The insecticidal activity of Streptomyces sp. KSF103 ethyl acetate (EA) extract against mosquitoes is known; however, the underlying mechanism behind this activity remains elusive. In this study, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was employed to investigate changes in the protein profile of Aedes aegypti larvae and adults treated with lethal concentrations of 50 (LC50) EA extract. By comparing the treated and untreated mosquitoes, this study aimed to identify proteins or pathways that exhibit alterations, potentially serving as targets for future insecticide development. Treatment with a lethal concentration of EA extract upregulated 15 proteins in larvae, while in adults, 16 proteins were upregulated, and two proteins were downregulated. These proteins were associated with metabolism, protein regulation/degradation, energy production, cellular organization and structure, enzyme activity, and catalysis, as well as calcium ion transport and homeostasis. Notably, ATP synthase, fructose-bisphosphate aldolase (FBA), and ATP citrate synthase were significantly expressed in both groups. Gene ontology analysis indicated a focus on energy metabolic processes. Molecular docking revealed a strong interaction between dodemorph, selagine (compounds from the EA extract), and FBA, suggesting FBA as a potential protein target for insecticide development. Further studies such as Western blot and transcriptomic analyses are warranted to validate the findings.
A new black fly species, Simulium (Gomphostilbia) wijiti, is described based on adult females, males, pupal exuviae and mature larvae from Mae Hong Son Province, Thailand. This new species is placed in the Simulium ceylonicum species-group. It is distinguished from four Thai members of the S. ceylonicum species-group [S. (G.) curtatum Jitklang et al., S. (G.) pangsidaense Takaoka, Srisuka & Saeung, S. (G.) sheilae Takaoka & Davies, and S. (G.) trangense Jitklang et al.], in the female by the short to medium long sensory vesicle; in the male by the large number of upper-eye (large) facets in 15 vertical columns and 15 or 16 horizontal rows; in the pupa by the dorsum of abdominal segments darkened; and in the larva by the antenna as long as or slightly shorter than the stem of the labral fan (longer than the stem of the labral fan in four other species). Phylogenetic analysis based on the COI gene sequences revealed that this new species is genetically closely related to S. leparense of the S. ceylonicum species-group, but is clearly separated from the latter species, and also from the three Thai related species (S. curtatum, S. sheilae and S. trangense) of the same species-group with interspecific genetic distances ranging from 9.65% to 12.67%. This is the fifth member of the S. ceylonicum species-group recorded from Thailand.
The control of mosquito breeding is an essential step towards the reduction of vector-borne disease outbreaks. Synthetic larvicidal agents produce resistance in vectors and cause safety concerns in humans, animals and aquatic species. The drawback of synthetic larvicides opened a new avenue for natural larvicidal agents, but poor dosage accuracy, need for frequent applications, low stability and sustainability are the major challenges with them. Hence, this investigation aimed to overcome those drawbacks by developing bilayer tablets loaded with neem oil to prevent mosquito breeding in stagnant water. The optimised batch of neem oil-bilayer tablets (ONBT) had 65%w/w hydroxypropyl methylcellulose K100M and 80%w/w ethylcellulose in its composition. After the completion of 4th week, 91.98 ± 0.871% azadirachtin was released from the ONBT, which was followed by a subsequent drop in the in vitro release. ONBT reported long-term larvicidal efficacy (>75%) and a good deterrent effect which was better than neem oil-based marketed products. The acute toxicity study on a non-target fish model (Poecilia reticulata), OECD Test No.203 confirmed the safety of the ONBT on non-target aquatic species. The accelerated stability studies predicted a good stability profile for the ONBT. The neem oil-based bilayer tablets can be used as an effective tool for the control of vector-borne diseases in society. The product may be a safe, effective and eco-friendly replacement for the existing synthetic as well as natural products in the market.
