Infected Insect Research Articles

Effects of fasting on the interplay between temperature and Trypanosoma cruzi infection on the life cycle of the Chagas disease vector Rhodnius prolixus

Rhodnius prolixus, a triatomine insect, is one of the most important vectors of Chagas disease in South America. Its interaction with the parasite Trypanosoma cruzi, the causative agent of this disease, is known to be deeply affected by ambient temperature and the nutritional status of the insect vector. In this study, we investigated how starvation affects the life cycle of R. prolixus and the population dynamics of the parasite inside the intestine of the vector at four temperatures ranging from 24°C to 30°C. The weights and molting times of chronically infected and uninfected insects were monitored through repeated 30-day fasting periods from first instar to adult stage, assessing their capacity to retain blood meal weight between developmental stages and tracking parasite concentrations in their urine. Our results demonstrate that ambient temperature is a crucial factor affecting the resistance of R. prolixus to starvation, as survival, body weight, and weight retention greatly decreased in high temperature treatments. Furthermore, we showed that temperature significantly influenced whether T. cruzi established an infection in early instars, with few insects developing infections at the lowest and highest temperature treatments. Additionally, we discovered that a fasting period of 30 days induces a steady decrease in parasite populations in the vector over its lifetime. Infection by T. cruzi had no effect on the survival, molting time, and nutritional factors monitored in our protocol. Our results highlight the importance of nutrition as a determining factor for the development of the vector and the parasite, providing valuable insights for elucidating the complex interplay between temperature and nutrition in shaping the epidemiology of Chagas disease in a changing climate.

McHDV VP60 Virus-like Particles Elicit Protective Immunity Against Moschus chrysogaster Hemorrhagic Disease in Rabbits

Moschus chrysogaster viral hemorrhagic disease (McVHD), caused by the Moschus chrysogaster hemorrhagic disease virus (McHDV), is an acute and highly fatal infectious disease of musk deer. At present, there is no prevention or treatment for this disease. In this study, we constructed a recombinant bacmid containing the McHDV VP60 gene and obtained the recombinant baculovirus rBac-McHDV VP60 by transfection into Sf9 (Spodoptera frugiperda) insect cells. The McHDV VP60 protein was successfully expressed in the insect cell-baculovirus expression system; furthermore, it was released in the supernatant of infected insect cells and spontaneously assembled to form virus-like particles (VLPs), which were structurally and immunologically indistinguishable from the Moschus chrysogaster viral hemorrhagic disease virion. Hypodermic vaccination of rabbits with the VLPs conferred complete protection in 14 days; this protection was found to be effective from the seventh day after VLP injection and was accompanied by a strong humoral response. This study is the first attempt to express the VP60 gene of McHDV using an insect baculovirus system, which provides an experimental basis for the virus-like particle vaccine of McVHD.

EUROPEAN PAPER BEES, POLISTES DOMINULA AND POLISTES NIMPHA (CHRIST, 1791) (HYMENOPTERA: VESPIDAE) PATHOGENS PRESENCE AND THEIR POTENTIAL INSECTICIDAL EFFECTS ON HONEYBEES ADULTS OF APIS MELLIFERA CAUCASIA (POLLMANN, 1889)

Honeybee (Apis mellifera) is an important element of biodiversity and terrestrial ecosystems. Any pathogenic infection in this beneficial insect can lead to major undesirable disasters. This study investigated the pathogenic bacteria and fungi from Polistes dominula and Polistes nimpha wasps and their potential insecticidal effects on Apis mellifera caucasia. For this purpose, bacteria and fungi were isolated from dead and diseased bees collected from Terme district of Samsun province in Türkiye in May and June 2020. In the study, Granulicatella adiacens, Staphylococcus xylosus, Sphingomonas paucimobilis bacteria and Cryptococcus laurentii and Candida famata fungi were obtained from the internal tissues and organs of Polistes dominula paper wasp adults. Staphylococcus xylosus and Sphingomonas paucimobilis were found to be common bacteria in both bee species. Serratia marcescens and Enterococcus faecalis bacterial species were found to have a very lethal effect on honeybees. Bioassay experiments were performed on the detected fungi, and it was observed that Cryptococcus laurentii and Candida famata fungi species also had lethal effects on honeybees. It has been revealed that entomopathogenic bacteria, which are known to be very effective in biological control against harmful insects, can cause unwanted infections in honeybees.

