Human Pathogen Research Articles

Draft genome sequence of a non-human primate-derived isolate of Candida parapsilosis.

Candida parapsilosis is a common human commensal and opportunistic fungal pathogen that is also found in non-human primates (NHPs). Here, we report the first draft sequence of C. parapsilosis NCYC 4418, a fecal isolate from an adult cynomolgus macaque.

ISOLATION, BIOCHEMICAL CHARACTERIZATION AND ANTIBIOTIC SENSITIVITY OF LACTOBACILLI IN QUAIL DROPPINGS

Background: Probiotics are microorganisms which are when given in a specific amount, have favorable effects on the host health. Objectives: The current study was conducted on quail’s droppings, which were collected from the various sailing points within district Dera Ismail Khan. The core aim of this field study was to isolate the Lactobacillus species from the droppings to identify and characterize them. Methods: Three species (lactobacillus brevis, lactobacillus salivarius and lactobacillus fermentum) were isolated on the basis of biochemical tests. Results: Lactobacillus salivarius was found more tolerant at pH 2 as compared to the other two species of lactobacillus, likewise at pH 3 lactobacillus brevis showed maximum growth, which was significantly higher than other species of lactobacillus. Similarly, all the three species of lactobacillus showed higher growth on 0.5 % ox gall as compared to the 1 % ox gall. Likewise antibiotic sensitivity testing of lactobacillus species shown sensitivity to erythromycin except L. fermentum and all three were resistance to tetracycline, kanamycin, ciprofloxacin and gentamycin. Conclusion: It is concluded that lactobacillus species have huge benefits like their tolerance to low pH and their growth on bile salts. Their use in diets will not only balance the gut microflora but also will inhibit the growth of pathogens in humans, animals and poultry species, as described by different studies which are conducted on above mentioned species.

Structural characterization of the full-length Hantaan virus polymerase.

Hantaviridae are a family of segmented negative-sense RNA viruses that contains important human and animal pathogens. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome. Here we establish the expression and purification polymerase from the Old World Hantaan virus and characterise the structure using Cryo-EM. We determine a series of structures at resolutions between 2.7 and 3.3 Å of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerases. We further describe structures with 5' vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including β-sheet, two helical arrangements, and nucleotide primed arrangement. The insights gained here guide future mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets.

The gE/gI complex is necessary for kinesin-1 recruitment during alphaherpesvirus egress from neurons.

Alphaherpesviruses include important human and veterinary pathogens that share a unique propensity to establish life-long latent infections in the peripheral nervous system. Upon reactivation, these viruses navigate back to body surfaces and transmit to new hosts. In this study, we demonstrate that the virus gE/gI-US9p membrane complex routes virus particles down this complex neuronal egress pathway by coordinating their association with multiple kinesin microtubule motors.

H3K4 Methylation and Demethylation in Fungal Pathogens: The Epigenetic Toolbox for Survival and Adaptation in the Host

Pathogenic fungi represent a diverse group of eukaryotic microorganisms that significantly impact human health and agriculture. In recent years, the role of epigenetic modifications, particularly histone modifications, in fungal pathobiology has emerged as a prominent area of interest. Among these modifications, methylation of histone H3 at lysine-4 (H3K4) has garnered considerable attention for its implications in regulating gene expression associated with diverse cellular processes. A body of literature has uncovered the pivotal roles of H3K4 methylation in multiple biological processes crucial for pathogenic adaptation in a wide range of fungal pathogens of humans and food crops. This review delves into the recent advancements in understanding the impact of H3K4 methylation/demethylation on fungal pathogenesis. We explore the roles of H3K4 methylation in various cellular processes, including fungal morphogenesis and development, genome stability and DNA repair, metabolic adaptation, cell wall maintenance, biofilm formation, antifungal drug resistance, and virulence. We also discuss the conservation of H3K4 methylation regulators and their potential as therapeutic targets to prevent fungal diseases. Collectively, this review underscores the intricate links between H3K4 methylation, fungal pathogenesis, and potential avenues for novel antifungal strategies.

