Wolbachia Interactions with Diverse Insect Hosts: From Reproductive Modulations to Sustainable Pest Management Strategies.

Effective in a variety of insect orders, including dipteran, lepidopteran, and hemipteran, Wolbachia-based control tactics are investigated, noting the importance of sterile and incompatible insect techniques. Encouraging approaches for controlling Aedes mosquitoes are necessary, as demonstrated by the evaluation of a new SIT/IIT combination and the incorporation of SIT into Drosophila suzukii management. For example, Wolbachia may protect plants from rice pests, demonstrating its potential for agricultural biological vector management. Maternal transmission and cytoplasmic incompatibility dynamics are explored, while Wolbachia phenotypic impacts on mosquito and rice pest management are examined. The importance of host evolutionary distance is emphasised in recent scale insect research that addresses host-shifting. Using greater information, a suggested method for comprehending Wolbachia host variations in various contexts emphasises ecological connectivity. Endosymbionts passed on maternally in nematodes and arthropods, Wolbachia are widely distributed around the world and have evolved both mutualistic and parasitic traits. Wolbachia is positioned as a paradigm for microbial symbiosis due to advancements in multiomics, gene functional assays, and its effect on human health. The challenges and opportunities facing Wolbachia research include scale issues, ecological implications, ethical conundrums, and the possibility of customising strains through genetic engineering. It is thought that cooperative efforts are required to include Wolbachia-based therapies into pest management techniques while ensuring responsible and sustainable ways.

Effect of tree height and spraying methods on Diaphorina citri kuwayama endosymbionts in the context of Huanglongbing disease management in citrus orchards.

BACKGROUND: Huanglongbing (HLB) (caused by Candidatus Liberibacter asiaticus) is the most damaging disease of citrus around the world. This study investigated the effects of citrus tree height on Diaphorina citri Kuwayama mortality, endosymbiont responses, and HLB distribution. RESULTS: The results reveal that the age of citrus trees plays a significant role in psyllid mortality. Interestingly, the cumulative mean mortality (%) of psyllids over the seven-day observation period was higher (31.50±0.03) when four-year-old (501A1, 502A2, 501A3) citrus trees were sprayed with a US-SMART mechanical sprayer. In contrast, the psyllids mortality was 0.09±0.23 for the 13-year-old citrus trees (104A2, 104A3, 104C1) sprayed with a US-SMART mechanical sprayer and 9.10±0.05 for 13-year-old (502A2, 502B2, 502D1) citrus trees sprayed with a fixed US-SMART mechanical sprayer. Our findings also revealed that psyllids from both four- and 13-year-old citrus trees carried Candidatus Carsonella ruddii species and Wolbachia, the primary and secondary endosymbionts, respectively. Surprisingly, infection rates of these endosymbionts remained consistent across different age groups, as confirmed by quantitative polymerase chain reaction analysis. Furthermore, our study highlights the significance of tree height as a proxy for tree age in influencing HLB occurrence. Specifically, four-year-old citrus trees subjected to the US-SMART mechanical sprayer for citrus psyllid control demonstrated effective disease management compared to 13-year-old (104A2, 104A3, 104C1) citrus trees sprayed with US-SMART mechanical sprayers. Additionally, the investigation explored the impact of tree height on HLB distribution. In four-year-old trees, no significant correlation between HLB disease and tree height was observed, potentially due to effective spray coverage with US-SMART mechanical sprayer. However, in 13-year-old (104A2, 104A3, 104C1) citrus tree sprayed with US-SMART mechanical sprayer, a positive correlation between tree height and HLB disease was evident. CONCLUSION: This research provides valuable insights into the complex interaction between citrus tree age, psyllid endosymbionts responses, and HLB distribution. These results emphasize effective HLB management strategies, especially in orchards with diverse tree age populations, ultimately contributing to the long-term sustainability of citrus cultivation. © 2023 Society of Chemical Industry.

Development of Loop-Mediated Isothermal Amplification Method for Rapid and Sensitive Identification of Hermetia illucens (Diptera: Stratiomyidae).

