Wolbachia strain diversity in a complex group of sympatric cryptic parasitoid wasp species.

BACKGROUND: Maternally-inherited symbionts can induce pre-mating and/or post-mating reproductive isolation between sympatric host lineages, and speciation, by modifying host reproductive phenotypes. The large parasitoid wasp genus Cotesia (Braconidae) includes a diversity of cryptic species, each specialized in parasitizing one to few related Lepidoptera host species. Here, we characterized the infection status of an assemblage of 21 Cotesia species from 15 countries by several microbial symbionts, as a first step toward investigating whether symbionts may provide a barrier to gene flow between these parasitoid host lineages. RESULTS: The symbiotic microbes Arsenophonus, Cardinium, Microsporidium and Spiroplasma were not detected in the Cotesia wasps. However, the endosymbiotic bacterium Wolbachia was present in at least eight Cotesia species, and hence we concentrated on it upon screening additional DNA extracts and SRAs from NCBI. Some of the closely related Cotesia species carry similar Wolbachia strains, but most Wolbachia strains showed patterns of horizontal transfer between phylogenetically distant host lineages. CONCLUSIONS: The lack of co-phylogenetic signal between Wolbachia and Cotesia suggests that the symbiont and hosts have not coevolved to an extent that would drive species divergence between the Cotesia host lineages. However, as the most common facultative symbiont of Cotesia species, Wolbachia may still function as a key-player in the biology of the parasitoid wasps. Its precise role in the evolution of this complex clade of cryptic species remains to be experimentally investigated.

Genetic diversity, population structure and ecological niche modeling of Thyrinteina arnobia (Lepidoptera: Geometridae), a native Eucalyptus pest in Brazil.

Thyrinteina arnobia (Lepidoptera: Geometridae) is a native American species. Despite its historical importance as an insect pest in Eucalyptus plantations, more information is needed regarding the population diversity, demography, and climatic variables associated with its distribution in different regions of Brazil. We used a phylogeographic approach to infer the genetic diversity, genetic structure, and demographic parameters of T. arnobia. We also conducted an ecological niche modeling (ENM) to predict suitable areas for T. arnobia occurrence in Brazil and other countries worldwide. Although T. arnobia populations have low genetic diversity in Brazil, we identified mitochondrial haplogroups predominating in different Brazilian regions and high ФST and ФCT values in AMOVA, suggesting a low frequency of insect movement among these regions. These results indicate that outbreaks of T. arnobia in Eucalyptus areas in different regions of Brazil are associated with local or regional populations, with no significant contribution from long-distance dispersal from different regions or biomes, suggesting that pest management strategies would be implemented on a regional scale. In Brazil, the demographic and spatial expansion signals of T. arnobia seem to be associated with the history of geographical expansion of Eucalyptus plantations, a new sustainable host for this species. ENM indicated that isothermality and annual rainfall are critical climatic factors for the occurrence of T. arnobia in tropical and subtropical areas in the Americas. ENM also suggested that T. arnobia is a potential pest in Eucalyptus areas in all Brazilian territory and in regions from Africa, Asia, and Oceania.

The Complete Mitochondrial Genome and Phylogenetic Analysis of Rhagastis binoculata (Matsumura, 1909) (Lepidoptera: Sphingidae).

The mitochondrial genome (mitogenome) Rhagastis binoculata (Matsumura, 1909), an endemic moth species in Taiwan, was sequenced and analyzed. The complete circular mitogenome of R. binoculata is 15,303 bp and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an AT-rich control region. The mitogenome has an overall nucleotide composition of 41.2% A, 11.9% C, 7.5% G, and 39.4% T, with an AT content of 80.6%. Of the protein-coding genes (PCGs), 12 start with ATG, ATT, and ATC, and COX1 starts with a "CGA" codon. All of the stop codons are "TAA, TAG, or T". Our phylogenetic analysis of 21 species of Sphingidae insects suggests that R. binoculata is clustered with Rhagastis mongoliana, which belongs to the subfamily Macroglossinae.