Wells Syndrome Associated with Fungal Infection

Introduction: Eosinophilic skin diseases encompass a broad spectrum, including Wells syndrome, which is characterized by histological findings of eosinophilic infiltration in the dermis. The etiology and pathogenesis of Wells syndrome are still unknown, but it has been reported that insect bites, infections, and medications can act as triggers. Fungal infections are common in clinical practice; however, when they are recurrent, have atypical characteristics, or are resistant to treatments, they must be recognized as potential triggers for hypersensitivity reactions such as Wells syndrome. Case Presentation: We present the clinical and histopathological characteristics of a case in which Wells syndrome was associated with a fungal infection. Conclusions: Wells syndrome is a rare skin disorder with polymorphic manifestations, making diagnosis challenging in clinical practice. Therefore, a high index of suspicion is required, and blood tests and skin biopsy are necessary to confirm the diagnosis.

Histone deacetylase HDAC3 regulates ergosterol production for oxidative stress tolerance in the entomopathogenic and endophytic fungus Metarhizium robertsii.

Oxidative stress is encountered by fungi in almost all niches, resulting in fungal degeneration or even death. Fungal tolerance to oxidative stress has been extensively studied, but the current understanding of the mechanisms regulating oxidative stress tolerance in fungi remains limited. The entomopathogenic and endophytic fungus Metarhizium robertsii encounters oxidative stress when it infects insects and develops a symbiotic relationship with plants, and we found that host reactive oxygen species (ROSs) greatly limited fungal growth in both insects and plants. We identified a histone H3 deacetylase (HDAC3) that catalyzed the deacetylation of lysine 56 of histone H3. Deleting Hdac3 significantly reduced the tolerance of M. robertsii to oxidative stress from insects and plants, thereby decreasing fungal ability to colonize the insect hemocoel and plant roots. HDAC3 achieved this by regulating the expression of three genes in the ergosterol biosynthesis pathway, which includes the lanosterol synthase gene Las1. The deletion of Hdac3 or Las1 reduced the ergosterol content and impaired cell membrane integrity. This resulted in an increase in ROS accumulation in fungal cells that were thus more sensitive to oxidative stress. We further showed that HDAC3 regulated the expression of the three ergosterol biosynthesis genes in an indirect manner. Our work significantly advances insights into the epigenetic regulation of oxidative stress tolerance and the interactions between M. robertsii and its plant and insect hosts.IMPORTANCEOxidative stress is a common challenge encountered by fungi that have evolved sophisticated mechanisms underlying tolerance to this stress. Although fungal tolerance to oxidative stress has been extensively investigated, the current understanding of the mechanisms for fungi to regulate oxidative stress tolerance remains limited. In the model entomopathogenic and plant symbiotic fungus Metarhizium robertsii, we found that the histone H3 deacetylase HDAC3 regulates the production of ergosterol, an essential cell membrane component. This maintains the cell membrane integrity to resist the oxidative stress derived from the insect and plant hosts for successful infection of insects and development of symbiotic associates with plants. Our work provides significant insights into the regulation of oxidative stress tolerance in M. robertsii and its interactions with insects and plants.

Immune modulation by dexketoprofen trometamol, a selective eicosanoid biosynthesis inhibitor of cellular immune response and phenoloxidase reaction in response to viral infection in Pimpla turionellae adults