Synthesis and Chemical Characterisation of Some Novel Substituted Flavone’s for It’s Anti Microbial Activity

The present study reports an experiment on some derivatives of 2-phenyl-chromen-4-one (flavone ring) have a proven inhibitory activity against a variety of human pathogens, including antibiotic-resistant Gram-positive and Gram-negative bacteria and antifungal activities screened against four human pathogenic bacteria and two phytopathogenic fungi. Flavanoid derivatives have been made through Algar– Flynn–Oyamada reaction from hydroxyacetophenones and aromatic-aldehydes. The structures of the synthesized compounds were elucidated by UV, IR and 1H NMR spectroscopic techniques, and elemental analysis. These compounds are potentially useful pharmacologically active compounds and are studied for biological activities like antibacterial and antifungal screens performed in vitro by the plate-hole diffusion method.

In vitro Antifungal Effects of Various Essential Oils against Aspergillus Crown Rot of Peanut

Synthetic fungicides that combat plant pathogenic fungi can enhance crop yields, ensuring stable crop production and market quality. However, the increase in the use of fungicides may cause to development of fungicide-resistant pathogen strains and the accumulation of fungicide residues in the food chain above safe limits. This situation underscores the need for improved fungal disease management through alternatives to synthetic fungicides. These alternatives include plant-derived compounds such as essential oils and extracts. Essential oils are known to be potent antifungal compounds against both human and plant pathogens. Aspergillus niger is a toxin-producing fungal disease agent that causes Aspergillus crown rot in peanuts. In this study, the antifungal activities of nine different essential oils from Foeniculum vulgare, Lippia citriodora, Origanum majorana, Origanum minutiflorum, Origanum onites, Origanum syriacum, Origanum vulgare, Salvia aramiensis and Thymus syriacus plants were evaluated against A. niger under in vitro conditions by using disc diffusion test. Among the nine essential oils tested, the highest antifungal activities were displayed by O. vulgare essential oil (with an inhibition zone diameter of 49.33 mm) which was followed by T. syriacus, O. onites, O. syriacum and O. minutiflorum essential oils (48.67, 47.00, 46.33 and 43.33 mm, respectively). The essential oils of F. vulgare, L. citriodora, and O. majorana showed relatively lower antifungal effects. The essential oil of S. aramiensis did not show antifungal effect against pathogen. The results indicated that plant essential oils could be valuable in promoting research aimed at developing new antifungal agent(s) for fungal disease management. However, further studies are needed to optimize the in vivo application conditions of essential oils against A. niger.

Biochar Reduced the Risks of Human Bacterial Pathogens in Soil via Disturbing Quorum Sensing Mediated by Persistent Free Radicals.

Biochar has great potential in reducing the abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) from soil. However, its efficiency in removing other biological pollutants, such as human bacterial pathogens (HBPs) and virulence factor genes (VFGs), is rarely studied. Herein, by pyrolyzing rice straw (RS) and pine wood (PW) at 350 and 700 °C, we prepared a series of biochar (RS350, RS700, PW350, and PW700) and investigated their impacts on the abundance and pathogenicity of HBPs. Compared with PW biochar, RS biochar effectively reduced the abundance of HBPs by 6.3-40.1%, as well as their pathogenicity, evidenced by an 8.2-10.1% reduction in the abundance of VFGs. Mechanistically, more persistent free radicals (PFRs) were formed in RS biochar than that of PW biochar during pyrolysis, and PFRs triggered the degradation of N-butyryl-l-homoserine lactone (C4-HSL) from 1.05 to 0.68 ng/kg, thereby disturbing the quorum sensing (QS) of HBPs. Once the QS was disturbed, the communications among HBPs were hindered, and their virulence factors were reduced, which ultimately lowered the abundance and pathogenicity of HBPs. Collectively, our study provides insights into the role of biochar in decreasing the risks of HBPs, which is significant in the development of biochar-based technologies for soil remediation.

SUMO-targeted Ubiquitin Ligases as crucial mediators of protein homeostasis in Candida glabrata.