Coral-associated organisms often exhibit a continuum of host specificities. We do not know whether the variation in host specificity is related to the settlement organs or preferential settlement behaviours of the larvae. We examined the morphology of attachment discs, the settlement and metamorphosis of coral barnacles-Pyrgoma cancellatum (lives in a single coral species), Nobia grandis (two families of corals), and Armatobalanus allium (six families of corals). Our results revealed that the attachment organ of all three species are spear-shaped with sparse villi, indicating that the morphology of the attachment organs does not vary among species with different host specificities. Larvae of P. cancellatum and N. grandis only settle on their specific hosts, suggesting that chemical cues are involved in the settlement. Cyprids of N. grandis display close searching behaviour before settlement. Cyprids of P. cancellatum settle immediately on their specific host corals, without any exploratory behaviour. The host specificity and exploratory behaviours of coral barnacle cyprids are results of adaptive evolution. We argue that there is a trade-off between exploration and energy conservation for metamorphosis processes. Coral barnacle metamorphosis is longer when compared to free-living species, likely because it involves the development of a tube-shaped base on the coral surface.
Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host - parasite interactions in black fly mermithids.
Simulium takahasii (Rubtsov), which was originally described from Japan, and recorded from Korea and China, is the first among the 19 species of the subgenus Wilhelmia Enderlein recorded from East Asia. It is striking in mating, blood-feeding and ovipositing in captivity and in experimentally transmitting Dirofilaria immitis (Leidy) and Brugia pahangi (Buckley & Edeson), and it is a severe biter of cattle and horses, rarely of humans. Nevertheless, updated information about its morphological characteristics was lacking, making comparisons with related species described from China difficult, since species of the subgenus Wilhelmia are almost indistinguishable from one another, in particular, in their female terminalia, male genitalia and most of larval features. In this study, as many morphological characteristics as possible of S. takahasii based on specimens from Japan are redescribed. New information about many features of this species including the length of the female sensory vesicle against the third palpal segment, number of male upper-eye (large) facets, arrangement of the eight pupal gill filaments, presence or absence of tiny dark setae on the dorsum of the larval abdomen and the number of rows and hooklets of the larval posterior circlet will be useful in evaluating the species status of several Wilhelmia species in China including the species regarded as S. takahasii.
The development of new alternatives strategies to synthetic insecticides aimed at reducing pest populations by developing pesticides based on plant extracts without negative effects in non target organisms and environment. The present study was undertaken in order to assess the insecticidal activity of the crude methanolic extract of the Algerian Asteraceae Cotula cinerea, against the larval and the pupal stage of Culex pipiens (Diptera: Culicidae). It is also to determine the chemical composition of the used extract, and to understand the mechanism of toxic action of the tested extract. Based on the preliminary tests, five concentrations of the crude methanolic extract of C. cinerea (0.62, 1.25, 2.50, 3.75, and 5 mg/mL) were tested for their insecticidal activity according to the protocol recommended by the World Health Organization. The chemical profile of the extract was also obtained by high performance liquid chromatography (HPLC). Histopathological effects and inhibition of acetylcholinesterase activity in treated mosquitoes with LC90 were examined to elucidate the mechanism of the toxic effect of the tested extract (48 h post treatment). Eight compounds have been identified by HPLC. That includes four flavonoids (rutin, quercetin, myrcetin and cathechin), three phenolic acids (benzoic acid, vanillic acid, p-coumaric acid) and one alkaloid (berberine). C. cinerea methanolic extract showed good larvicidal and pupicidal activities with LC50 and LC90 values of 1.10 and 4.37 mg/mL respectively against pupae, 24h post treatment and 1.26, 2.35 mg/mL respectively against the fourth instar larvae. Data of enzymatic assay performed on LC50 and LC90 pupae and larvae revealed prominent neurotoxic effects. C. cinerea extract reduced the activity of acetylcholinesterase (AChE) enzyme in a concentration dependent manner. Obtained inhibition percentages, 48 h after treatment, were 35.11 ± 7.44 and 51.83 ± 4.04% for pupal stage and 30.98 ± 2.97 % and 48.77 ± 4.72% for the fourth instar larvae for LC50 and LC90 values respectively. Treated larvae and pupae showed also histopathological damages in the pupal cuticle and larval midgut. The results of this study showed that C. cinerea crude methanolic extract could be considered as an eco-friendly alternative for mosquito control.