Hemp regulates the fitness of corn earworm (Lepidoptera, Noctuidae) and its tachinid (Diptera) parasitoids.

Pest management on hemp is still in its infancy, and biological control options are limited. Helicoverpa zea (corn earworm) is one of the key pests of hemp cultivated outdoors, especially on cultivars grown for cannabinoids and grain. In a three-year study, we assessed the effect of diet on the performance of H. zea and its tachinid parasitoids. Parasitized (bearing fly eggs) and unparasitized (without eggs) H. zea larvae were fed on hemp flowers or an artificial diet. Five tachinid species parasitized H. zea larvae, but the most abundant species were Winthemia rufopicta (68.8%) and Lespesia aletiae (28.3%). Overall, 55.2% of H. zea larvae bearing tachinid eggs died, while the mortality of unparasitized larvae reached 24.7%. The success of tachinids increased by 2-fold when the host larvae were fed on an artificial diet. Our results demonstrated that high protein food (artificial diet), intensity of parasitism, and caterpillar size play a role in the fitness of both the herbivores (H. zea) and its tachinid parasitoids. These findings have important implications for understanding biological control mechanisms and open new insights into the impact of landscape variation on plant-herbivore-parasitoid interactions. This study contains supporting evidence that makes both Winthemia rufopicta and Lespesia aletiae excellent candidates for biological control programs against H. zea, a key pest of hemp in the United States.

Semiochemical-baited traps as a new method supplementing light traps for faunistic and ecological studies of Macroheterocera (Lepidoptera).

Attractivity and selectivity of two types of traps with synthetic, long-lasting, bisexual generic attractants were compared to conventional light traps to promote their wider use, as an easy-to-use standardised method for entomology. The targeted herbivorous Macroheterocera species playing important role in ecosystems as food source for higher trophic levels (e.g. predatory arthropods, birds and mammals), while other hand they can cause significant economic loss in agriculture. Data on their population dynamic and composition of their assemblages are necessary for both nature conservation and efficient pest management. Light- and semiochemical-baited traps with semisynthetic- (SBL = the acronym stands for semisynthetic bisexual lure) and synthetic lures (FLO = the acronym stands for floral lure of synthetic floral compounds) were used in species rich area of West Ukraine, and in all 10,926 lepidopterans trapped were identified. The attractivity of the light trap was highest with 252 species caught, while traps with semiochemicals captured 132 species including 28 exclusively caught only by them. The qualitative selectivity of light vs. semiochemical-baited traps differed considering both taxa and habitat preferences in such a way that they completed each-other. Differences in quantitative selectivity were also proved even in case of pest species. The parameters of methods varied depending on the phenological phases of the studied assemblages. Considering the revealed attractivity and selectivity, the parallel use of the two methods can offer improved reliable data for conservation biology and pest management.

Comparative Proteomics and Metabonomics Analysis of Different Diapause Stages Revealed a New Regulation Mechanism of Diapause in Loxostege sticticalis (Lepidoptera: Pyralidae).

Histone acetylation is an important epigenetic mechanism that has been shown to play a role in diapause regulation. To explore the physiological and molecular mechanisms of histone deacetylase in the diapause process, LC-MS/MS analysis was used to perform TMT proteomic and metabolomic analysis on non-diapause (ND), pre-diapause (PreD), diapause (D), cold treatment (CT), and post-diapause (RD) stages of the meadow moth. A total of 5367 proteins were identified by proteomics, including 1179 differentially expressed proteins. We found 975 (602 up-regulated and 373 down-regulated), 997 (608 up-regulated and 389 down-regulated), 1119 (726 up-regulated and 393 down-regulated), 1179 (630 up-regulated and 549 down-regulated), 94 (51 up-regulated and 43 down-regulated), 111 (63 up-regulated and 48 down-regulated), 533 (243 up-regulated and 290 down-regulated), 58 (31 up-regulated and 27 down-regulated), and 516 (228 up-regulated and 288 down-regulated) proteins in ND and PreD, ND and D, ND and CT, ND and RD, PreD and D, PreD and CT, PreD and RD, D and CT, D and RD, and CT and RD stages, respectively. A total of 1255 differentially expressed metabolites were annotated by metabolomics. Through KEGG analysis and time series analysis of differentially expressed metabolites, we found that phospholipids were annotated in significantly different modules, demonstrating their important role in the diapause process of the meadow moth. Using phospholipids as an indicator for weighted gene co-expression network analysis, we analyzed the most relevant differentially expressed proteins in the module and found that ribosomal 40s and 60s subunits were the most relevant proteins for diapause. Because there have been studies that have shown that histone deacetylase is associated with the diapause of meadow moths, we believe that histone deacetylase regulates the 40s and 60s subunits of ribosomes, which in turn affects the diapause of meadow moths. This finding expands our understanding of the regulation of meadow moth diapause and provides new insights into its control mechanism.