Candida glabrata is an opportunistic human pathogen, capable of causing severe systemic infections that are often resistant to standard antifungal treatments. To understand the importance of protein SUMOylation in the physiology and pathogenesis of C. glabrata, we earlier identified the components of SUMOylation pathway and demonstrated that the deSUMOylase CgUlp2 is essential for pathogenesis. In this work we show that the CgUlp2 is essential to maintain protein homeostasis via the SUMO-targeted ubiquitin ligase pathway. The dual loss of deSUMOylase and specific ubiquitin ligase, CgSlx8, results in heightened protein degradation, rendering the cells vulnerable to various stressors. This degradation affects crucial processes such as purine biosynthesis and compromises mitochondrial function in the mutants. Importantly, the absence of these ubiquitin ligases impedes the proliferation of C. glabrata in macrophages. These findings underscore the significance of SUMOylation and SUMO-mediated protein homeostasis as pivotal regulators of C. glabrata physiology and capacity to survive in host cells. Understanding these mechanisms could pave the way for the development of effective antifungal treatments.

β-1,6-Glucan plays a central role in the structure and remodeling of the bilaminate fungal cell wall.

The cell wall of human fungal pathogens plays critical roles as an architectural scaffold and as a target and modulator of the host immune response. Although the cell wall of the pathogenic yeast Candida albicans is intensively studied, one of the major fibrillar components in its cell wall, β-1,6-glucan, has been largely neglected. Here, we show that β-1,6-glucan is essential for bilayered cell wall organization, cell wall integrity, and filamentous growth. For the first time, we show that β-1,6-glucan production compensates the defect in mannan elongation in the outer layer of the cell wall. In addition, β-1,6-glucan dynamics are also coordinated by host environmental stimuli and stresses with wall remodeling, where the regulation of β-1,6-glucan structure and chain length is a crucial process. As we point out that β-1,6-glucan is exposed at the yeast surface and modulate immune response, β-1,6-glucan must be considered a key factor in host-pathogen interactions.

High Occurrence of ST131 Among Extraintestinal Pathogenic Escherichia coli in Humans and Dogs in Sri Lanka.

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important human and animal pathogen. In particular, the ST131 of ExPEC is a widely distributed clone, resistant to extended-spectrum cephalosporins and fluoroquinolones. We investigated the occurrence of ST131 among ExPEC from humans and dogs in Sri Lanka and determined its antimicrobial resistance. A total of 215 ExPEC isolates were collected from humans (n = 179) and dogs (n = 36) from two different cities in Sri Lanka. Antimicrobial resistance was tested by the disk diffusion method, and the presence of resistance-encoding genes (blaTEM, blaSHV and blaCTX-M) and ST131 clades/subclades was tested by PCR. The majority of the isolates were (61.8%) ST131, with 55.8% in ST131-clade C. Of the clade C isolates, 44.3%, 27.8%, 15.7% and 6.7% were in clades C2, C other, C1-non-M27 and C1-M27, respectively. Approximately 11% of the ST131 isolates were carbapenem resistant. ExPEC from dogs showed comparable resistance rates to human isolates except for resistance to amoxicillin-clavulanate and amikacin. We observed a high occurrence of ST131 and its clade C, with more than 11% exhibiting resistance to carbapenems in Sri Lanka. Furthermore, ST131-C1-M27, with high resistance to both quinolones and extended-spectrum cephalosporins, was also present. Our results emphasise the importance of the One Health approach in the monitoring of antimicrobial-resistant E. coli in different regions of Sri Lanka to gain a better understanding of their prevalence over time, contributing to effective antimicrobial stewardship.

Exploring the Gastrointestinal Microbiome of Eurasian Griffon Vultures (Gyps fulvus) Under Rehabilitation in Portugal and Their Potential Role as Reservoirs of Human and Animal Pathogens

The Eurasian griffon vulture (Gyps fulvus), a widely distributed scavenger, plays a crucial role in ecosystem health by consuming decomposing carcasses. Scavengers have adapted to avoid disease from the rotting carrion they feed on, probably through a specialized gut microbiome. This study aimed to characterize the gut microbiome of G. fulvus (n = 8) present in two rehabilitation centers in mainland Portugal and evaluate their potential as reservoirs of pathogens. Samples were studied through high-throughput 16S rDNA amplicon sequencing of the hypervariable V3-V4 regions and further analyzed using the Qiime2 bioinformatics platform. Our results showed that factors such as sex, location, and time of sampling did not significantly affect the gut microbiome of the griffon vulture. Its composition was highly similar to that of phylogenetically closed animals. However, several potential human and veterinary pathogens were identified. In conclusion, the gut microbiome of Gyps fulvus in rehabilitation centers is not significantly altered by stress associated with captivity. Its composition is similar to that of other vultures and scavengers due to their identic diet and needs, suggesting a well-conserved functional gut microbiome, which seems to be influenced by season. The potential risks posed by the identified pathogens to humans and other animals should be further investigated.

