We reported a new microsporidium, Daphniaspora nanhuensis n. gen. n. sp., from the intestine of Moina micrura collected from Lake Nanhu in Wuhan City, Hubei Province, China. Infected daphnids generally appeared to have hypertrophied and opaque intestines under light microscopy. Electron microscopy revealed all developmental stages residing in direct contact with the host cell cytoplasm. The earliest stages observed were uninucleate meronts with a round nucleus. Multinucleate sporogonial plasmodia developed into uninucleate sporoblasts by rosette-like budding. Mature spores were dumbbell-shaped, measuring 4.57 ± 0.40 (3.75-5.54) µm long and 1.79 ± 0.18 (1.40-2.23) wide. The bipartite polaroplast was composed of tightly packed anterior lamellae and wide posterior lamellae. Isofilar polar filaments coiled 5-6 turns and arranged in one row. The exospore consisted of three layers. BLASTn searches of obtained SSU rDNA sequence showed the highest similarity (78.52 %) with Liebermannia dichroplusae (GenBank accession number: EF016249). Phylogenetic analysis based on the obtained SSU rDNA sequence indicated that the present species represented an independent branch but formed a sister relationship with the genus Liebermannia. Based on the morphological characteristics, ultrastructural features, and molecular data, we propose the establishment of a novel genus (Daphniaspora n. gen.) and species (Daphniaspora nanhuensis n. sp.) to accommodate the present species.

Sexual precocity is a common problem in the aquaculture of Chinese mitten crabs (Eriocheir sinensis), and can reduce the aquaculture performance. The healthy intestines play a vital role in sustaining aquaculture performance. Therefore, this study investigated the effects of sexual precocity on the feeding behavior, histopathology, and intestinal microbiota of E. sinensis. Based on the data obtained, precocious crabs showed a longer feeding response time (136.51 ± 41.51 min) and a shorter feeding duration (36.46 ± 11.86 s) than non-precocious crabs (13.37 ± 3.96 min and 49.33 ± 21.95 s). Meanwhile, histological abnormalities were also observed in the intestines of precocious crabs. The histopathological results of the precocious crabs' intestine revealed a larger intestinal diameter (1.113 ± 0.071 mm) and luminal area (0.621 ± 0.037 mm2), a thicker peritrophic membrane (13.870 ± 3.236 μm), as well as broken-down hindgut glands and disordered-arranged columnar epithelial cells. 16S rRNA gene sequencing analysis revealed deeper changes related to intestinal health in precocious crabs, with greater abundance of potentially pathogenic bacteria such as Vibrio and Roseimarinus. The potentially pathogenic bacteria in precocious crabs' intestine such as Mycoplasmataceae and Alphaproteobacteria were found in greater abundance, while beneficial bacteria including Rhizobiales and ZOR0006 were lower in abundance. Univariate correlation analysis revealed that Rhizobiales and ZOR0006 had negative correlations with various potentially pathogenic bacteria including Vibrio and Roseimarinus. Based on the determination of microbiota in the water environment and SourceTrack analysis, the results indicated a decline in the proportion of intestinal microbiota derived from the water environment in precocious crabs. In conclusion, precocious crabs exhibit poorer intestinal health, which exacerbates aquaculture risks.

Microsporidiosis, a disease caused by microsporidia, affects many animals, with symptoms ranging from diarrhea to death, especially in immunocompromised individuals. Current treatments, such as the drugs albendazole and fumagillin, are limited in efficacy. To address this problem, we utilized Caenorhabditis elegans infected with its naturally occurring microsporidian parasite Nematocida parisii to evaluate 4,080 structurally diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library. From this screen, we identified 34 compounds that restored the reproductive capacity of C. elegans. We chose 17 compounds for additional validation experiments and all of them prevented N. parisii infection in C. elegans, with 10 of these capable of suppressing microsporidia invasion by inactivating mature spores. Additionally, five of the identified compounds were effective against Pancytospora epiphaga, a species related to human-infecting microsporidia. Together this work identifies and characterizes compounds that inhibit microsporidia infection.