Nodulation is the first immune defence mechanism related to melanisation in response to microbial infections in insects. Adult parasitoid insects have been hypothesised to produce nodules with melanisation in response to viral infections and, eicosanoids, to mediate nodulation reactions and phenoloxidase (PO) activation in this type of infections. To test this hypothesis, endoparasitoid Pimpla turionellae adults were first inoculated with a novel generation nonsteroidal anti-inflammatory drug (NSAID) dexketoprofen trometamol (DT) (5 μg/adult), which is a selective cyclooxygenase-1 (COX-1) inhibitor. These adults were then immediately injected with intrahaemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1) as a model insect-virus interaction. Additionally, adults were fed on artificial diet with increasing concentrations of DT (0.001, 0.01, or 0.1 g/100 ml diet) per os prior to intrahaemocoelic injection of BHSV-1 (2 × 103 PFU/adult) and nodulation and PO activity were recorded at 2 h post inoculation (PI). BHSV-1-treated newly emerged adults fed with inhibitors showed low levels of nodulation and increased PO enzyme activity. DT-treated Pimpla adults produced significantly fewer nodules (approximately nine nodules/adult), whereas viral infection provoked nodules (approximately 33 nodules/adult) in comparison with needle (vehicle)-treated controls (approximately five nodules/adult). Increasing dietary dexketoprofen trometamol concentrations decreased nodulation (by 12-fold at the highest concentration) and increased PO reactions (by approximately 3-fold at the highest concentration) to BHSV-1 injection. Compared with control adults, adults orally fed on the lowest DT concentration (0.001 %) significantly increased PO activity (1.22 ± 0.23–2.74 ± 0.31 unit/min/mg protein) while nodules significantly decreased (43.19 ± 4.26–17.84 ± 2.19) in response to virus infections. These findings suggest that eicosanoid biosynthesis, at least in the context of prostaglandins (PGs) formed by COX-1, mediates nodulation reactions and PO activation in response to viral infection in adults of this endoparasitoid. This is the first demonstration that the immune response of P. turionellae adults to viral pathogens is modulated by DT, which initiates haemolymph PO activation.

Development of Fusion-Based Assay as a Drug Screening Platform for Nipah Virus Utilizing Baculovirus Expression Vector System.

Nipah virus (NiV) is known to be a highly pathogenic zoonotic virus, which is included in the World Health Organization Research & Development Blueprint list of priority diseases with up to 70% mortality rate. Due to its high pathogenicity and outbreak potency, a therapeutic countermeasure against NiV is urgently needed. As NiV needs to be handled within a Biological Safety Level (BSL) 4 facility, we had developed a safe drug screening platform utilizing a baculovirus expression vector system (BEVS) based on a NiV-induced syncytium formation that could be handled within a BSL-1 facility. To reconstruct the NiV-induced syncytium formation in BEVS, two baculoviruses were generated to express recombinant proteins that are responsible for inducing the syncytium formation, including one baculovirus exhibiting co-expressed NiV fusion protein (NiV-F) and NiV attachment glycoprotein (NiV-G) and another exhibiting human EphrinB2 protein. Interestingly, syncytium formation was observed in infected insect cells when the medium was modified to have a lower pH level and supplemented with cholesterol. Fusion inhibitory properties of several compounds, such as phytochemicals and a polysulfonated naphthylamine compound, were evaluated using this platform. Among these compounds, suramin showed the highest fusion inhibitory activity against NiV-induced syncytium in the baculovirus expression system. Moreover, our in silico results provide a molecular-level glimpse of suramin's interaction with NiV-G's central hole and EphrinB2's G-H loop, which could be the possible reason for its fusion inhibitory activity.

Automated Detection of Plant Diseases and Pests Using Advanced Image Processing and Neural Networks

Pests and viral plant diseases have proliferated in agriculture. Rats spread plant diseases. Hard to detect viral infections or insect infestations that impair plant growth and yield. It decreases crop size, quality, and marketability, incurring major economic losses. Strong, disease-resistant crops are needed for agriculture. Bacterial, fungal, and insect/pest diseases destroy 70% and 15% of crops. Above 85% of disease severity destroys crops. Early detection systems track pests and diseases. High-definition equipment is costly for rural farmers. Correct diagnosis permits quick infection severity and type assessment and control. In precision agriculture, neural networks and image processing eliminate human error. Sustainable agriculture and organic agricultural cultivation must start early. The proposed system classifies diseases and pests for plant safety. A healthy, fast-growing plantation is our goal. Early detection of viral diseases and insect infestations boosts crop productivity. Plant disease diagnostics focuses on leaves and buds. Stem and insect infections are covered. The suggested method classifies plant health and disease automatically. Peanut leaves develop Bacterial Blight from contagious illnesses like Cercospora Leaf Peanut. Boll Weevils, European Corn Borers, and Fall Army Worms attack cotton. The conditions are categorised. Image accuracy depends on lighting, resolution, location, and backdrop complexity. Various low-resolution pictures are enriched to better classification. Complex leaf intensity variations cause the system to misdiagnose illness or insect infestation. To avoid misclassification, the system segments the sample image using various methods to find lesions. Multilevel Segmentation (MS) improves Deep neural network feature extraction using approximate outlines. This method protects important pixels, eliminates unneeded pixels, and amplifies the lesion to control segmentation and reduce insufficient and excessive segmentation.