Evaluation of endophytic colonization and establishment of entomopathogenic fungi against Tuta absoluta (Lepidoptera: Gelechiidae) in tomato plants.

Background: The tomato, Solanum lycopersicum L., is one of the most important horticultural crops that can be consumed fresh or after being processed worldwide. The tomato leaf miner (Tuta absoluta) is one of the most devastating pest to tomato plants due to its mine-feeding nature in the mesophyll tissue of the plant. Fungal entomopathogens can exist naturally in plants as an asymptote. This study aimed to detect the endophytic colonization of Beauveria bassiana and Metarhizium robertsii within tomato plants via artificial inoculation and their virulence effects on Tuta absoluta. Methods: Isolates with the highest percent germination and virulence against T. absoluta were selected for endophytic evaluation within tomato plants by different artificial inoculation techniques. Results: This study revealed that, isolates with the highest percent germination and virulent to Tuta absoluta had the potential to colonize tomato plants. The result showed that, the maximum mortality rate (97.5%) of Tuta absoluta larvae was achieved by Metarhizium robertsii isolate K-61 at a concentration of 1x10 8conidial/ml at 7 days post inoculated. However, the highest cumulative mortality (100%) was recorded by Beauveria bassiana isolate APPRC-27 at 10 days post inoculated through the direct contact method. The highest endophytic colonization was registered by isolate APPRC-27 (76.67%) at 7 days post-inoculated using the leaf spray technique, but it declined to 11.67% after 28 days of inoculated. In the case of the seedling inoculation technique, the highest endophytic colonization was obtained in the root tissues of tomatoes at 28 days of inoculated by isolate K-61. Conclusions: This study revealed that the leaf spray inoculation technique was the most effective method, followed by seedling inoculation, for the deployment of Beauveria bassiana and Metarhizium robertsii endophytes in tomato plant tissues. Therefore, virulent Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of Tuta absoluta if deployed as endophytes.

Chromosome-level Genome Assembly of Theretra japonica (Lepidoptera: Sphingidae).

Theretra japonica is an important pollinator and agricultural pest in the family Sphingidae with a wide range of host plants. High-quality genomic resources facilitate investigations into behavioral ecology, morphological and physiological adaptations, and the evolution of genomic architecture. However, chromosome-level genome of T. japonica is still lacking. Here we sequenced and assembled the high-quality genome of T. japonica by combining PacBio long reads, Illumina short reads, and Hi-C data. The genome was contained in 95 scaffolds with an accumulated length of 409.55 Mb (BUSCO calculated a genome completeness of 99.2%). The 29 pseudochromosomes had a combined length of 403.77 Mb, with a mapping rate of 98.59%. The genomic characterisation of T. japonica will contribute to further studies for Sphingidae and Lepidoptera.

Genome-Wide Gene Birth-Death Dynamics Are Associated with Diet Breadth Variation in Lepidoptera.

