Wax Moth Larvae Research Articles

Host-foraging strategies of five local entomopathogenic nematode species in South Africa

Entomopathogenic nematodes (EPNs) are obligate parasites of soil-dwelling insects and are used as biological control agents for many insect pests. These nematodes have a free-living third growth stage called infective juveniles (IJs), which are responsible for foraging and infecting suitable insect hosts. Infective juveniles exhibit three host-foraging strategies: cruising, ambushing, and intermediate foraging strategies. The foraging strategy of EPNs is important for successful infection but is poorly understood. The current study investigated the host-foraging strategies of five local South African EPN species including Heterorhabditis noenieputensis, H. safricana, Steinernema fabii, S. jeffreyense, and S. yirgalemense by assessing their dispersal behavior. Of the five EPN species, H. noenieputensis, H. safricana, S. jeffreyense, and S. yirgalemense showed a positive response to the presence of the wax moth larvae, whereas S. fabii showed a negative response. The four EPN species that showed a positive response to the presence of the host also caused 100% mortality of wax moth larvae that were buried in sand at a depth of 10 cm, whereas S. fabii caused the lowest mortality of 34%. The average distance traveled by all five EPN species decreased on rough textured substrate compared with smooth textured substrate. The observed behavioral patterns suggested that H. noenieputensis, H. safricana, S. jeffreyense, and S. yirgalemense use a cruiser foraging strategy whereas S. fabii uses an ambusher foraging strategy.

A Diet with Amikacin Changes the Bacteriobiome and the Physiological State of Galleria mellonella and Causes Its Resistance to Bacillus thuringiensis.

Environmental pollution with antibiotics can cause antibiotic resistance in microorganisms, including the intestinal microbiota of various insects. The effects of low-dose aminoglycoside antibiotic (amikacin) on the resident gut microbiota of Galleria mellonella, its digestion, its physiological parameters, and the resistance of this species to bacteria Bacillus thuringiensis were investigated. Here, 16S rDNA analysis revealed that the number of non-dominant Enterococcus mundtii bacteria in the eighteenth generation of the wax moth treated with amikacin was increased 73 fold compared to E. faecalis, the dominant bacteria in the native line of the wax moth. These changes were accompanied by increased activity of acidic protease and glutathione-S-transferase in the midgut tissues of larvae. Ultra-thin section electron microscopy detected no changes in the structure of the midgut tissues. In addition, reduced pupa weight and resistance of larvae to B. thuringiensis were observed in the eighteenth generation of the wax moth reared on a diet with amikacin. We suggest that long-term cultivation of wax moth larvae on an artificial diet with an antibiotic leads to its adaptation due to changes in both the gut microbiota community and the physiological state of the insect organism.

PARASITISATION POTENTIAL OF EGG AND LARVAL PARASITOIDS AGAINST LESSER WAX MOTH Achroia grisella F. (LEPIDOPTERA: PYRALIDAE) UNDER STORED CONDITION

The study conducted to evaluate the parasitisation potential of egg (Trichogramma chilonis Ishii) and larval parasitoids (Bracon brevicornis Wesmael and Apanteles galleriae Wilkinson) on the developmental stages of lesser wax moth, Achroia grisella F. The results indicated that a maximum parasitisation of 46.67% was noticed on egg masses released with seven pairs of T. chilonis, with an overall mean of 28.89 % and also the adult emergence was recorded about 59.49 %. Among the two larval parasitoids taken, the per cent parasitisation of lesser wax moth larvae by A. galleriae was maximum (38.68% overall mean) than by B. brevicornis (33.14%) with a mean adult emergence of 71.16% and 64.78% respectively.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

Selection on plastic adherence leads to hyper-multicellular strains and incidental virulence in the budding yeast.