The black soldier fly (BSF) is well known for its ability to biologically convert organic waste into insect biomass, including protein and oil, which can be utilised as animal feed. Since raw BSF products, such as BSF powder, are difficult to differentiate from other biological raw materials, therefore new analytical approaches are required. In this study, we have developed a new and fast method based on loop-mediated isothermal AMPlification (LAMP) reaction that can diagnose black soldier fly larvae and BSF byproducts with high accuracy, specificity and sensitivity. Species-specific primers for BSF were designed based on targeting the mitochondrial cytochrome C oxidase I (COI) gene. The assay was able to detect as low as 820 fg/L of BSF DNA in 60 min at 65 °C, which was a hundredfold higher than the detection limit of classical polymerase chain reaction and did not show cross-reactivity. In conclusion, the LAMP assay demonstrated excellent sensitivity and specificity to detect BSF and BSF byproducts, with a sampling-to-result identification time of 60 min.

Saliva-Mediated Contrasting Effects of Two Citrus Aphid Species on Asian Citrus Psyllid Feeding Behavior and Plant Jasmonic Acid Pathway.

While herbivorous insect saliva plays a crucial role in the interaction between plants and insects, its role in the inter-specific interactions between herbivorous insects has received little attention. Pre-infestation of citrus plants with Aphis spiraecola Patch and Aphis (Toxoptera) citricidus (Kirkaldy) exhibited positive and negative effects on the performance (feeding and reproduction) of Diaphorina citri Kuwayama. We explored the role of saliva in this plant-mediated interaction by infiltrating fresh and boiled aphid saliva into plants and detecting D. citri feeding behavior and citrus plant defense response. Leaf infiltration of A. spiraecola saliva disrupted the subsequent feeding of D. citri, indicated by prolonged extracellular stylet pathway duration and decreased phloem sap ingestion duration. By contrast, infiltration of A. citricidus saliva decreased the duration of the extracellular stylet pathway and phloem sap ingestion of D. citri. Furthermore, gene expression analysis showed that several salicylic acid (SA)- and jasmonic acid (JA)-pathway-related genes were activated by A. spiraecola saliva infiltration. However, two SA-pathway-related genes were activated and three JA-pathway-related genes were suppressed following A. citricidus saliva infiltration. Treatment with boiled saliva did not similarly impact D. citri feeding behavior or plant defense response. This study suggests that salivary components (those that can be inactivated by heating) from two citrus aphid species differently affect plant defenses and that they were responsible for the contrasting plant-mediated effects of two citrus aphids on the feeding behavior of D. citri. This study indicates a novel three-way citrus aphid-plant-citrus psyllid interaction.

Vermicompost Amendments Disrupt Feeding Behavior of Diaphorina citri Kuwayama and Boost Activities of Salicylic Acid and Jasmonic Acid Pathway-Related Enzymes in Citrus.

Plants grown with vermicompost amendments are known to be harmful to sap-sucking insects, but the underlying mechanism remains to be determined. Here we investigated the feeding behavior of Diaphorina citri Kuwayama on Citrus limon (L.) Burm. F using the electrical penetration graph technique. Plants were grown in soil with different vermicompost rates (0%, 20%, 40%, and 60% w/w). Additionally, plants were tested for the activity of salicylic acid (SA) and jasmonic acid (JA) pathway-related enzymes. When compared to the control, vermicompost treatments (40% and 60%) decreased duration of phloem sap feeding and increased duration of the pathway phase of D. citri, and the 60% vermicompost made it more difficult for D. citri to reach and gain access to phloem sap. Enzymatic assays indicated that the 40% amendment rate increased phenylalanine ammonia lyase (involved in the SA pathway) and polyphenol oxidase (involved in the JA pathway), while the 60% amendment rate increased -1,3-glucanases (involved in the SA pathway) and lipoxygenase (involved in the JA pathway). The 20% amendment rate had no effect on feeding or enzyme activities. This study revealed that vermicompost amendments can reduce the efficiency of D. citri feeding, which may result from increased plant resistance via the SA and JA pathway.

Plant jasmonic acid mediated contrasting effects of two citrus aphid species on Diaphorina citri Kuwayama.