As an important component of innate immunity in insects, the Toll pathway is a conserved signaling cascade that activates humoral immunity through NF-κB-related pathways, contributing to the production of antimicrobial peptides (AMPs) and effectors in response to fungal, bacterial and viral infections. Although extensive advances have been made in the identification and function of the Toll pathway in insects, knowledge of the regulatory mechanisms of this pathway in heteropteran insects, for which the available knowledge is rare, is essential for exploring their developmental and immune functions to exploit species-specific and environmentally friendly bioinsecticides for control of heteropteran pests. In addition, increasing attention has been given to the activation, signal transduction and regulation of the Toll pathway in response to various stimuli in heteropteran insects because of the absence of some components of the immune deficiency (IMD) pathway and interaction with gut symbionts. In this review, we outline the current research progress on the functional roles of the Toll pathway in several heteropteran insects, such as Riptortus pedestris, Rhodnius prolixus, Triatoma pallidipennis, Cimex lectularius, Plautia stali, and others, which are involved in the responses to changes in feeding conditions, pathogenic infection, embryogenesis and wing polyphenism, as well as interactions with gut symbionts. Ultimately, we propose a framework for future research on the identification and functional characterization of the Toll pathway to expand the current data and address potential limitations on the investigation and application of this pathway in the design of pioneering pest control strategies for heteropteran pests.

Glycosylation, a key post-translational protein modification, has been found central to host-pathogen interactions, underpinning numerous critical interactions mediated by carbohydrate structures (glycans). Glycan-dependent interactions regulate pathogen adhesion, recognition, invasion, and immune evasion. Invertebrates represent the largest and most diverse group of species on Earth, from established classical model organisms such as Drosophila and Caenorhabditis, to medically relevant parasites and disease-carrying vectors. However, glycan-mediated interactions between invertebrates and their pathogens remain far less studied compared to vertebrate systems. This review summarizes current knowledge on the role of invertebrate glycosylation, including N- and O-glycans and glycosaminoglycans, in defense against diverse pathogens, and the immune-evasive role of glycosylation employed by invertebrate (or invertebrate-borne) pathogens during host infection. While current research highlights the critical importance of glycosylation in these interactions, some key gaps persist: a lack of comprehensive glycomic and glycoproteomics analysis across representative invertebrate species, and poor understanding of receptor molecules and signaling mechanisms for these glycans and glycoproteins. We hope this review will stimulate further research into this critical yet underexplored facet of invertebrate-pathogen interactions.

Soil salinity varies widely across geographies both due to natural factors and human activities, including agriculture, road salt application, sea level rise, and desertification. Increases in soil salinity may affect organisms widely and particularly impact soil food webs. As parasites, entomopathogenic nematodes (EPNs) occupy crucial links in soil food webs and are important for agriculture as biological control agents of insect pests. Previous research found that the EPN Steinernema carpocapsae may exhibit higher salt tolerance than several of its congeners. We recently identified that S. carpocapsae uniquely evolved two amino acid substitutions in the first extracellular loop of the sodium pump (Na/K-ATPase). Here, we tested whether these substitutions explain S. carpocapsae's reported lower sensitivity to salt. Our results confirm that S. carpocapsae exhibits higher salt tolerance and show that it can more effectively locate and infect insect hosts than its congeners S. feltiae and S. hermaphroditum in highly saline environments. We then retraced the evolution of the two amino acid substitutions in S. carpocapsae by introducing them alone and in combination in Caenorhabditis elegans using CRISPR genome engineering. We found that C. elegans mutants with single substitutions showed improved salt tolerance. However, this improvement disappeared in the double mutant, whose sodium pump mimicked that of S. carpocapsae. This pattern of negative epistasis between the amino acid substitutions suggests they are not responsible for variation in salt tolerance between Steinernema species. Sodium pump evolution in S. carpocapsae might instead be driven by encounters with cardiac glycosides, which are released into soil by several clades of plants including milkweeds, sequestered by some of this EPN's herbivorous insect hosts, and known to target the first extracellular loop of the sodium pump. Our findings provide valuable insights into EPN adaptation to changes in environmental sodium levels and may have implications for their use in biological control.

We investigated the occurrence of density-dependent prophylactic resistance (DDPR) in the South American locust, Schistocerca cancellata, by exposing gregarious-phase nymphs to the microsporidium Paranosema locustae across a range of densities. Infection prevalence and intensity were highest at low densities and declined sharply at intermediate densities. At high densities, infections were virtually absent. A threshold density for the expression of DDPR may exist, likely above 130 nymphs/m. These findings support the presence of DDPR in S. cancellata and suggest that P. locustae has limited potential as a biocontrol agent for managing gregarious populations during outbreaks.