Many disease-causing microbes are not obligate pathogens; rather, they are environmental microbes taking advantage of an ecological opportunity. The existence of microbes whose life cycle does not require a host and are not normally pathogenic, yet are well-suited to host exploitation, is an evolutionary puzzle. One hypothesis posits that selection in the environment may favor traits that incidentally lead to pathogenicity and virulence, or serve as pre-adaptations for survival in a host. An example of such a trait is surface adherence. To experimentally test the idea of 'accidental virulence', replicate populations of Saccharomyces cerevisiae were evolved to attach to a plastic bead for hundreds of generations. Along with plastic adherence, two multicellular phenotypes- biofilm formation and flor formation- increased; another phenotype, pseudohyphal growth, responded to the nutrient limitation. Thus, experimental selection led to the evolution of highly-adherent, hyper-multicellular strains. Wax moth larvae injected with evolved hyper-multicellular strains were significantly more likely to die than those injected with evolved non-multicellular strains. Hence, selection on plastic adherence incidentally led to the evolution of enhanced multicellularity and increased virulence. Our results support the idea that selection for a trait beneficial in the open environment can inadvertently generate opportunistic, 'accidental' pathogens.

Comparison of virulence of some strains of microorganisms on the larva model of a large wax moth and a mouse model. Is replacement possible?

To date, conducting experiments involving animals is difficult due to the high cost, the complexity of reproducibility, as well as the ethical standards of modern society. The 3R concept developed in the last century encourages the development of alternative test systems. Among other alternative test systems, the wax moth larva occupies a special place. The use of larvae of the great wax moth (Galleria melonella) as a test system for determining the virulent properties of microorganisms is a promising direction and makes it possible to reduce the cost of research. However, not in all experiments, researchers could register the same semilethal doses for selected strains when comparing the Galleria melonella test system and mammals. Previously, a study was conducted, where, among other things, the semilethal doses of some strains were determined using the Galleria melonella test system. The aim of this study was to compare the semilethal doses of selected strains when administered to G. mellonella and mice. For the experiment, the animals were injected intraperitoneally with suspensions of reference strains of Escherichia coli ATCC 25922 (VKPM B - 8076), Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 6303 and clinical isolates of Escherichia coli ENV 194, Staphylococcus aureus SA 0077 (MRSA), Streptococcus pneumoniae 1068 19 A After infection, the animals were observed daily throughout the experiment. During daily clinical observation, visually noted (if any) clinical signs indicating the development of an infectious process, and also recorded body weight. After determining the LD50, the results were compared with the data obtained in the experiment with the larvae of the great wax moth. As a result of comparing the semi-lethal doses of the selected strains, it can be concluded that there is no clear correlation between the data obtained in the two experiments.

Morphological and biological characteristics of Galleria mellonella L. (Lepidoptera: Pyralidae) reared on different artificial diets

The greater wax moth (Galleria mellonella L.) is a common insect used as alternative host for mass rearing and research of natural enemies. Finding appropriate artificial diets can aid in increasing the efficiency of mass rearing. The study evaluated three different artificial diets by determining some biological and morphological characteristics of greater wax moths in laboratory of Plant Protection Department, Agronomy Faculty, Nong Lam University, Ho Chi Minh City, Vietnam. The experimental results showed that the artificial diet CT2 was the most effective one of the three treatments. Its larval stage was shortest, with an average of 27.6 ± 4.2 days. The greater wax moth larvae reared on CT2 grew fast and had large size. Over a period of 20 - 30 days of age, the length and width of greater wax moth larvae ranged from 11.9 ± 5.2 to 16.3 ± 6.2 mm and 2.7 ± 1.4 to 3.9 ± 1.8 mm, respectively. The female greater wax moths laid 819.0 ± 175.5 to 1,008.1 ± 354.6 eggs on average. When greater wax moths were reared on three different artificial diets, there was no difference in fecundity

Pathogenicity and other characteristics of the endophytic Beauveria bassiana strain (Bals.) (Hypocreales: Cordycipitaceae)