Black soldier fly (Hermetia illucens (Linnaeus, 1758)) larvae inhibit oviposition of house fly (Musca domestica Linnaeus, 1758) by releasing a semiochemical, although in some situations, inhibition is only partial. We hypothesized that there is a certain period in the life cycle of black soldier fly when it can cause antibiosis of the house fly. Choice and non-choice tests were used to separately examine the effectiveness of each developmental stage of black soldier fly (egg, larval (phase I, II, and III), prepupal and pupal stages) and crude extract of larvae on house fly oviposition. Tests using each developmental stage were compared with controls lacking black soldier fly. The effects of black soldier fly on the number of newly hatched house fly larvae were evaluated and there was a significant difference between the test and control in the number of eggs laid by house fly for all phases of the black soldier fly larval stage. Strong inhibition was found in some black soldier fly larval phases. Significant differences in the numbers of house fly eggs oviposited in food containers treated with crude extract were found when compared with a control, confirming that chemicals from black soldier fly larvae resulted in inhibition of oviposition of house fly. The results from experiment also indicated that chemicals from black soldier larvae influenced the number of house fly larvae newly hatched from eggs.
This case study reports on two unrecorded Coleopteran species found together on a human corpse in Malaysia. The mummified human remains were discovered in a house in Selangor, Malaysia. The pathologist confirmed that the death was due to a traumatic chest injury. Maggots, beetles, and fly pupal casings were found mainly on the front part of the body. Empty puparia of Diptera were collected during the autopsy and identified later as the muscid Synthesiomyia nudiseta (van der Wulp, 1883) (Diptera: Muscidae). Also, the insect evidence received included larvae and pupae of Megaselia sp. (Diptera: Phoridae). According to the insect development data, the minimum postmortem period was estimated by the time to reach the pupal developmental stage (in days). The entomological evidence included the first record of Dermestes maculatus De Geer, 1774 (Coleoptera: Dermestidae) and Necrobia rufipes (Fabricius, 1781) (Coleoptera: Cleridae), which have not previously been recorded on human remains in Malaysia.
Mosquitoes are infectious vectors for a wide range of pathogens and parasites thereby transmitting several diseases including malaria, dengue, Zika, Japanese encephalitis and chikungunya which pose a major public health concern. Mostly synthetic insecticides are usually applied as a primary control strategy to manage vector-borne diseases. However excessive and non-judicious usage of such chemically derived insecticides has led to serious environmental and health issues owing to their biomagnification ability and increased toxicity towards non-target organisms. In this context, many such bioactive compounds originating from entomopathogenic microbes serve as an alternative strategy and environmentally benign tool for vector control. In the present paper, the entomopathogenic fungus, Lecanicillium lecanii (LL) was processed to make the granules. Developed 4% LL granules have been characterized using the technique of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The developed formulation was also subjected to an accelerated temperature study at 40 °C and was found to be stable for 3 months. Further, GCMS of the L. lecanii was also performed to screen the potential biomolecules present. The developed formulation was found to be lethal against Anopheles culicifacies with an LC50 value of 11.836 µg/mL. The findings from SEM and histopathology also substantiated the mortality effects. Further, the SEM EDX (energy dispersive X-ray) studies revealed that the treated larvae have lower nitrogen content which is correlated to a lower level of chitin whereas the control ones has higher chitin content and healthy membranes. The developed LL granule formulation exhibited high toxicity against Anopheles mosquitoes. The granule formulations can be used as an effective biocontrol strategy against malaria-causing mosquitoes.