Expression, purification, and function of baculovirus infected insect cells‐derived hookworm AIP‐1 and AIP‐2 proteins

AbstractAnti‐inflammatory protein (AIP)‐1 and AIP‐2, identified as parasite excretory/secretory (ES) proteins, play a crucial role in promoting the survival of the parasite and evading the host immunological response. Both proteins inhibit inflammatory reactions, induce apoptosis in effector cells, and influence the phenotype of the immune response. Numerous parasite‐derived proteins have shown promise as therapeutic targets for inflammatory and allergic diseases. Despite this, the precise biological roles, and molecular characteristics of many such proteins remain unclear.In this study, AIP‐1 and AIP‐2 were produced in the baculovirus‐insect cell expression system (BEVS). The multiplicity of infection (MOI) and the duration of infection conditions for AIP‐1 and AIP‐2 protein expression were successfully optimized to induce the highest expression levels of AIP‐1 and AIP‐2 proteins in insect cells. The insect cell‐derived AIP‐1 and AIP‐2 exhibited inhibitory functions against human matrix metalloproteinases (MMPs). This study demonstrates that insect cell‐derived AIP‐1 and AIP‐2 have the potential as therapeutic proteins for MMP‐TIMP (Metalloprotease‐Tissue inhibitor of metalloprotease) axis related disease.

OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

Characterization of an envelope protein 118L in invertebrate iridescent virus 6 (IIV6).

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication.

Expression of a viral ecdysteroid UDP-glucosyltransferase enhanced the insecticidal activity of the insect pathogenic fungus Beauveria bassiana.

Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20-hydroxyecdysone (20E). In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP-glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT-1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT-1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT-1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT-1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry.

The Balance between Protealysin and Its Substrate, the Outer Membrane Protein OmpX, Regulates Serratia proteamaculans Invasion.

Serratia are opportunistic bacteria, causing infections in plants, insects, animals and humans under certain conditions. The development of bacterial infection in the human body involves several stages of host-pathogen interaction, including entry into non-phagocytic cells to evade host immune cells. The facultative pathogen Serratia proteamaculans is capable of penetrating eukaryotic cells. These bacteria synthesize an actin-specific metalloprotease named protealysin. After transformation with a plasmid carrying the protealysin gene, noninvasive E. coli penetrate eukaryotic cells. This suggests that protealysin may play a key role in S. proteamaculans invasion. This review addresses the mechanisms underlying protealysin's involvement in bacterial invasion, highlighting the main findings as follows. Protealysin can be delivered into the eukaryotic cell by the type VI secretion system and/or by bacterial outer membrane vesicles. By cleaving actin in the host cell, protealysin can mediate the reversible actin rearrangements required for bacterial invasion. However, inactivation of the protealysin gene leads to an increase, rather than decrease, in the intensity of S. proteamaculans invasion. This indicates the presence of virulence factors among bacterial protealysin substrates. Indeed, protealysin cleaves the virulence factors, including the bacterial surface protein OmpX. OmpX increases the expression of the EGFR and β1 integrin, which are involved in S. proteamaculans invasion. It has been shown that an increase in the invasion of genetically modified S. proteamaculans may be the result of the accumulation of full-length OmpX on the bacterial surface, which is not cleaved by protealysin. Thus, the intensity of the S. proteamaculans invasion is determined by the balance between the active protealysin and its substrate OmpX.