BackgroundAgriculture crops such as tomatoes and wheat are frequently targeted by insect pests which have a significant negative impact on the agricultural economies. The deployment of entomopathogenic fungi (EPF) for the control of the insect pests is an important alternative to synthetic insecticides. The EPF, Beauveria bassiana (Balsamo) Vuillemin, has been reported widely as a suitable biological control agent of many agricultural pests.ResultsIn this study, B. bassiana SS-1 was isolated from local plant crops and its pathogenicity was assessed against the greater wax moth larvae Galleria mellonella (L.). The development of the pathogenic B. bassiana SS-1 on the insect was visualized using scanning electron microscopy (SEM). Results showed the ability of B. bassiana SS-1 to secrete extracellularly the important enzymes essential for insect cuticle penetration. B. bassiana SS1 recorded the maximum mean lipase (5.3 U/ml), protease (32.13 U/ml), and chitinase activities (2.95 U/ml). The endophytic pathogenic fungus B. bassiana SS-1 demonstrated pathogenicity against the fourth instar larvae of G. mellonella showing LC50 at 2.47 × 102 conidia/ml and LC95 at 3.98 × 105 conidia/ml. The SEM results showed physical contact with B. bassiana SS-1 hyphae on the surface of the G. mellonella larvae. Thus, the isolated EPF B. bassiana SS-1, even endophytic, could be a promising biocontrol agent to manage agricultural insect pests.ConclusionThis study provided a comprehensive characterization of the pathogenicity of B. bassiana SS-1 with its microbiological characteristics. Future studies are needed to focus on the detection of highly virulent isolates against different insect pests and to assess their field contribution as a favorable biological control agent.

Novel Entomopathogenic Fungi of Tamil Nadu Soils and Their Pathogenicity on Waxmoth Larva (Galleria mellonella)

Background: Biological plant protection with entomopathogenic fungi is a vital component of sustainable pest management. The most widely used entomopathogenic fungi (EPF) are Beauveria bassiana, Metarhizium anisopliae, Verticillium sp. And Isaria sp., though there are several other genera of EPF whose potential correlates to that of commonly used fungi for reducing insect pests. The primary goal of this study was to assess the efficiency of a few novel fungal isolates against insect pests. Methods: Two concentrations of four different entomopathogenic fungi were evaluated for their potency against wax moth larvae, Galleria mellonella at Insectary, Tamil Nadu Agricultural University, Coimbatore. Result: Mortality was observed in both the concentrations of four fungi, among which Penicillium simplicissimum performed well on par with Clonostachys rosea and Purpureocillium lilacinum. Hence, these fungi could be formulated and utilized in biological pest control.

Staphylococcus aureus From an Acute Fracture-related Infection Displays Important Bacteriological and Histopathologic Differences From a Chronic Equivalent in a Murine Bone Infection Model.