Gastrointestinal Shedding of Rubulaviruses from Egyptian Rousette Bats: Temporal Dynamics and Spillover Implications

Bats are recognized as reservoirs for diverse paramyxoviruses, some of which are closely related to known human pathogens or directly implicated in zoonotic transmission. The emergence of the zoonotic Sosuga virus (SOSV) from Egyptian rousette bats (ERBs), which caused an acute febrile illness in a reported human case in Africa, has increased the focus on the zoonotic potential of the Rubulavirinae subfamily. Previous studies identified human parainfluenza virus 2 (HPIV2)- and mumps (MuV)-related viruses in ERBs from South Africa, with HPIV2-related viruses restricted to gastrointestinal samples, an underexplored target for rubulavirus biosurveillance, suggesting that sample-type bias may have led to their oversight. To address this, we performed a longitudinal analysis of population-level fecal samples from an ERB maternity roost for rubulavirus RNA, employing a broadly reactive hemi-nested RT-PCR assay targeting the polymerase gene. We detected HPIV2- and MuV-related viruses in addition to numerous pararubulaviruses, highlighting significant viral diversity. Temporal analysis of three major clades revealed peaks in rubulavirus shedding that correlated with seasonal environmental changes and host reproductive cycles, although shedding patterns varied between clades. These findings identify specific periods of increased risk for the spillover of bat-associated rubulaviruses to humans, providing critical information for developing targeted mitigation strategies to minimize zoonotic transmission risk within the local community.

Genetic diversity of populations of Simulium reptans s.l. (Diptera: Simuliidae) in the territory of Russia and Northern Kazakhstan.

The presence of sibling (cryptic) species among bloodsucking black flies can be a serious problem in assessing both their diversity and the role of individual species in the transmission of dangerous human and animal pathogens. The peculiarities of molecular genetic structure of populations and ecology of black flies of Simulium reptans s.l. (complex of Simulium reptans (Linnaeus) and Simulium reptantoides Carlsson) in European and Asian parts of Russia and Northern Kazakhstan in comparison with populations from Central and Northern Europe were investigated. Using restriction analysis of the COI gene and subsequent sequencing, we searched for unique mtDNA variants that could belong to previously undescribed forms of the studied group. In the north of the European part of Russia (north of Eastern Europe), only S. reptans A was registered, and in the Asian part of Russia (Siberia) and Northern Kazakhstan, only S. reptans B was found. Using phylogenetic analysis of the total data, three branches of S. reptans B were revealed and named in accordance with the areas of their distribution-East European, Eurasian and Asian. Simulium reptantoides Carlsson has currently been found only in Europe. The peculiarities of distribution and possible adaptations to different habitats of S. reptantoides and representatives of individual groups within S. reptans are discussed. Based on the characteristics of the sampling sites of the studied specimens, as well as the samples available in the NCBI database, the ecological and geographical features of the habitats of the molecular forms of Simulium reptans A and B were revealed. To obtain more accurate information on the distribution of S. reptans A and B forms in Eurasia, taking into account the elevational zonation, it is necessary to conduct additional studies in the mountainous regions of South-Western Europe, Siberia and Northern China. Based on the sequences presented in the DNA database, a method (AS-PCR COI variant) for separating the cryptic species-Simulium reptans (Linnaeus) and S. reptantoides Carlsson-was proposed. During testing of this method on the material collected in Russia and Northern Kazakhstan, only S. reptans was identified (S. reptantoides was absent), which completely coincided with the sequencing results. To further confirm the effectiveness of the method, it is necessary to conduct testing on material including representatives of the both species.