Translucent post-larvae disease (TPD) is a lethal syndrome causing high mortality in post-larvae of Litopenaeus vannamei. This study investigated the protective efficacy of gallic acid (GA), a non-antibiotic compound, against TPD induced by a field isolate Vibrio parahaemolyticus TS-GE (V. para. TS-GE). Immersion challenge assays confirmed the high virulence of V. para. TS-GE, as it caused 100 % mortality in post-larvae within 24 h at 2.82 × 10 CFU/mL. Whole-genome sequencing revealed its genome comprised two chromosomes (3.50 Mb and 1.92 Mb) and three plasmids (69.7 kb, 60.7 kb, 60.5 kb). The 69.7-kb plasmid harbored TPD-associated virulence genes vhvp1 and vhvp2, while chromosomal genes encoded 40 type III secretion system components and thermolabile hemolysin. Pangenome analysis revealed the open genome nature of V. parahaemolyticus strains. Multi-locus sequence typing identified V. para. TS-GE as ST2621. In vitro, GA exhibited growth-inhibitory activity against V. para. TS-GE. In vivo, 200 μg/mL GA significantly reduced cumulative mortality (P < 0.01) from 100 % to 18.3 %, preserving hepatopancreatic epithelium and midgut structure in V. para. TS-GE infected post-larvae. Mechanistic investigations revealed GA disrupted bacterial cell wall/membrane integrity, inhibited swimming motility, and suppressed biofilm formation. Molecular docking simulations predicted favorable binding of GA to virulence proteins VHVP1 (-6.3 kcal/mol) and VHVP2 (-7.8 kcal/mol), suggesting dual antibacterial and anti-virulence activities. These findings highlight GA as a promising antibiotic alternative for TPD control. Genomic data provide insights into the pathogenic adaptation of V. para. TS-GE in aquaculture.

Entomopathogenic viruses affect insect populations by reducing fitness and influencing other measures of performance such as development and longevity. Given the huge diversity of viruses, studies are needed to understand the mechanisms underlying how viruses affect their insect hosts. The Junonia coenia densovirus (JcDV) infects a wide range of Lepidoptera and can cause death in the larval and pupal stages. In this study, we used the Melissa blue butterfly (Lycaeides melissa: Lycaenidae) to describe infection progression at the individual level, the effects of infection on development, survival, and longevity, and whether plant diet influenced these factors. Caterpillars were infected with JcDV and reared on either a low-quality host plant (Medicago sativa) or one of two high-quality host plants (Astragalus canadensis or Lotus nevadensis). We found that average viral load was similar across six days post-inoculation with a slight peak on day four. Viral load was highest among individuals reared on the low-quality host plant. Infection resulted in faster development time compared to uninfected individuals, and diet-specific effects were evident in survival to adulthood, with the native host plant (A. canadensis) offering optimal conditions for survival. None of the infected individuals that reached adulthood successfully eliminated JcDV, but viral loads were much lower in adults than larvae, indicating individuals reduced or maintained low viral loads between life stages. These results suggest diet-dependent response to JcDV infection and have implications for understanding the mechanisms that facilitate or impede the colonization of novel host plants.

Entomopathogenic protozoans are, to a large extent, host specific, and through acute or chronic infections they negatively alter host reproductive fitness, metabolism, immune response, juvenile hormonal balance, and host development. Hence, these entomopathogens are well-suited to reduce populations of stored product insect pests. The first step towards achieving successful suppression, natural or applied, of storage insect pest populations is the detection for these entomopathogenic protozoans in their habitats. Therefore, a preliminary survey of naturally occurring protozoan infections in stored-grain insect pests was carried out across some Governorates in Lower and Upper Egypt. The protozoan-natural mortality rates among the subject insect pests were recorded. Based on morphological characteristics, particularly spore or oocyst morphology, five entomopathogenic protozoans were taxonomically identified, at the genus level (i.e., four apicomplexans, Adelina sp., Farinocystis sp., Mattesia sp., and Gregarina sp., as well as one microsporidian or fungal pathogen, Nosema sp.). Observations on the morpho-pathological, physio-pathological, and behavioural changes induced by protozoan infections in beetles of Cryptolestes turcicus, Rhyzopertha dominica, Tribolium castaneum, and moths of Plodia interpunctella were recorded, as well. Among the interesting findings, a behavioural abnormality was induced by Nosema infection in P. interpunctella moths; viz., the complete failure of copulated pairs to be separated after copulation (i.e., frequent occurrence of ca., 66.70-73.70 %). Additionally, an increased abundance, by ca., 2.40-fold, of the total protein content has been quantified in Adelina-infected or Farinocystis-infected T. castaneum beetles compared to the uninfected beetles. The pathological changes observed in this study may provide new insights into the interaction between the subject entomopathogenic protozoans and their insect hosts.