Staphylococcus aureus is the leading pathogen in fracture-related infection. Previous in vitro experiments, in vivo testing in wax moth larvae, and genomic analysis of clinical S. aureus isolates from fracture-related infection identified low-virulence (Lo-SA5464) and high-virulence (Hi-SA5458) strains. These findings correlated with acute fracture-related infection induced by Hi-SA5458, whereas Lo-SA5464 caused a chronic fracture-related infection in its human host. However, it remains unclear whether and to what extent the causative pathogen is attributable to these disparities in fracture-related infections. Are there differences in the course of infection when comparing these two different clinical isolates in a murine fracture-related infection model, as measured by (1) clinical observations of weight loss, (2) quantitative bacteriology, (3) immune response, and (4) radiographic and histopathologic morphology? Twenty-five (including one replacement animal) female (no sex-specific influences expected), skeletally mature C57Bl/6N inbred mice between 20 and 28 weeks old underwent femoral osteotomy stabilized by titanium locking plates. Fracture-related infection was established by inoculation of high-virulence S. aureus EDCC 5458 (Hi-SA5458) or low-virulence S. aureus EDCC 5464 (Lo-SA5464) in the fracture gap. Each of these groups consisted of 12 randomly assigned animals. Mice were euthanized 4 and 14 days postsurgery, resulting in six animals per group and timepoint. The severity and progression of infection were assessed in terms of clinical observation of weight loss, quantitative bacteriology, quantitative serum cytokine levels, qualitative analysis of postmortem radiographs, and semiquantitative histopathologic evaluation. For clinical observations of weight change, no differences were seen at Day 4 between Hi-SA5458- and Lo-SA5464-infected animals (mean -0.6 ± 0.1 grams versus -0.8 ± 0.2 grams, mean difference -0.2 grams [95% CI -0.8 to 0.5 grams]; p =0.43), while at 14 days, the Hi-SA5458 group lost more weight than the Lo-SA5464 group (mean -1.55 ± 0.2 grams versus -0.8 ± 0.3 grams; mean difference 0.7 grams [95% CI 0.2 to 1.3 grams]; p = 0.02). Quantitative bacteriological results 4 days postoperatively revealed a higher bacterial load in soft tissue samples in Hi-SA5458-infected animals than in the Lo-SA5464-infected cohort (median 6.8 x 107 colony-forming units [CFU]/g, range 2.2 x 107 to 2.1 x 109 CFU/g versus median 6.0 x 106 CFU/g, range 1.8 x 105 to 1.3 x 108 CFU/g; difference of medians 6.2 x 107 CFU/g; p = 0.03). At both timepoints, mice infected with the Hi-SA5458 strain also displayed higher proportions of bacterial dissemination into organs than Lo-SA5464-infected animals (67% [24 of 36 organs] versus 14% [five of 36 organs]; OR 12.0 [95% CI 3.7 to 36]; p < 0.001). This was accompanied by a pronounced proinflammatory response on Day 14, indicated by increased serum cytokine levels of interleukin-1β (mean 9.0 ± 2.2 pg/mL versus 5.3 ± 1.5 pg/mL; mean difference 3.6 pg/mL [95% CI 2.0 to 5.2 pg/mL]; p < 0.001), IL-6 (mean 458.6 ± 370.7 pg/mL versus 201.0 ±89.6 pg/mL; mean difference 257.6 pg/mL [95% CI 68.7 to 446.5 pg/mL]; p = 0.006), IL-10 (mean 15.9 ± 3.5 pg/mL versus 9.9 ± 1.0 pg/mL; mean difference 6.0 pg/mL [95% CI 3.2 to 8.7 pg/mL]; p < 0.001), and interferon-γ (mean 2.7 ± 1.9 pg/mL versus 0.8 ± 0.3 pg/mL; mean difference 1.8 pg/mL [95% CI 0.5 to 3.1 pg/mL]; p = 0.002) in Hi-SA5458-infected compared with Lo-SA5464-infected animals. The semiquantitative histopathologic assessment on Day 4 revealed higher grades of granulocyte infiltration in Hi-SA5458-infected animals (mean grade 2.5 ± 1.0) than in Lo-SA5464-infected animals (mean grade 1.8 ± 1.4; mean difference 0.7 [95% CI 0.001 to 1.4]; p = 0.0498). On Day 14, bone healing at the fracture site was present to a higher extent in Lo-SA5464-infected animals than in Hi-SA5458-infected animals (mean grade 0.2 ± 0.4 versus 1.8 ± 1.2; mean difference -1.6 [95% CI -2.8 to -0.5]; p = 0.008). Similar to septic infection in a human host, infection with Hi-SA5458 in this murine model was characterized by a higher bacterial load, more-pronounced systemic dissemination, and stronger systemic and local inflammation. Thus, there is strong support for the idea that pathogenic virulence plays a crucial role in fracture-related infections. To confirm our observations, future studies should focus on characterizing S. aureus virulence at the genomic and transcriptomic levels in more clinical isolates and patients. Comparing knockout and wildtype strains in vitro and in vivo, including the S. aureus strains studied, could confirm our findings and identify the genomic features responsible for S. aureus virulence in fracture-related infections. For translational use, virulence profiles of S. aureus may be useful in guiding treatment decisions in the future. Once specific virulence targets are identified, one approach to fracture-related infections with high-virulence strains might be the development of antivirulence agents, particularly to treat or prevent septic dissemination. For fracture-related infections with low virulence, prolonged antimicrobial therapy or exchange of an indwelling implant might be beneficial owing to slower growth and persistence capacity.

Efficiency of the Iraqi Isolates of Beauveria Bassiana &amp; Metarhizium Anisopliae in Controlling the Greater Wax Moth Galleria Mellonella L.