BACKGROUND: Herbivores may influence each other directly and through plant mediated inter-specific interactions. The Asian citrus psyllid (Diaphorina citri Kuwayama) and citrus aphids are key pests that can co-exist on citrus, but their plant-mediated interaction between them is unknown. Here we investigated plant-mediated effect of two citrus aphid species, the polyphagous Aphis spiraecola Patch and the oligophagous Aphis (Toxoptera) citricidus (Kirkaldy) on the feeding behavior and reproduction of Diaphorina citri, and explored the underlying mechanisms. RESULTS: In comparison with those on aphid free plants, Diaphorina citri had decreased reproduction and reduced phloem feeding on Aphis spiraecola pre-infested plants, while the reproduction and feeding efficiency were increased on Aphis citricidus pre-infested plants. Jasmonic acid (JA) dependent defense was significantly activated by Diaphorina citri feeding on Aphis spiraecola pre-infested plants, but was suppressed by Diaphorina citri feeding on Aphis citricidus pre-infested plants compared with that on aphid free plant. By contrast, only one tested marker gene in salicylic acid (SA) signaling was activated by Diaphorina citri feeding on Aphis spiraecola pre-infested plants. Furthermore, exogenous application of methyl jasmonate, but not SA, conferred resistance against Diaphorina citri in our citrus trials. CONCLUSION: Our results indicate that pre-infestation by two citrus aphid species differentially altered Diaphorina citri induced citrus JA dependent defense, which resulted in different effect on subsequent Diaphorina citri performance. © 2022 Society of Chemical Industry.

Assessing the Bacterial Communities Composition from Differently Treated Agarwood via 16S rRNA Gene Metabarcoding.

Agarwood (Aquilaria sinensis) is one of the most important resin-containing plants used to produce agar around the world and it is a precious herbal medicine usually combined with other herbs. In this study, we used the Illumina sequencing technique to explore the agarwood bacterial community structure from four different incense formations of agarwood, including healthy agarwood, drilling agarwood, liquid fermentation agarwood, and insect attack agarwood. Our results showed that 20 samples of three different incense-formation methods of agarwood and healthy agarwood acquired 1,792,706 high-quality sequences. In-depth investigation showed that when the diversity of agarwood bacterial species was higher, the agarwood incense quality was higher as well. Among healthy agarwood, drilling agarwood, fermentation agarwood, and insect attack agarwood, the bacterial community structure had significant changes. Natural agarwood, such as insect attack agarwood, kept more bacterial community structure, and the incense quality was better. Furthermore, we observed that in the healthy agarwood, Amnibacterium and Delftia were the predominant bacteria. Actinoplanes, Bordetella, and Sphingobacterium were the dominant bacteria in the drilling agarwood. Additionally, Pelagibacterium and Methylovirgula were some of the main bacteria in the fermentation liquid agarwood and the insect attack agarwood, while Cellulomonas and Aeromicrobium were the dominant bacteria. This research provides a basis for further research into the underlying mechanisms of incense production, as well as the bacterial community applications of agarwood production.

First description of males of Ensiferothrips wallacei Mound amp; Tree (Thripidae: Dendrothripinae) with variation in fore wing setae.

The genus Ensiferothrips now includes four described species, it is similar to Dendrothrips in the structure of the abdominal tergites and hind coxae, also the position of the fore wing cilia, but the wing apex bears a stout seta, and the costal setae are very stout. It was originally known from New Caledonia based on a single species, E. primus Bianchi, but subsequently E. secundus Mound was described from Lord Howe Island on Smilax australis [Smilacaceae](Mound 1999). More recently, Mound and Tree (2016) described two further species, E. lamingtoni from Queensland, Australia on Quintinia sieberi [Grossulariaceae] and E. wallacei from Indonesia without host plant information. A single specimen of the later species has also been recorded from Hainan, China (Zhang et al. 2018). The genus is clearly tropical in distribution, from south China to northern Australia, revealing the strong connection between south China, southeast Asia and northern Australia. Little biological information is known of these species, though they seem to live on hard mature leaves (Mound Tree 2020). In this study, a series of specimens of E. wallacei was found from Hainan on Smilax sp. The first description of the male of this species is presented here, together with interesting variation in the chaetotaxy of the fore wings.

Thysanoptera as predators: their diversity and significance as biological control agents.

