enzymes. We examined venom phenotypes in two closely related and co-occurring centipede species, Scolopendra morsitans (n=28) and S. hardwickei (n=11), in peninsular India to determine whether their venoms are similar or dissimilar. An integrated proteo-transcriptomic approach was used to characterise the venom phenotypes of the two species across multiple individuals in peninsular India. We used species occurrence records and species distribution models to assess the distributional overlap among these species within the peninsular Indian region. The species showed significant overlap in their current and projected geographical ranges, corresponding with their co-occurrence. We characterised the venom profiles of both species and found that the venoms were cocktails of enzymes, {beta}-pore-forming toxins, and neurotoxins comprising 110 and 84 proteins in S. morsitans and S. hardwickei, respectively. However, the venom composition of both species differed significantly in toxin abundance and species-specific protein repertoires. This indicates trait divergence in venom phenotypes, suggesting that distinct venom compositions may facilitate coexistence among ecologically similar predatory centipedes. The observed variation in venom phenotypes among co-distributed species opens up important avenues for future research into their ecological roles and functional significance. In this study, we provided a detailed account of venom composition across multiple individuals from the species geographic range and highlighted the importance of investigating the role of venom as a trait that could influence species interactions and shape communities in these diverse tropical forests.