Haemonchus contortus (Hc) is a hematophagous parasite affecting the health and productivity of flocks. The administration of chemical anthelmintic drugs (AH) is the common method of deworming; however, generates resistance in the parasites to AH and it is a public health risk due to drug residues in milk, meat and sub-products. Natural compounds from plants are explored to diminish this parasitosis, improving their health and productivity, without the negative effects of AH. Ipomoea genus is a group of climbing plants belonging to the Convulvulaceae family possessing perennial leaves and tuberous roots. Medicinal properties has been attributed to this plant including nutritional agents, emetics, diuretics, diaphoretics, purgatives and pesticides. The objective of this study was assessing the in vitro nematocidal activity of a hydroalcoholic extract (HA-E) obtained from Ipomoea pauciflora (Cazahuate) flowers against Hc infective larvae (L3) and to identify its phytochemical profile (PhC-P). The assay was carried out using microtiter plates (MTP). Four HA-E concentrations were assessed and Ivermectin and distilled water were used as positive and negative control groups, respectively. Approximately 100 Hc L3 were deposited in each well (n=12) and incubated at 25-35°C for 7 days. Data were analyzed using ANOVA and a General Linear Model (GLM) followed by Tukey test (P<0.05). The treatments showing a concentration-dependent effect (CDE) were analyzed to identify their 50% and 90% lethal concentrations (CL50, 90) via a Probit Analysis. The highest mortality was observed at 50 mg/mL (82.64 ± 0.71%) and the lowest at 6.25 mg/mL (56.46 ± 2.49%), showing a CDE with increasing mortality from 6.25 to 50 mg/mL. The PhC-P revealed the presence of alkaloids, coumarins, flavonoids, tannins and triterpenes/ sterols. A HA-E from flowers of I. pauciflora will be considered to assess its potential use in the control of haemonchosis in small ruminants.
Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka, both from the Nansei Islands, Japan, were morphologically reexamined and genetically analysed by using the COI gene sequences. The female, male, pupa and mature larva of the two species are redescribed. Morphological reexamination shows that both species are more similar to species in the S. asakoae species-group than to those in the S. ceylonicum species-group, by having a medium-long female sensory vesicle, yellow tuft hairs (S. (G.) okinawense) or yellow tuft hairs mixed with a few to several dark hairs (S. (G.) tokarense) at the base of the radial vein in the female and male, and medium-long larval postgenal cleft. However, the body of the male ventral plate (viewed ventrally) is parallel-sided (S. (G.) okinawense) or parallelsided or slightly narrowed (S. (G.) tokarense) and not emarginated basally, differing from those of most species in the S. asakoae species-group. Our genetic analysis shows that S. (G.) tokarense is in the S. asakoae species-group, and S. (G.) okinawense formed a separate sister clade with other members of the S. asakoae species-group with high bootstrap support. From the results of morphological and genetic analysis combined, S. (G.) okinawense and S. (G.) tokarense are transferred from the S. ceylonicum species-group to the S. asakoae species-group.
The estimation of the postmortem interval (PMI) is an essential information in death investigations. It is necessary to know the developmental data of the most important necrophagous insect species in every geographical area. Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) is one of the most common species associated with human body decomposition, especially in the tropics, so a precise knowledge of its life cycle is crucial. However, despite its ubiquity, developmental data in a range of tropical temperatures is scarce. For this reason, the aim of this study is to provide the developmental data of the blowfly, C. megacephala, in a range of tropical temperatures and to apply these data in forensic entomology. Four temperatures were examined (i.e., 27.0°C 29.5°C, 32.0°C and 34.5°C) and the time of developments from egg to adult were recorded. To build the growth curves, five larvae per day were measured with a digital caliper. Accumulated degree-days were calculated and the isomorphen diagram for this species was plotted. As we predicted, the results showed that the life cycle of this species was affected by the increasing temperature. The implications of these new data for determining the minimum PMI in forensic entomology were discussed.
The female, male, pupa and mature larva of Simulium (Simulium) poolpholi sp. nov. collected from a large and seasonal stream in northeastern Thailand are described. This new species is placed in the S. striatum species-group by the female sternite 8 concave posteromedially in form of the inverted-U shape, ovipositor valve with a ventrally produced process along its inner margin, male ventral plate saddle-shaped, style with a basal protuberance, and pupal gill with 10 filaments. It is morphologically similar to S. (S.) phraense Takaoka, Srisuka & Saeung from northern Thailand by lacking annular ridges on the surface of pupal gill filaments but it is distinguished from the latter species by the arrangement of pupal gill filaments. Molecular analysis using the fast-evolving nuclear big zinc finger (BZF) gene shows that S. (S.) poolpholi sp. nov. formed a distinct clade, being separated from the seven other Thai species of this species-group by a genetic distance of 1.84-4.55%. Our discovery increases the number of species in the S. striatum species-group in Thailand to nine.