The current study was conducted in the laboratories of the Plant Protection Department/College of Agriculture and Forestry/University of Mosul during the year 2019-2020. The aim of this study was to evaluate the efficiency of the Iraqi isolate of the biological control fungi Beauveria bassian and Metarhizium anisopliaeas chemical pesticides alternatives in controlling different stages of the greater wax moth Galleria mellonella. The results of the biological effect of the fungi Beauveria bassiana and Metarhizium anisopliae on the greater wax moth life stages showed that the cumulative mortality of eggs increased gradually with increasing concentration used. However, Beauveria bassiana was more efficient in achieving higher mortality rates than M. anisopliae in general. The highest cumulative mortality rate for eggs was at the highest concentration (7.5g/l) for both fungi. On the other hand, it was found that the highest mortality rate in wax moth larvae was inB. bassianacombined treatmentwhere spraying the larvae and the feed. The third and fourth larval instars were more sensitive than the sixth and seventh larval instars, which were not affected by the lower concentrations of the pesticide (2.5 and 5 g/L), regardless of pesticide type. The results also showed that the mortality rate in all phases increases with the increase in the post-treatment period. The results of the study indicated the superiority of B. bassiana in all treatments, concentrations and periods after treatment and led to the highest mortality rate compared with using M. anisopliae. The cumulative mortality rate in the greater wax moth was directly proportional to the increase in treatment methods and the increase in concentration and the time period after treatment.

An invertebrate model using Galleria mellonellato study the innate immune response to Staphylococcus haemolyticus

Abstract Staphylococcus haemolyticus is a normal inhabitant of healthy skin and is also an emerging cause of nosocomial infections in the immunocompromised. S. haemolyticus infections often lead to bacteremia, septicemia, peritonitis, endocarditis and various other outcomes. The goal of this project was to establish an invertebrate infection model for studying the innate immune response to Staphylococcus haemolyticus infections using the wax moth larva (Galleria mellonella). We have found that injecting wax worm larvae with 10 6to 10 7cfu/μL of S. haemolyticus consistently induced a rapid response and larval death within 24 hours at the higher dose range. We have characterized the general histopathological changes associated with infection and have started to characterize the general innate immune response as well as the hemocyte response to S. haemolyticus. Our findings support the usefulness of this model as an initial screening tool for systemic microbial infections. Supported by grants from the Research Council and the College of Arts &amp; Sciences at Texas A&amp;M University-San Antonio.

The use of a Habrobracon blunted to protect bee honeycomb

The research aimed to evaluate the effectiveness of using the entomophagous of the Habrobracon in honeycombs as a means of biological protection of the land from wax moths. We have conducted studies on the possibility of using organic beekeeping to protect honeycombs from wax moths through Habrobracon. Since the timing of seasonal colonization of wax moths in biological protection is paramount, to get the maximum benefit from using entomophagous, we conducted a two-factor experiment, taking into account the diversity of treatments and the amount of entomophagous used. The group produced entomophagous; 60-70% accounted for females and 30-40% for males. During our research, we formed eleven groups of 19 frames with dry. The authors placed a structure of freshly woven honeycombs with honey residues in the middle of the hive to provide nutrition for imago-entomophagous. The formed groups were identified in 20 frame hives that filled the land. As a result of the peculiarities of the entomophagous to penetrate various holes and cracks, the hives were located apart from each other at a distance allowing to prevent the entomophagous from entering from another pack. No protective treatments were carried out in the control group, and when setting a negative experience along with the dry, frames were placed in the hive, already affected by larvae and wax moth pupae. During the season, the authors visually noted the degree of damage to combs by wax moths. The effectiveness of using Habrobracon as a biological protection of combs was determined by the number of natural frames that flew out, parasitic caterpillars and adults of the wax moth. Assessing the degree of damage to honeycombs by wax moth, it was noted that in variants where the placement of entomophagous was seven pieces or more per m2 with the subsequent settlement of their population according to the research scheme, there were practically no cell lesions. However, it should be noted in some hives, the presence of wax moth larvae is affected by entomophagous. The pest affected the entire land by July-August simultaneously in both the control and group where the negative experience was laid. Our studies convincingly prove the practicality of using the bee’s cells to combat the larva of the wax moth. The entomophagous of the Habrobracon blunted in the amount of 7 pcs/m2 , with the population settling during the season by a 4-fold release of the entomophagous.

Insecticidal Activity of Some Plant Extract Against Greater Wax Moth Larvae (Galleria mellonella L.)