The gut microbiota-independent virulence of noninvasive bacterial pathogen Citrobacter rodentium.

Attaching and effacing (A/E) bacterial pathogens consist of human pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli and their murine equivalent Citrobacter rodentium (CR). Emerging evidence suggests that the complex pathogen-microbiota-host interactions are critical in conferring A/E pathogen infection-induced severe symptoms and lethality in immunocompromised hosts; however, the precise underlying mechanisms remain enigmatic. Here we report that CR infection causes severe colitis and mortality in interleukin 22 knockout (Il22-/-) and Rag1 knockout (Rag1-/-) mice under germ-free (GF) conditions. In a gut microbiota-independent manner, CR colonizes in GF Il22-/- and Rag1-/- animals, triggers colonic epithelial tissue damage and systemic dissemination of CR, and results in lethal infections. Pretreatment with cefoxitin, a broad-spectrum antibiotic, exacerbates CR-induced colitis and lethality in specific-pathogen-free (SPF) Il22-/- and Rag1-/- mice. Together our results reveal that CR possesses a gut microbiota-independent virulence, which is better illustrated during infections in immunocompromised hosts associated with severe outcomes.

Assessing the potential risk of human pathogen resistance to medical antifungal treatments arising from agricultural use of fungicides with the same mode of action

AbstractA mechanistic basis is described for assessment of resistance risk to medical antifungal treatments from agricultural use of fungicides of the same mode of action. The following need to occur in landscape environments for a risk to be posed by dual use: (i) emergence, whereby a resistant strain emerges by mutation and invasion; (ii) selection, whereby a mutation conferring a fitness advantage is selected for in the presence of fungicide; and (iii) exposure of humans to resistant strains from the landscape, potentially resulting in invasive fungal infection. We identify 20 human pathogens for which there is evidence that all three processes above could, in principle, occur. A model is derived to explore what determines resistance emergence and selection in human pathogens in landscape environments. Knowledge gaps are identified in key parameters. The analysis suggests that emergence and selection were particularly affected by fitness cost associated with the resistance mutation(s) and fungicide concentration. Emergence was also determined by the amount of pathogen reproduction (related to pathogen population size). If fungicide resistance is associated with even a small fitness cost, then environments with low fungicide concentrations, such as field soils, may not be conducive to resistance emergence or selection. These general findings were related to a specific case of observational data from the Netherlands for Aspergillus fumigatus. The analysis supports previous work that compost is towards the high‐risk end of the spectrum for this species. Agricultural soils, nonagricultural land and grassland were lower risk.

Lack of TYK2 signaling enhances host resistance to Candida albicans skin infection

Candida albicans is the most common human fungal pathogen, causing diseases ranging from local to life-threating systemic infections. Tyrosine kinase 2 (TYK2), a crucial mediator in several cytokine signaling pathways, has been associated with protective functions in various microbial infections. However, its specific contribution in the immune response to fungal infections has remained elusive. In this study, we show that mice lacking TYK2 or its enzymatic activity exhibit enhanced resistance to C. albicans skin infections, limiting fungal spread and accelerating wound healing. Impaired TYK2-signaling prompted the formation of a distinctive layer of necrotic neutrophils around the fungal pathogens. Transcriptomic analysis revealed TYK2’s pivotal role in regulating interferon-inducible genes in neutrophils, thereby impacting their antifungal capacity during infection. Furthermore, we show that TYK2-dependent interferon-gamma (IFNγ) production contributes to fungal dissemination from the skin to the kidneys. Our study uncovers a hitherto unrecognized detrimental role of TYK2 in cutaneous C. albicans infections.