Comparative analyses of gene birth-death dynamics have the potential to reveal gene families that played an important role in the evolution of morphological, behavioral, or physiological variation. Here, we used whole genomes of 30 species of butterflies and moths to identify gene birth-death dynamics among the Lepidoptera that are associated with specialist or generalist feeding strategies. Our work advances this field using a uniform set of annotated proteins for all genomes, investigating associations while correcting for phylogeny, and assessing all gene families rather than a priori subsets. We discovered that the sizes of several important gene families (e.g. those associated with pesticide resistance, xenobiotic detoxification, and/or protein digestion) are significantly correlated with diet breadth. We also found 22 gene families showing significant shifts in gene birth-death dynamics at the butterfly (Papilionoidea) crown node, the most notable of which was a family of pheromone receptors that underwent a contraction potentially linked with a shift to visual-based mate recognition. Our findings highlight the importance of uniform annotations, phylogenetic corrections, and unbiased gene family analyses in generating a list of candidate genes that warrant further exploration.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Resistance of Lepidopteran Pests to Bacillus thuringiensis Toxins: Evidence of Field and Laboratory Evolved Resistance and Cross-Resistance, Mode of Resistance Inheritance, Fitness Costs, Mechanisms Involved and Management Options.

Bacillus thuringiensis (Bt) toxins are potential alternatives to synthetic insecticides for the control of lepidopteran pests. However, the evolution of resistance in some insect pest populations is a threat and can reduce the effectiveness of Bt toxins. In this review, we summarize the results of 161 studies from 20 countries reporting field and laboratory-evolved resistance, cross-resistance, and inheritance, mechanisms, and fitness costs of resistance to different Bt toxins. The studies refer mainly to insects from the United States of America (70), followed by China (31), Brazil (19), India (12), Malaysia (9), Spain (3), and Australia (3). The majority of the studies revealed that most of the pest populations showed susceptibility and a lack of cross-resistance to Bt toxins. Factors that delay resistance include recessive inheritance of resistance, the low initial frequency of resistant alleles, increased fitness costs, abundant refuges of non-Bt, and pyramided Bt crops. The results of field and laboratory resistance, cross-resistance, and inheritance, mechanisms, and fitness cost of resistance are advantageous for predicting the threat of future resistance and making effective strategies to sustain the effectiveness of Bt crops.

Effect of temperature on the biological parameters of pink bollworm, Pectinophora gossypiella Saunders (Lepidoptera: Gelechiidae).

Pink bollworm (PBW) Pectinophora gossypiella is an important pest cotton worldwide. There are multiple factors which determines the occurrence and distribution of P. gossypiella across different cotton growing regions of the world, and one such key factor is 'temperature'. The aim was to analyze the life history traits of PBW across varying temperature conditions. We systematically explored the biological and demographic parameters of P. gossypiella at five distinct temperatures; 20, 25, 30, 35 and 40 ± 1 °C maintaining a photoperiod of LD 16:8 h. The results revealed that the total developmental period of PBW shortens with rising temperatures, and the highest larval survival rates were observed between 30 °C and 35 °C, reaching 86.66% and 80.67%, respectively. Moreover, significant impacts were observed as the pupal weight, percent mating success, and fecundity exhibited higher values at 30 °C and 35 °C. Conversely, percent egg hatching, larval survival, and adult emergence were notably lower at 20 °C and 40 °C, respectively. Adult longevity decreased with rising temperatures, with females outliving males across all treatments. Notably, thermal stress had a persistent effect on the F1 generation, significantly affecting immature stages (egg and larvae), while its impact on reproductive potential was minimal. These findings offer valuable insights for predicting the population dynamics of P. gossypiella at the field level and developing climate-resilient management strategies in cotton.

Mating behavior and oviposition of Palpita forficifera (Lepidoptera: Crambidae).