A potentially novel actinobacterium isolated from forest soil, Streptomyces sp. KSF103 was evaluated for its insecticidal effect against several mosquito species namely Aedes aegypti, Aedes albopictus, Anopheles cracens and Culex quinquefasciatus. Mosquito larvae and adults were exposed to various concentrations of the ethyl acetate (EA) extract for 24 h. Considerable mortality was evident after the EA extract treatment for all four important vector mosquitoes. Larvicidal activity of the EA extract resulted in LC50 at 0.045 mg/mL and LC90 at 0.080 mg/mL for Ae. aegypti; LC50 at 0.060 mg/mL and LC90 at 0.247 mg/mL for Ae. albopictus; LC50 at 2.141 mg/mL and LC90 at 6.345 mg/mL for An. cracens; and LC50 at 0.272 mg/mL and LC90 at 0.980 mg/mL for Cx. quinquefasciatus. In adulticidal tests, the EA extract was the most toxic to Ae. albopictus adults (LD50 = 2.445 mg/mL; LD90 = 20.004 mg/mL), followed by An. cracens (LD50 = 5.121 mg/mL; LD90 = 147.854 mg/mL) and then Ae. aegypti (LD50 = 28.873 mg/mL; LD90 = 274.823 mg/mL). Additionally, the EA extract exhibited ovicidal activity against Ae. aegypti (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), Ae. albopictus (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), and An. cracens (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), evaluated up to 168 h post-treatment. It displayed no toxicity on the freshwater microalga Chlorella sp. Beijerinck UMACC 313, marine microalga Chlorella sp. Beijerinck UMACC 258 and the ant Odontoponera denticulata. In conclusion, the EA extract showed promising larvicidal, adulticidal and ovicidal activity against Ae. aegypti, Ae. albopictus, An. cracens, and Cx. quinquefasciatus (larvae only). The results suggest that the EA extract of Streptomyces sp. KSF103 has the potential to be used as an environmental-friendly approach in mosquito control. The current study would serve as an initial step toward complementing microbe-based bioinsecticides for synthetic insecticides against medically important mosquitoes.
Oil-in-water (o/w) emulsion is utilized as an insecticide delivery system for mosquito control. However, evaporation inhibition adjuvant is needed to prevent fog drift, inhibit release of insecticidal actives and prolong suspension time. In the current study, we evaluated the effect of different short-chain alcohols, namely, propylene glycol, 1,3-propanediol, glycerol and crude glycerol, as adjuvants on the physicochemical properties of d-phenothrin o/w emulsion system. The bioactivity of optimized formulations containing 20 wt% glycerol (D1), 20 wt% propylene glycol (D2) and without added alcohol (negative control) were tested against larvae, pupae and adult Aedes aegypti (Ae. aegypti). It was found that propylene glycol produced smaller droplets at lower concentrations but poor long-term stability at higher concentrations, whereas glycerol had an appreciable effect on initial droplet size and stability with increasing concentration. According to the dose-response bioassays and room size chamber testing, the highest larvicidal, pupicidal and adulticidal activities were observed with D2, followed by D1 and negative control. Overall, the above study demonstrated improved emulsion stabilities and potency against Ae. aegypti larvae, pupae and adults using glycerol as adjuvant for effective mosquito control.