Autophagy is a conserved cellular process with dual roles in antiviral defense and viral utilization that plays a crucial role in host-pathogen interactions. Here, we investigated the function and related mechanisms of Bmced6 in domestic silkworm, Bombyx mori, during the infestation of B. mori nucleopolyhedrovirus (BmNPV). At first, immunofluorescence indicated that Bmced6 was mainly localized in the cytoplasm. And overexpression of Bmced6 significantly enhanced the proliferation of BmNPV, as evidenced by the increased expression level of the viral capsid protein, VP39, and the enhanced viral fluorescence intensity of BmNPV-GFP. Moreover, siRNA-mediated knockdown of Bmced6 inhibited viral infection and transmission. Furthermore, we found that the positive effect of Bmced6 on viral infestation was associated with enhanced virus-induced autophagy , including increased autophagosome formation, ATG8 lipidation, Atg8 puncta formation and up-regulation of autophagy-related genes. Meanwhile, abnormal expression of Bmced6 disrupted mitochondrial homeostasis, leading to ultrastructural damage, decreased mitochondrial membrane potential (MMP), and dysregulated reactive oxygen species (ROS) production. These findings establish that Bmced6 facilitates BmNPV infection through the coupling of autophagy activation and mitochondrial dysfunction, providing new insights into the molecular mechanisms underlying viral infection in insects.

Acute hepatopancreatic necrosis disease (AHPN) is a lethal shrimp disease caused by Vibrio parahaemolyticus carrying virulent pir genes in extrachromosomal plasmids. However, the limitations of current polymerase chain reaction (PCR) methods pose challenges for on-site AHPND diagnosis. This study aimed to develop a real-time Loop-Mediated Isothermal Amplification (LAMP) assay for diagnosing AHPND at shrimp farming sites. Two primer sets were designed to target a 300 bp region within the pir gene on the pVPA3-1 plasmid of the reference strain 13-028/A3. Using a Genie II machine, an AHPND-specific primer set was selected to optimize a LAMP reaction mixture (LAMP-mixture I) and reaction conditions. The LAMP-mixture I provided clear and accurate results at 65 °C within approximately 50 min. The detection limit (100 fg) was comparable to that of duplex PCR developed for AHPND detection and was 100 times more sensitive than conventional PCR with existing LAMP primers (10 pg), though less sensitive than AP4 nested PCR. To enhance the feasibility of the LAMP assay in shrimp farms, a portable real-time LAMP machine developed by SMTION (Daejeon, Korea) was employed. The LAMP products were analyzed using SYBR Green I and calcein detection methods. Both methods produced positive results and showed no cross-reactivity with non-AHPND strains. The real-time LAMP calcein method demonstrated high diagnostic specificity, positive predictive value, and 78 % accuracy when evaluated with field samples. Hence, the real-time LAMP calcein method developed here offers potential for rapid and reliable AHPND diagnosis in shrimp farming sites, in comparison to other PCR-based strategies in simplicity and specificity.

Bonamia ostreae is a protozoan parasite of significant concern in oyster aquaculture due to its impact on flat oyster species. In this study, we screened 30 wild flat oysters (Ostrea denselamellosa) collected from the western coastal region of South Korea for B. ostreae infection using two molecular assays. Species-specific BOSTRE-F/R primers targeting the ITS region detected 14/30 (46.67 %) positive samples, while Bonamia genus-specific Bo/Boas primers targeting the SSU rRNA region yielded 19/30 (63.33 %) positives. Although the higher detection rate with Bo/Boas primers raised the possibility of other Bonamia species, further PCR using B. exitiosa-specific primers confirmed the absence of B. exitiosa. To enhance species identification and phylogenetic analysis, extended SSU rDNA sequences (1,094 bp) were amplified using two primer sets, one newly developed for this study. Phylogenetic analysis demonstrated that the South Korean isolate clustered tightly within the B. ostreae clade alongside strains from Europe and North America, suggesting low genetic diversity and a possible recent introduction via human-mediated oyster movements. Histological examination and in situ hybridization (ISH) confirmed the presence of B. ostreae microcells localized within hemocytes and surrounding connective tissues. Despite successful detection, the infection intensity was low, and histopathological changes were mild, suggesting that O. denselamellosa may be relatively less susceptible to B. ostreae, though further investigation is needed to assess its role in the parasite's transmission dynamics. These findings highlight the need for enhanced molecular surveillance and international biosecurity measures, as this study represents the first confirmed report of B. ostreae infection in O. denselamellosa and the first record of this parasite in East Asia.