Some species of thrips have evolved predatory behaviours, with their reported prey range including thrips, mites, scale-insects, whitefly, psyllid nymphs, and eggs of moths and beetles. However, our current understanding of the biology and potential of these thrips as biological control agents is insufficient, limiting our understanding of their role in pest management. This paper assesses published information on the diversity of predatory thrips and the available biological data on their diet breadth. About 100 species within three different families - Aeolothripidae, Phlaeothripidae and Thripidae - seem likely to be predatory, although some conclusions are speculative. Both facultative and obligate predators can be found among the species with prey records. Obligate predators feed primarily on arthropod tissues, whereas facultative predators consume plant tissues in addition to prey. Quantitative information regarding developmental biology, longevity and fecundity, was obtained for only 11 species. Obligate predators, particularly members of Scolothrips and Franklinothrips, are efficient biological control agents. To better augment the use of predatory thrips in pest management, further increased investment in mass rearing, life history and ecological studies, including interactions with other organisms and their habitat, are needed on these natural enemies. © 2022 Society of Chemical Industry.

A Review of Franklinothrips vespiformis (Thysanoptera: Aeolothripidae): Life History, Distribution, and Prospects as a Biological Control Agent.

Predatory species comprise a small but important and often overlooked component of the Thysanoptera. A case in point, the ant-mimicking Franklinothrips are widely distributed in the tropics and are considered important generalist natural enemies for thrips and some other small arthropod prey. Franklinothrips present an addition to biocontrol applications, i.e., greenhouse or commercial application for certain target pests and situations. Current knowledge, including distribution, biological features, life history pa rameters, prey specificity, host plant associations and lass production is yet insufficient to decide to what extent Franklinothrips could contribute for biological control programs. In this review, we summarized the geographical background, morphology, and prey associations, with a focus on F. vespiformis, the most widely distributed species of predatory thrips. This literature review serves as the basis for future research into the use of Franklinothrips as biocontrol agents for economically significant insect and mite pests in China and elsewhere.

Two new synonyms among Panchaetothripinae (Thysanoptera: Thripidae) with three species newly recorded from China.

Helionothrips mube and Panchaetothrips bifurcus are here recognized as new synonyms of Helionothrips aino and Panchaetothrips timonii based on large numbers of specimens and continuous morphological differences. Three species, Caliothrips insularis, Hercinothrips femoralis and Phibalothrips rugosus are newly recorded from China and the previous record of Caliothrips indicus from China is here considered incorrect. Illustrations of males and larvae of Hercinothrips femoralis are also provided.

Aquilaria Species (Thymelaeaceae) Distribution, Volatile and Non-Volatile Phytochemicals, Pharmacological Uses, Agarwood Grading System, and Induction Methods.

Agarwood is a highly valuable fragrant wood of Aquilaria spp. (Thymelaeaceae) which has been widely utilized in traditional medicine, religious rites, and cultural activities. This study summarizes a review on the identification of Aquilaria cultivars, volatile and non-volatile phytochemicals, pharmacological uses, and agarwood grading system to determine its quality, and different agarwood induction methods. Due to the highly demanding and depleted natural resources, the research on agarwood is still insufficient, and it has broad research and development prospects in many industries. However, due to the significant scientific nature of agarwood application, developing high-quality products and drugs from agarwood have become highly important, while no one has discussed in detail the phytochemicals uses and provided a summary until now. The main phytochemicals of agarwood include terpenoids, dominated by sesquiterpenes. For centuries, terpenoids have been used in traditional Chinese medicine and have been shown to possess various pharmacological properties, including bacteriostatic, antibacterial, sedation, analgesia, anti-inflammation, anti-asthmatic, hypoglycemic, antidepressant, and many others. Alongside biological activity screening, phytochemical advances and pharmacological research have also made certain progress. Therefore, this review discusses the research progress of agarwood in recent years and provides a reference basis for further study of Aquilaria plants and agarwood.

An uncharacterized protein from the metagenome with no obvious homology to known lipases shows excellent alkaline lipase properties and potential applications in the detergent industry.

A novel lipase, Lip486, which has no obvious homology with known lipases, was discovered using functional metagenomics technology. Phylogenetic tree analysis suggested that the enzyme belongs to a new subfamily called lipolytic enzyme family II. To explore the enzymatic properties, lip486 was expressed heterologously and efficiently in Escherichia coli. The recombinant enzyme displayed the highest activity on the substrate p-nitrophenyl caprate with a carbon chain length of 10, and its optimum temperature and pH were 53 °C and 8.0, respectively. The recombinant Lip486 showed good activity and stability in strong alkaline and medium-low-temperature environments. The results of compatibility and soaking tests showed that the enzyme had good compatibility with 4 kinds of commercial detergents, and an appropriate soaking time could further improve the enzyme activity. Oil stain removal test results for a cotton cloth indicated that the washing performance of commercial laundry detergent supplemented with Lip486 was further improved. In addition, as one of the smallest lipases found to date, Lip486 also has the advantages of high yield, good stability and easy molecular modification. These characteristics reflect the good application prospects for Lip486 in the detergent and other industries in the future.