Galleria mellonella L. (Lepidoptera: Pyralidae) is a significant international pest and the most devastating pest throughout the world of beeswax. Natural pesticides must be replaced with synthetic materials since they are more ecologically friendly. This study aims to determine alternative methods of controlling the larger wax moth by assessing the efficacy of several plant-based biocides for this purpose various ethanolic plant extracts such as rosemary (Rosmarinus officinalis L), Clove (Syzygium aromaticum), Eucalyptus (Eucalyptus sp) and Cinnamon (Cinnamomum verum) where used with four extraction rates (5,10,15 and 20) against the late instar larvae of Galleria mellonella in vitro after 24,48 and 72 hr. Obtained results revealed that the pupal mortality increased when the concentration increased and the highest accumulative mortality percentage of wax moth larvae during the experiment, it was demonstrated that all studied plants were efficient at suppressing Galleria mellonella larvae. The result shows that three days after application treatment of rosemary 20% gave the maximum total mortality percentage (%100) followed by (%96.58, %92.66 and %64.86) for Eucalyptus, Cinnamon and clove after 24,48 and 72 hr. respectively. It may be suggested that spraying natural products is a good option on new wax combs to protect them against infestation by wax moth larvae

Gamma Irradiation as a Possible Phytosanitary Treatment against the Greater Wax Moth, Galleria mellonella, Mature Larvae

Larvae of greater wax moth, Galleria mellonella L., can be a devastating pest of honeybee combs in storage. Current control measures may not be adequate or with some drawbacks. Treatment with ionizing radiation could be a possible alternative. Determining the dose, however, requires a detailed study on the radiosensitivity of this stage.&#x0D; In this study, the sensitivity of the greater wax moth mature larvae to ionizing radiation was examined. Larvae were exposed to gamma radiation dosages (50 to 400 Gy) and effects on rate of development, pupation and pupal eclosion (adult emergence) were investigated. In addition, large scale confirmatory tests were conducted to validate treatment efficacy at statistical confidence levels needed for phytosanitary (quarantine and disinfestations) security. Results showed that radiation sensitivity of G. mellonella mature larvae increased, as expected, with increasing radiation dose. The magnitude of the effect, however, depends on the criterion used for measuring effectiveness. Both pupation and pupal eclosion were significantly affected at 50 Gy dose and no emergence was recorded at 300 Gy. Results of large scale confirmatory trials (&gt;30,000 mature larvae) supported the results of the small scale studies. According to probit analysis of dose mortality data for pupation and pupal eclosion, the LD99 for preventing development of mature larvae to pupae and adults is 576 and 318 Gy, respectively. In addition, the developmental rate of mature larvae to adults was negatively affected. This suggests that a dose of 250 Gy may be adequate for disinfesting honeybee products infested with greater wax moth larvae.

Low dose γ-radiation induced effects on wax moth (Galleria mellonella) larvae

Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative in vivo bioassays to rodents for studying microbial virulence, antibiotic development, and toxicology. In the current study, our aim was to assess the putative adverse effects of background gamma radiation levels on G. mellonella. To achieve this, we exposed larvae to low (0.014 mGy/h), medium (0.056 mGy/h), and high (1.33 mGy/h) doses of caesium-137 and measured larval pupation events, weight, faecal discharge, susceptibility to bacterial and fungal challenges, immune cell counts, activity, and viability (i.e., haemocyte encapsulation) and melanisation levels. The effects of low and medium levels of radiation were distinguishable from the highest dose rates used – the latter insects weighed the least and pupated earlier. In general, radiation exposure modulated cellular and humoral immunity over time, with larvae showing heightened encapsulation/melanisation levels at the higher dose rates but were more susceptible to bacterial (Photorhabdus luminescens) infection. There were few signs of radiation impacts after 7 days exposure, whereas marked changes were recorded between 14 and 28 days. Our data suggest that G. mellonella demonstrates plasticity at the whole organism and cellular levels when irradiated and offers insight into how such animals may cope in radiologically contaminated environments (e.g. Chornobyl Exclusion Zone).