Palpita forficifera Munroe, 1959 (Lepidoptera: Crambidae) is considered the main pest of the olive tree (Olea europaea L., Oleaceae) in Brazil and Uruguay. The aim of this work was to study the mating and oviposition behavior of P. forficifera in the field and laboratory. In the field, the sex emitting the mating pheromone was determined and in the laboratory, the rate of emergence of males and females; the age, time and duration of mating; number of copulations and oviposition time of P. forficifera were recorded. The field results showed that it was possible to capture up to five males per trap in just one night in traps with the presence of female P. forficifera. Copulation occurs between the seventh and twenty-third day of life and is most frequent during the third and sixth hours of scotophase. The average duration of the first copulation was 174 min, with 35% of couples recopulating, and there were cases of up to five copulations. Oviposition times were concentrated between 20:00 and 02:00. The results obtained provide insight into the reproductive behavior of P. forficifera and are useful for future studies aimed at identifying the sex pheromone to improve monitoring of the pest in olive orchards.

Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

A new species of Diolcogaster (Hymenoptera: Braconidae) from Brazil of potential use in the biological control of the pest Hypercompe brasiliensis (Stoll) (Lepidoptera: Erebidae).

Species of Diolcogaster parasitize Lepidoptera pests of commercial plants. The diversity of this genus is high, but few species of Diolcogaster have been described. The description of a new Diolcogaster species provides information for the biological control using this insect. This study presents the description and key notes on the biology of a new Diolcogaster parasitoid wasp. This species was reared from a caterpillar of Hypercompe brasiliensis collected after feeding on a Gloxinia perennis plant important to floriculture. Two complementary identification analyzes were performed on Diolcogaster adult bodies. The first was the analyses of its external morphology and the second its molecular analysis (mitochondrial DNA). The morphological analysis defined the insect as a new species of Diolcogaster, named Diolcogaster joanesi sp. nov. A maximum-likelihood (ML) analysis partially confirmed the morphological analysis, placing D. joanesi within a cluster including a previously identified species (Diolcogaster choi) and seven other morphospecies. The proximity of D. joanesi to D. choi is discussed and an updated key for all New World species of the xanthaspis group is provided. Twenty-eight adult wasps were obtained (22 females and six males) out of 50 cocoons which larvae emerged from the caterpillar host. The findings contribute to the broader understanding of Diolcogaster in the Neotropics and its potential for the biological control of lepidopteran defoliators.

Effects of the insecticide flupyradifurone on Anticarsia gemmatalis caterpillar and its predator Podisus nigrispinus.

The caterpillar Anticarsia gemmatalis (Lepidoptera: Noctuidae) is a prevalent pest in soybean plantations, managed using both natural and synthetic chemical products. However, the emergence of resistance in some populations emphasizes the need to explore alternative insecticides. Flupyradifurone, a neurotoxic insecticide, has not been previously used for controlling A. gemmatalis. This study evaluated the potential of flupyradifurone in the management of A. gemmatalis. Initially, the toxicity and anti-feeding effects, as well as histopathological and cytotoxic impacts, of flupyradifurone on A. gemmatalis were evaluated. Subsequently, the indirect effects of flupyradifurone on the midgut and fat body of the predator Podisus nigrispinus (Hemiptera: Pentatomidae) were verified. The results indicate the susceptibility of caterpillars to flupyradifurone, with an LC50 of 5.10 g L-1. Furthermore, the insecticide adversely affects survival, induces an anti-feeding response, and inflicts damage on the midgut of the caterpillars. However, flupyradifurone also leads to side effects in the predator P. nigrispinus through indirect intoxication of the caterpillars, including midgut and fat body damage. While flupyradifurone demonstrates toxicity to A. gemmatalis, suggesting its potential for the chemical control of this pest, the indirect negative effects on the predator indicate the need for its controlled use in integrated pest management programs with the insecticide and the predator.

Minimally destructive hDNA extraction method for retrospective genetics of pinned historical Lepidoptera specimens.