The effective control of Aedes mosquitoes using traditional control agents is increasingly challenging due to the presence of insecticide resistance in many populations of key mosquito vectors. An alternative strategy to insecticides is the use of toxic sugar baits, however it is limited due to short-term efficacy. Alginate-Gelatin hydrogel beads (AGHBs) may be an effective alternative by providing longer periods of mosquito attraction and control, especially of key vectors of dengue viruses such as Aedes aegypti and Aedes albopictus. Sodium alginate (ALG) and gelatin (GLN) are natural polymers, which can be a potential candidate to develop the AGHBs baits due to their biodegradability and environmental safety. Here we provide an assessment of the preparation of AGHBs optimized by varying the concentrations of ALG, GLN, and its cross-linking time (TIME). Fourier transform infrared spectroscopy (FTIR) analysis results in the determination of liquid bait loaded in the AGHBs. The evaluation of AGHBs' effectiveness as the potential baiting tool based on the mortality rate of mosquitoes after the bait consumption. The 100 % percent mortality of Aedes mosquitoes was obtained within 72 h of bait consumption. The field evaluation also justifies the applicability of AGHBs for outdoor applications. We conclude that the AGHBs are applicable as a baiting tool in carrying liquid bait in achieving mosquito mortality.
Control of ticks and tick-borne pathogens is a priority for human and animal health. Livestock-holders extensively rely on acaricide applications for tick control. Different groups of acaricides including cypermethrin and amitraz have been consistently used in Pakistan. There has been a gap in understanding the susceptibility or resistance of Rhipicephalus microplus, the most prevalent tick in Pakistan, to acaricides. The present study aimed to molecularly characterize cypermethrin and amitraz targeted genes such as voltage-gated sodium channel (VGSC) and octopamine tyramine (OCT/Tyr) of R. microplus ticks in Khyber Pakhtunkhwa (KP), Pakistan to monitor the acaricides resistance. Tick specimens were collected from cattle and buffaloes in northern (Chitral, Shangla, Swat, Dir, and Buner), central (Peshawar, Mardan, Charsadda, Swabi, and Nowshera), and southern districts (Kohat, Karak, Lakki Marwat, Tank, and Dera Ismail Khan) of KP, Pakistan. Different concentrations of commercially available cypermethrin (10%) and amitraz (12.5%) were prepared for in vitro larval immersion tests (LIT). In LIT, the average mortality rate of immersed larvae was recorded that was increased gradually with an increase in the concentration of specific acaricide. The larvae's highest mortality rates (94.5% and 79.5%) were observed at 100-ppm of cypermethrin and amitraz, respectively. A subset of 82 R. microplus ticks was subjected to extract genomic DNA, followed by PCR to amplify partial fragments of VGSC (domain-II) and OCT/Tyr genes. The BLAST results of the consensus sequence of VGSC gene (domain-II) showed 100% identity with the acaricides susceptible tick sequence from the United States (reference sequence). Obtained identical sequences of OCT/Tyr genes showed maximum identity (94-100%) with the identical sequences reported from Australia (reference sequence), India, Brazil, Philippines, USA, South Africa, and China. Thirteen single nucleotide polymorphisms (10 synonymous and three non-synonymous) were observed at various positions of partial OCT/Tyr gene fragments. The SNP at position A-22-C (T-8-P) in OCT/Tyr gene has been linked to amitraz resistance in R. microplus ticks. Molecular analysis and LIT bioassay's findings indicate the availability of resistant R. microplus ticks in the KP region. To our understanding, this is the first preliminary study to monitor cypermethrin and amitraz resistance via molecular profiling of cypermethrin and amitraz targeted genes (VGSC and OCT/Tyr) in combination with in vitro bioassays (LIT) in R. microplus ticks from Pakistan.
Galleria mellonella larvae have been increasingly used in research, including microbial infection studies. They act as suitable preliminary infection models to study host-pathogen interactions due to their advantages, such as the ability to survive at 37°C mimicking human body temperature, their immune system shares similarities with mammalian immune systems, and their short life cycle allowing large-scale studies. Here, we present a protocol for simple rearing and maintenance of G. mellonella without requiring special instruments and specialized training. This allows the continuous supply of healthy G. mellonella for research purposes. Besides, this protocol also provides detailed procedures on the (i) G. mellonella infection assays (killing assay and bacterial burden assay) for virulence studies and (ii) bacterial cell harvesting from infected larvae and RNA extraction for bacterial gene expression studies during infection. Our protocol could not only be used in the studies of A. baumannii virulence but can also be modified according to different bacterial strains.