The opportunities of using a natural adaptogen from the &lt;i&gt;Galleria mellonella&lt;/i&gt; larvae in veterinary medicine

A comparative analysis of the adaptation effects of the Porsolt test of fractionated solutions obtained from the products of the larvae of the large wax moth (Galleria mellonella) on biological systems was performed. The method of X-ray photoelectron spectroscopy determined that in the obtained light fraction of the products of larval life there is a high-energy component of N1s spectra (~402.0 eV) and bond energy for carbon in the C-N bond (~286.0 eV), which indicates the presence of protonated amino groups in the samples. Absorption at 1700 cm-1 can be attributed to the presence of dicarboxylic amino acids (glutamic and aspartic) and other organic acids, which are much less in the heavy fraction. During oral administration of the studied extracts, their multidirectional effect on the intestines of laboratory mice was found. The negative effect of heavy fraction on the microbiocenosis of the intestine by the qualitative composition of the microflora was detected. When the light fraction is used, the intestinal microflora is close to the control. Analysis of the number of leukocytes of the small intestine when the fractions were injected reflected the local immune response of the body. The content of these cells on the mucosal plate of mice injected with the lung fraction was 67.2% higher than that of the control mice (p ≤ 0.05). The content of leukocytes on the own plate of the mucosa of mice injected with the heavy fraction was 2.7 times higher compared to the control, indicating an inflammatory reaction. The difference between the experimental groups is significant (p ≤ 0.001). The comparative effect of the fractions showed that their oral administration had a different effect on the swimming time compared to the control. After 20 days of the experiment, mice injected with the heavy fraction had significantly increased their swimming time compared to the first day (p ≤ 0.05). However, a negative adaptogenic effect compared to the control group is noticeable. A significant difference was found between the results on the 30th day between the experimental groups, which indicates a pronounced difference in the results obtained.

Virulent and necrotrophic strategies of Bacillus thuringiensis in susceptible and resistant insects, Galleria mellonella.

Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant (R) populations at ∼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48h post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.

Steinernema bakwenae n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from South Africa

Summary Steinernema bakwenae n. sp. isolate SF857 has been isolated from a soil sample from the North West province of South Africa. The infective juveniles were isolated using Cobb’s decanting and sieving method, hand-picked from the nematode population and inoculated directly onto wax moth larvae. Morphological and molecular characterisation provides evidence for placing the new species in the Khoisanae-clade, adding a fifth member to this all-African clade. The new species is characterised by the following morphological features: third-stage infective juveniles with a body length of 1106 (1013-1233) μm, a distance from the anterior end to the excretory pore of 90 (81-103) μm, and a tail length of 87 (76-103) μm. The first-generation males can be recognised by a spicule length of 80 (66-89) μm and by a gubernaculum length of 51 (44-56) μm. The new species was confirmed by means of crossbreeding and characterised by means of sequences of the internal transcribed spacer (ITS), the partial 28S regions (D2-D3) of the ribosomal DNA and the mitochondrial cytochrome oxidase subunit (COI). Phylogenetic analyses show that S. innovationi and S. tophus are the closest relatives to S. bakwenae n. sp. in the Khoisanae-clade.

The Cell Wall Deacetylases Spy1094 and Spy1370 Contribute to Streptococcus pyogenes Virulence

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a strictly human pathogen that causes a wide range of diseases, including skin and soft tissue infections, toxic shock syndrome and acute rheumatic fever. We have recently reported that Spy1094 and Spy1370 of S. pyogenes serotype M1 are N-acetylglucosamine (GlcNAc) deacetylases. We have generated spy1094 and spy1370 gene deletion mutants in S. pyogenes and gain-of-function mutants in Lactococcus lactis. Similar to other cell wall deacetylases, our results show that Spy1094 and Spy1370 confer lysozyme-resistance. Furthermore, deletion of the genes decreased S. pyogenes virulence in a human whole blood killing assay and a Galleria mellonella (Greater wax moth) larvae infection model. Expression of the two genes in L. lactis resulted in increased lysozyme resistance and survival in whole human blood, and reduced survival of infected G. mellonella larvae. Deletion of the spy1370, but not the spy1094 gene, decreased resistance to the cationic antimicrobial peptide cecropin B, whereas both enzymes increased biofilm formation, probably resulting from the increase in positive charges due to deacetylation of the cell wall. In conclusion, Spy1094 and Spy1370 are important S. pyogenes virulence factors and might represent attractive targets for the development of antibacterial agents.