Decapod iridescent virus 1 (DIV1) is a newly identified pathogen responsible for significant disease outbreaks and high mortality rates in farmed crustaceans. While previous studies have reported the susceptibility of mud crab Scylla paramamosain, a species of considerable commercial importance in Asia, to DIV1, detailed investigations into its infection by DIV1, particularly in situ confirmation evidence, are still lacking. This study experimentally confirms that S. paramamosain is susceptible to DIV1, demonstrating that the virus can infect and induce disease through both intramuscular injection and oral routes. The infection resulted in high mortality, especially in the high-dose injection group, while oral exposure led to slower disease progression. Quantitative PCR analysis revealed high viral loads in hepatopancreas, gonads, and gills. Histopathological examination identified typical signs of infection, including eosinophilic inclusions and nuclear pyknosis. Furthermore, in situ DIG-labeling loop-mediated isothermal amplification (ISDL) provided direct evidence of the widespread distribution of DIV1 across various tissues, including the gonads, suggesting potential implications for reproduction. Transmission electron microscopy (TEM) revealed the presence of icosahedral viral particles with typical iridovirus morphology in infected cells, further confirming DIV1 replication in S. paramamosain. The study underscores the importance of the route of exposure in determining disease progression and mortality, with oral exposure resulting in lower mortality compared to injection. These findings establish S. paramamosain as a susceptible species for DIV1, expanding the known host range of the virus and offering valuable insights into its pathogenesis, tissue tropism, and potential impacts on aquaculture.

Procambarus clarkii is an economically important cultivated freshwater crayfish species in China. Intensive aquaculture and challenging environmental conditions cause diseases that kill crayfish and impact productivity. Clarification of immune mechanisms could assist the breeding of disease-resistant crayfish and improve survival rate. Here, a tumor necrosis factor alpha-induced protein 8-like gene (TNFAIP8L) encoding a 187 amino acid protein in P. clarkii was characterized, and phylogenetic analysis revealed high homology with genes in other crustaceans. Recombinant TNFAIP8L protein was successfully expressed and purified, and pull-down, mass spectrometry, molecular docking, and western blotting identified hemocyanin B and heat shock protein 60 (HSP60) as TNFAIP8L-interacting proteins. Enzyme-linked immunosorbent assay experiments revealed a higher affinity for hemocyanin B than for HSP60. Expression patterns of TNFAIP8L in different tissues and under immune challenge were determined by real-time PCR. TNFAIP8L was expressed in all tissues examined with highest levels in hemocytes, gills, and intestines. Following immune challenge, TNFAIP8L was down-regulated in hemocytes and gills. RNA interference and overexpression of TNFAIP8L induced the expression of immune-related genes Toll, Serpin, B-cell lymphoma (Bcl), Lectin, Defensin, Crustin, and anti-lipopolysaccharide factor (ALF) in hemocytes and gills. Together, the results suggest that TNFAIP8L mediates immune responses in P. clarkii.