A fungal pathogen secretes a cell wall-associated β-N-acetylhexosaminidase that is co-expressed with chitinases to contribute to infection of insects.

β-N-acetylhexosaminidases (HEXs) are widely distributed in fungi and involved in cell wall chitin metabolism and utilization of chitin-containing substrates. However, details of the fungal pathogens-derived HEXs in the interaction with their hosts remain limited. An insect nutrients-induced β-N-acetylhexosaminidase, BbHex1, was identified from the entomopathogenic fungus Beauveria bassiana, which was involved in cell wall modification and degradation of insect cuticle. BbHex1 was localized to cell wall and secreted, and displayed enzyme activity to degrade the chitinase-hydrolyzed product (GlcNAc)2. Disruption of BbHex1 resulted in a significant decrease in the level of cell wall chitin in the presence of insect nutrients and during infection of insects, with impaired ability to penetrate insect cuticle, accompanying downregulated cell wall metabolism-involved and cuticle-degrading chitinase genes. However, the opposite phenotypes were examined in the gene overexpression strain. Distinctly altered cell wall structures caused by BbHex1 mutation and overexpression led to the easy activation and evasion (respectively) of insect immune response during fungal infection. As a result, BbHex1 contributed to fungal virulence. Bioinformatics analysis revealed that promoters of some co-expressed chitinase genes with the BbHex1 promoter shared conserved transcription factors Skn7, Msn2 and Ste12, and CreA-binding motifs, implying co-regulation of those genes with BbHex1. These data support a mechanism that the fungal pathogen specifically expresses BbHex1, which is co-expressed with chitinases to modify cell wall for evasion of insect immune recognition and to degrade insect cuticle, and contributes to the fungal virulence against insects. © 2024 Society of Chemical Industry.

Evaluating Mosquito Biocontrol Effectiveness by Isolating and Characterizing Some Fungi Against Culex Pipiens and Anopheles Stephensi in Sothern Part of Iraq

Objectives: Isolation Aspergillus tamarii, Cladosporium herbarum, and Verticillium lecanii fungi from naturally infected Culex pipiens and Anopheles stephensi insects were morphologically and molecularly identified. Methods: In this study, populations of mosquitoes were cultured and examined to determine fungal infection and evaluated as potential agents against C. pipiens and A. stephensi. Results: A variety of fungal isolates demonstrated differing degrees of pathogenicity 24 hours after treatment against C. pipiens and A. stephensi eggs, four-instar mosquito larvae, and adults. and as a biological control, it was found that the fungal suspension of each of the used fungi affected the life roles of the two mosquitoes. as it was more than the fungus suspension A. tamarii, Cl. herbarum on V. lecanii. The highest percentage of eggs mortality were (36.72, 48.97) %, (29.14, 42.25) %, and (24.45, 35.70) % of C. pipiens and A. stephensi when using the highest concentration of 1*105 spore/ml, 3*105 spore/ml and 2*105 spore/ml of fungicide A. tamarii, C. herbarum, V.lecanii and respectively after 24 hours. The highest mortality rate was for the fourth larval stages, and the mosquito A. stephensi is more sensitive to type C. pipiens infection., as, in order (75.6, 67.19, 56.8) %, (74.18, 59.81, 50) %, and (65, 52,42.71) %. Conclusions: Results highlight the significance of a mosquito's natural fungal opponent. All isolates had an impact on adults and larvae, although they were less successful against eggs. Both have the potential to develop, particularly against the larvae of the primary arbovirus, malaria, and lymphatic filariasis vectors, A. stephensi, and C. pipiens.