The millions of specimens stored in entomological collections provide a unique opportunity to study historical insect diversity. Current technologies allow to sequence entire genomes of historical specimens and estimate past genetic diversity of present-day endangered species, advancing our understanding of anthropogenic impact on genetic diversity and enabling the implementation of conservation strategies. A limiting challenge is the extraction of historical DNA (hDNA) of adequate quality for sequencing platforms. We tested four hDNA extraction protocols on five body parts of pinned false heath fritillary butterflies, Melitaea diamina, aiming to minimise specimen damage, preserve their scientific value to the collections, and maximise DNA quality and yield for whole-genome re-sequencing. We developed a very effective approach that successfully recovers hDNA appropriate for short-read sequencing from a single leg of pinned specimens using silica-based DNA extraction columns and an extraction buffer that includes SDS, Tris, Proteinase K, EDTA, NaCl, PTB, and DTT. We observed substantial variation in the ratio of nuclear to mitochondrial DNA in extractions from different tissues, indicating that optimal tissue choice depends on project aims and anticipated downstream analyses. We found that sufficient DNA for whole genome re-sequencing can reliably be extracted from a single leg, opening the possibility to monitor changes in genetic diversity maintaining the scientific value of specimens while supporting current and future conservation strategies.

Selection of host plants for production of Clanis bilineata (Lepidoptera: Sphingidae).

Clanis bilineata Walker (Lepidoptera: Sphingidae), a burgeoning edible insect, is experiencing rising demand in China and other regions. Despite this interest, larval production is currently constrained by the limitations of artificial production technologies, particularly the selection of optimal host plants. This study rigorously evaluated the performance of C. bilineatha larvae on four main host plants: round-leaf soybean, pointed-leaf soybean, black locust, and kudzu. Preference tests demonstrated that the larvae were most attracted to black locust (34.76 ± 4.65%), with subsequent preferences for kudzu (25.00 ± 6.12%), round-leaf soybean (23.17 ± 2.79%), and pointed-leaf soybean (14.02 ± 4.74%). No significant preference differences were noted between round-leaf soybean and either black locust or kudzu. In feeding assays, the larvae exhibited a marked preference for round-leaf soybean (37.36 ± 0.81 g, total feeding amount for larvae), followed by kudzu (37.26 ± 0.82 g), pointed-leaf soybean (35.38 ± 1.31 g), and black locust (28.53 ± 0.81 g). When the larvae were fed on round-leaf soybean, they exhibited significantly higher survival rate (39.33 ± 0.90%), body weight (9.75 ± 0.07 g), total biomass (383.43 ± 7.35 g), pupation rate (87.78 ± 1.73%), and egg production (189.80 ± 1.06 eggs/female) compared to other hosts. These findings uncovered that round-leaf soybean significantly enhances larval performance, suggesting its potential for improving C. bilineata larval production and sustainability in cultivation systems.

Transfer of micro-organisms from dry surface biofilms and the influence of long survival under conditions of poor nutrition and moisture on the virulence of Staphylococcusaureus.

BACKGROUND: Biofilms on dry hospital surfaces can enhance the persistence of micro-organisms on dry harsh clinical surfaces and can potentially act as reservoirs of infectious agents on contaminated surfaces. AIM: This study was conducted to quantify the transfer of viable Staphylococcus aureus cells from dry biofilms through touching and to investigate the impact of nutrient and moisture deprivation on virulence levels in S. aureus. METHODS: Dry biofilms of S. aureus ATCC 25923 and a defective biofilm-forming ability mutant, S. aureus 1132, were formed in 24-well plates under optimized conditions mimicking dry biofilm formation on clinical surfaces. Microbial cell transfer was induced through the touching of the dry biofilms, which were quantified on nutrient agar. To investigate the impact of nutrient and moisture deprivation on virulence levels, dry and standard biofilms as well as planktonic cells of S. aureus ATCC 25923 were inoculated into Galleria mellonella and their kill rates compared. FINDINGS: Results of this study showed that viable cells from dry biofilms of S. aureus ATCC 25923 were significantly more virulent and readily transferrable from dry biofilms through a touch test, therefore representing a greater risk of infection. The biofilm-forming capability of S. aureus strains had no significant impact on their transferability with more cells transferring when biofilm surfaces were wet. CONCLUSIONS: These findings indicate that dry biofilms on hospital surfaces may serve as a reservoir for the dissemination of pathogenic micro-organisms in hospitals, thus highlighting the importance of regular cleaning and adequate disinfection of hospital surfaces.