One of the causes of the decline in distribution and abundance of the endangered white-clawed crayfish Austropotamobius pallipes complex throughout Europe is the invasion of alien crayfish, and the associated spread of infectious diseases, primarily the crayfish plague caused by Aphanomyces astaci. Another relevant disease is microsporidiosis (porcelain disease), caused by Astathelohania contejeani and Nosema austropotamobii. Between 2021 and 2024, we conducted a monitoring survey, aimed at mapping the distribution of A. astaci, A. contejeani and N. austropotamobii in wild populations of A. pallipes and in two non-indigenous species in Trentino (North-East Italy). We applied a non-invasive sampling method (cuticular swabs) in 33 populations of A. pallipes, 2 populations of Procambarus clarkii and 4 populations of Faxonius limosus, to investigate the presence of A. astaci. Aphanomyces astaci was detected in eight thriving populations of A. pallipes, and the presence of a low virulence genotype (genotype group A) was confirmed in one of them. Aphanomyces astaci was detected in one of the two populations of P. clarkii, as well as in one of the four populations of F. limosus. No mortality outbreaks in populations of A. pallipes were recorded in Trentino during the study period. Specimens with macroscopic signs of porcelain disease were found in thirteen populations of A. pallipes, abdominal muscle tissues were collected and subjected to molecular evaluation; microsporidiosis was detected in all the tested crayfish: seven populations were infected only by A. contejeani, and in six populations both microsporidia were detected. Crayfish from one population of P. clarkii and one of F. limosus were tested for microsporidiosis as well, but the results were negative. Based on the presence of chronically infected but thriving populations across the Trentino territory, we provide suggestions for the management of A. pallipes populations in conservation actions which require the movement of specimens (translocations, rearing in captivity).

Fish farming may pose a risk to adjacent octopus farms due to pathogen transmission. Moreover, the immune defense mechanisms of cephalopods are still not fully understood. This study aimed to determine changes in total protein concentration and hemolysis activity of Octopus vulgaris hemolymph, after intramuscular (IM) or intravenous (IV) challenges with aquaculture fish pathogens (either Photobacterium damselae subsp. piscicida or damselae or Vibrio alginolyticus or anguillarum O1) at two temperatures (21 ± 0.5 ℃ and 24 ± 0.5 ℃). Results showed that Octopus vulgaris exhibited a mean total protein concentration of 173.93 ± 69.37 mg/mL across all experimental conditions, markedly exceeding values reported for other mollusks, such as the bivalves Chamelea gallina (0.75-1.66 mg/mL) and Mytilus galloprovincialis (0.59-1.60 mg/mL). Patterns of total protein concentration, related to the genera of the pathogen used for the challenges, were observed. Four-way ANOVA revealed significant main effects of bacterium (F(3, 144) = 54.360, p < 0.001) and temperature (F(1, 144) = 10.014, p = 0.002) on total protein, along with multiple significant interaction effects, including bacterium × temperature, route × time, and bacterium × route × temperature × time (all p < 0.001). Hemolysis remained at low levels across both experimental temperatures, challenge routes, and pathogens, not exceeding 25 % in any case. Values above 15 % and up to 20 % were recorded in specific conditions, such as Photobacterium damselae subsp. damselae at 24 ± 0.5 °C on Day 3 in CIM-, IM-, and IV- control and challenged groups respectivelly; V. alginolyticus at 24 ± 0.5 °C on Day 3 in IM-challenged groups; and Vibrio anguillarum O1 at 21 ± 0.5 °C on Day 3 and Day 7 in IM-challenged groups. ANOVA for hemolytic activity showed significant main effects of bacterium (F(3, 144) = 22.032, p < 0.001) and temperature (F(1, 144) = 4.083, p = 0.045), with multiple significant interactions, including bacterium × temperature, route × time, and bacterium × route × temperature × time (all p < 0.001). These results indicate that the route of challenge may play a major role in hemolysis activity, with temperature and time post-challenge also exerting significant effects, possibly through a complex synergistic interaction. Our results may assist in elucidating common octopus defense mechanisms against common fish pathogens and provide important information to the scientific community and the marine aquaculture sector.

Acute hepatopancreatic necrosis disease (AHPND) poses a major threat to global shrimp aquaculture, especially impacting Penaeus vannamei. Given the high cost of prevention and the absence of effective treatments, the most efficient way to control AHPND is through early detection to quickly identify the pathogen. This study presents the development and optimization of a low-cost, portable diagnostic platform utilizing loop-mediated isothermal amplification (LAMP) for the rapid detection of AHPND. This platform integrates fluorescence detection with a smartphone-compatible device, providing a convenient and effective solution for on-site diagnosis. We also screened fluorescent nucleic acid dyes with optimal adaptability and developed an efficient, user-friendly tool to improve the performance of the isothermal amplification method. In addition, a deep learning-based infection detection algorithm was developed for automated diagnosis. The visualization technology showed high specificity and sensitivity for detecting AHPND in shrimp samples, with a detection limit of 1 copies/µL. These results highlight the potential of this method in resource-limited environments and provide a valuable tool for early detection and management of AHPND in shrimp aquaculture.