Metabolomics-based approaches in unraveling virus infections in insects: revealing unknown hidden interactions

Metabolomics-based approaches in unraveling virus infections in insects: revealing unknown hidden interactions

Nonlinear dynamics and optimal control strategies of a novel fractional-order lumpy skin disease model

Fractional order differential equations are often employed to better and accurately model several natural and physical processes with memory characteristics than the integer-order models. In this work, a proposed fractional-order model is presented to simulate one of the most dangerous viral cattle diseases. The present fractional-order Lumpy Skin Disease model considers the interactions between different compartments of cattle and virus vectors. The solution of the model is examined in terms of existence, uniqueness, positivity and boundedness. Then, the stability analysis of the different equilibrium points in the model is carried out. The basic reproduction number, R0, has been obtained too. The dependence of stability regions on the values of key parameters is demonstrated and the sensitivity of R0 to different parameters in the system is also investigated. Finally, a proposed optimal control scheme is adopted to successfully diminish the disease spread. It is found that the stability of disease-free equilibrium point is verified at small values of the infection rate of cattle caused by infected cattle or insects. In addition, the disease-free equilibrium point maintains its stability in cases where the recovery rate of cattle or the vaccination rate of cattle are increased. Moreover, the high-efficiency vaccination program helps in stabilizing the disease-free equilibrium point. Also, the fractional-order parameter is found to provide additional degree of freedom in the model to represent different scenarios of evolution for solution orbits. The optimal control parameters of the model are examined in different scenarios to limit or suppress the spread of the disease. It is demonstrated that two control parameters are sufficient to achieve the goals of disease elimination and stabilization of an infection-free steady state. Numerical simulations are verified to confirm our theoretical findings and estimations.

Detection and Differentiation of Entomopathogenic Serratia spp. to Inform Reintroduction of the Critically Endangered Lord Howe Island Stick Insect Dryococelus australis.

Once rodents have been successfully eradicated from Lord Howe Island, Australia, the critically endangered Lord Howe Island stick insect (Dryococelus australis (Montrouzier)) may be reintroduced, a century after it was thought to have become extinct. In captive populations of D. australis, elevated mortalities have been associated with bacterial pathogens. To better define the infectious risk posed by entomopathogens to the reintroduction program, we investigated the bacteria isolated from captive D. australis kept at Melbourne Zoo and on Lord Howe Island and from environmental samples and free-living invertebrates collected on various parts of the island. At Melbourne Zoo, Serratia and Pseudomonas spp. were the bacteria most frequently isolated between 2013 and 2019. Serratia spp. were also the organisms most frequently isolated from insects sampled in April 2019 from the captive population on Lord Howe Island. In addition, Serratia spp. were isolated from a range of environmental samples collected on Lord Howe Island during March-April 2019. These environmental isolates had a broader range of biochemical and molecular characteristics than those obtained from the captive insect populations. A large proportion of these isolates were urease positive and had biochemical profiles previously not described for Serratia spp. This study highlights the need for better surveillance for potential pathogens in understudied regions and sites. We conclude that infections caused by Serratia spp. might pose a problem to the captive breeding program for D. australis but that the risk of introducing novel pathogens to Lord Howe Island through infected insects is low. Our study explores some of the potential risks involved in captive breeding and provides a valuable example of using pathogen surveillance to better inform an invertebrate conservation program.

49-year-old male with infected insect bite

49-year-old male with infected insect bite

Galleria mellonella Invertebrate Model Mirrors the Pathogenic Potential of Mycoplasma alligatoris within the Natural Host

Most mycoplasmal infections result in chronic, clinically silent disease. In direct contrast, Mycoplasma alligatoris elicits a fulminant, multisystem disease in the natural host, Alligator mississippiensis (American alligator). The goals of the study were to better understand the disease in the natural host and to determine if the invertebrate model G. mellonella could serve as a surrogate alternate host. The survival of alligators infected intratracheally was dose dependent (p=0.0003), ranging from no mortality (102 CFU) to 100% mortality (108 CFU), with 60% mortality at the 104 and 105 CFU infectious dose. Microbial load in blood, joints, and brain was dose dependent, regardless of whether alligators were infected intratracheally or intravenously (p <0.002). Weight loss was similarly impacted (p <0.001). Experimental infection of the invertebrate Galleria mellonella mirrored the result in the natural host. In a dose response infection study, both larval survival curves and successful pupation curves were significantly different (p≤0.0001) and dose dependent. Infected insects did not emerge as moths (p <0.0001). Here, we describe the first study investigating G. mellonella as a surrogate model to assess the pathogenic potential of M. alligatoris. G. mellonella survival was dose dependent and impacted life stage outcome.