Role of Health Education Activities in Reducing the Spread of Parasitic Pathogens in the Population

The purpose of the research is to emphasize the role of health education as an important factor in reaching out to the population to reduce and spread parasitic pathogens.Materials and methods. By analyzing the statistical data of Annual Reports on parasitic diseases of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare in Kemerovo Region - Kuzbass, the most common parasitic diseases in the population in the region have been established. Sanitary and educational activities were conducted with pre-school and school-age children (students of Grades 8–11) in the direction of bringing to the audience information about the types of parasites of medical importance registered in children and adults in Kuzbass and how to protect themselves from them. Oral and visual information methods were used (conversation, message, lecture, quiz, game, information leaflets and stands, leaflets, brochures, videos).Results and discussion. It is important for a medical student from the first year of the educational process to understand the role of sanitary and educational activities in his future profession as a doctor, and the relevance of personal prophylaxis measures against human parasitic pathogens. First-year students who won a grant in the contest of student ESG projects in the field of ecology and social orientation from Sberbank PJSC on social issues related to the population of Kuzbass with the project “Sanitary and Educational Activities in Parasitosis” implemented their ideas on preventive measures in educational institutions of Kuzbass. The educational activities were aimed at informing the educational audience about the causative agents of parasitic diseases registered by medical institutions among the child population of the region. The “NoParasites” team held talks and lectures on personal preventive measures against parasitic diseases. Brochures were developed and an information stand for parents was installed in the kindergarten. From the point of view of the department's activity the implemented student project has an educational orientation, forms worthy professional and personal qualities in future doctors.

Single nucleotide polymorphisms are associated with strain-specific virulence differences among clinical isolates of Cryptococcus neoformans

Studies across various pathogens highlight the importance of pathogen genetic differences in disease manifestation. In the human fungal pathogen Cryptococcus neoformans, sequence type (ST) associates with patient outcome. We performed a meta-analysis of four genomic studies and identified overlapping gene regions associated with virulence, suggesting the importance of these gene regions in cryptococcal disease in diverse clinical isolates. We explored the relationship between virulence and strain genetic differences using the cryptococcosis mouse model and a closely related library of ST93 clinical isolates. We identified four in vivo virulence phenotypes: hypervirulence, typical virulence with CNS disease, typical virulence with non-CNS disease, and latent disease. Hypervirulent isolates were clade specific and associated with an interferon gamma (IFNγ) dominated immune response. Using a genome wide association study (GWAS), we identified nine genes with polymorphisms associated with IFNγ production, including the inositol sensor ITR4. The itr4Δ mutant recapitulated the hypervirulence phenotype and ITR4 affects expression of two IFNγ associated genes. Finally, we showed that IFNγ production is associated with SNPs that downregulate ITR4 and with SNP accumulation in other IFNγ associated genes. These data highlight the complex role of pathogen genetics in virulence and identify genes associated with hypervirulence and IFNγ in Cryptococcus neoformans.

Environmental Modulators on the Development of the Raccoon Roundworm (Baylisascaris procyonis): Effects of Temperature on the Embryogenesis

Members of the Ascarididae family are common zoonotic pathogens in humans and play an economic role in domestic and livestock animal husbandry. This family includes the obligatorily parasitic nematodes of the genus Baylisascaris, with the raccoon roundworm Baylisascaris procyonis being the most well-known representative. B. procyonis uses the raccoon (Procyon lotor) as its primary host and can utilize a broad range of mammals as paratenic hosts. Sexual reproduction of the adult nematodes occurs in the small intestine. Eggs are excreted into the environment through feces, where they develop into the infectious stage under suitable conditions within a few days to weeks. Infection of primary and paratenic hosts occurs through the oral ingestion of these infectious eggs. Raccoons can also become infected by ingesting infected paratenic hosts. Humans serve as accidental hosts and can suffer significant damage to organ tissues, the visual system, and the central nervous system after ingesting infectious eggs. The aim of the study was to investigate the effects of ambient temperature on embryonic development and to document the morphological changes during embryogenesis. Live specimens were collected from the raccoon intestine and incubated. Single-celled eggs were collected during this process. The eggs were decorticated and then preserved. To test the effects of ambient temperature, the eggs were incubated at 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, and 38°C and monitored at 24-hour intervals for their developmental stages. Detailed photographic documentation of the developmental stages was conducted. An increase in ambient temperature led to a reduction in development time. The temperature range within which embryogenesis proceeded to the L1 larval stage was between 10°C and 30°C. Incubation at 5°C did not produce L1 larvae even after 11 months. Incubation at 35°C and 38°C resulted in the complete degeneration of the eggs before reaching the L1 larval stage.