Melissococcus plutonius, the agent of European foulbrood (EFB), has been well studied in Apis mellifera but its epidemiology in Apis cerana remains less understood. We surveyed 37 apiaries across Guangxi, China, and detected M. plutonius in 32.4%, 10.8%, and 27.0% of larvae, adult bees, and honey samples, respectively, all originating from asymptomatic colonies. Duplex PCR revealed frequent co-detection of typical and atypical strains. Viable isolates were recovered from 58.3% of PCR-positive larval samples. Multi-locus sequence typing (MLST) showed six isolates (T1, X16, L20, Q21, Q22, L32) clustering with the atypical reference strain DAT561, and one isolate (P6) clustering with the typical reference strain ATCC 35311. Our findings highlight the high prevalence and strain diversity of M. plutonius in A. cerana in subtropical Guangxi and emphasize the need for region-specific surveillance strategies.

Honey bees are often exposed to and infected by multiple viruses, including deformed wing virus (DWV) and Israeli acute paralysis virus (IAPV). These pathogens are major contributors to colony failure, threatening the supply of pollination services in agriculture. However, understanding and modeling the infection and transmission dynamics of these viruses is hampered by gaps in knowledge about their infection timelines, specifically how virus load changes post-exposure. Moreover, while honey bees frequently experience co-occurring infections from multiple viruses, little is known about how co-infection affects virus-induced mortality and replication dynamics. We hypothesized that, while both DWV and IAPV are known to be highly infectious and can cause honey bee mortality, each presents a distinct infection timeline and peak infection intensity time window. In addition, we hypothesized that co-infection would result in increased mortality compared to individual virus infections due to increased pathogen-induced stress. To test these hypotheses, we exposed day-old honey bee workers to DWV, IAPV, or a combination of DWV and IAPV via carefully controlled experimental injections and tracked mortality and virus levels over time. After exposure, bees were maintained in the laboratory for 10 days and sampled daily for virus quantification. We observed a similar response pattern to both honey bee viruses in the first 24 h post-infection, where virus levels rapidly increased following inoculation. While DWV infection had persistently high virus levels and a delayed mortality response after peak DWV load was achieved, IAPV infection was rapidly followed by either mortality or a decline in virus load as bees recovered from infection. Co-infected bees showed some variation in mortality, although there were no significant differences in virus load between DWV or IAPV within co-infected bees compared to bees infected with the individual viruses. These results help answer fundamental questions related to the pathology of DWV and IAPV within honey bees that help clarify how these pathogens interact and persist within honey bee colonies.

Ecytonucleospora (=Enterocytozoon) hepatopenaei (EHP) is an intracellular parasitic microsporidian, Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a linear single-stranded DNA virus. Both of them are widespread pathogens in shrimp farming, causing slow growth in shrimp and leading to significant economic losses. Litopenaeus vannamei (L. vannamei), the dominant species in shrimp farming, can be co-infected by EHP and IHHNV. According to our prior research, EHP and IHHNV exhibit synergistic interactions during infection. However, the underlying mechanisms of their co-infection remain unclear. In this study, L. vannamei from four experimental groups (EHP infected group, IHHNV infected group, EHP and IHHNV co-infected group, healthy group) were compared by transcriptome sequencing to analyze the synergistic pathogenic mechanisms of IHHNV and EHP. The results showed that co-infection induced more pronounced changes in the expression of genes related to metabolism, growth, and immunity compared to single infections. Specifically, EHP infection had a greater impact on shrimp physiology and immune responses, suggesting a dominant role in co-infection. Meanwhile, IHHNV infection disrupted metabolic processes, inhibited molting, and exacerbated the immune suppression caused by EHP. The synergistic pathogenic mechanism of IHHNV and EHP may involve EHP weakening the immune defenses of L. vannamei, facilitating IHHNV infection and proliferation. Additionally, the metabolic dysregulation induced by IHHNV may further enhance consumption of cellular nutrients and energy by EHP. These findings preliminarily reveal the synergistic pathogenic mechanism of EHP and IHHNV in L. vannamei, offering a theoretical foundation for understanding disease outbreaks in shrimp farming and guiding strategies against multi-pathogen infections.