Diamondback Moth Larvae Trigger Host Plant Volatiles that Lure Its Adult Females for Oviposition.

The diamondback moth (DBM) is a destructive pest of crucifer crops. In this study, DBM larvae shown to herbivore induced plant volatiles (HIPVs) that were attractive to adult females exposed in a Y-tube olfactometer. Our results showed that olfactory responses of adult females to HIPVs induced by third instar larvae feeding on Barbarea vulgaris were significantly higher (20.40 ± 1.78; mean moths (%) ± SD) than those induced by first instar larvae (14.80 ± 1.86; mean moths (%) ± SD). Meanwhile, a significant concentration of Sulphur-containing isothiocyanate, 3-methylsulfinylpropyl isothiocyanate, and 4-methylsulfinyl-3-butenyl isothiocyanate were detected in HIPVs released by third instar larvae compared to those released by first instar larvae while feeding on B. vulgaris. When the DBM females were exposed to synthetic chemicals, singly and in blend form, a similar response was observed as to natural HIPVs. Our study demonstrated that the relationship between isothiocyanates acting as plant defense compounds, host plant cues emission and regulation of the DBM adult female behavior due to key volatile triggered by the DBM larvae feeding on B. vulgaris.

Asymmetric Interaction between Aphis spiraecola and Toxoptera citricida on Sweet Orange Induced by Pre-Infestation.

Indirect interactions between herbivorous insects that share the same host have been focused on insects feeding on herbaceous plants, while few studies investigate similar interactions on woody plants. We investigated performance and feeding behavior of two citrus aphids, Aphis spiraecola Patch and Toxoptera citricida Kirkaldy, on sweet orange as affected by prior infestation of conspecifics and heterospecifics. Results showed that pre-infestation-induced interactions between A. spiraecola and T. citricida were asymmetric, with A. spiraecola gaining more fitness. In detail, pre-infestation by A. spiraecola decreased adult weight, enhanced survival rate and accelerated phloem sap acceptance of conspecifics. However, A. spiraecola pre-infestation did not affect performance or feeding behavior of T. citricida. In another infestation sequence, the pre-infestation of T. citricida did not affect conspecifics, but positively affected heterospecifics, indicated as a decreased pre-reproductive period, enhanced survival rate, adult weight, fecundity, and feeding efficiency, i.e., faster access and acceptance of phloem sap, and longer phloem sap ingestion duration. Furthermore, we found A. spiraecola pre-infestation enhanced amino acid concentration, amino acid to sugar ratio, activated salicylic acid and jasmonic acid marker gene expression, while T. citricida pre-infestation only depressed jasmonic acid marker gene expression. Changes in nutrient and phytohormone-dependent defense probably underlie the asymmetric effect.

[Molecular cloning and characterization of a novel metagenome-derived bacterial perhydrolase].

The aim of this study is to obtain bacterial perhydrolases with chlorination activity, expands the resources of perhydrolases, and lays a foundation for it's industrial applications. We constructed a metagenomic library using environmental DNA isolated from sludge samples of a paper mill of Tanghe county, and identified a per822 gene encoding a bacteria perhydrolase via activity-based functional screening. Then, we overexpressed Per822 heterologously in Escherichia coli, and characterized the recombinant enzyme after purification. Finally, we further investigated the ability of Per822 to produce peracetic acid (PAA). Sequence analysis revealed that per822 encoded a protein of 273 amino acids. The recombinant Per822 had the activity of peroxidase, esterase and halogenase respectively, and thus was regarded as a typical representative of multifunctional enzymes. The purified Per822 exhibited maximal chlorination activity (hyperhydrolysis) at 55 °C and pH 4.5 with monochlorodimedone as substrate, and the enzyme was stable in the pH range of 3.5-8.0 and below 70 °C. Also, the chlorination activity of this enzyme could be activated by Fe²âº. In addition, the enzyme displayed high ability to generate PAA using ethyl acetate as cosubstrate. The highly soluble expression, catalytic versatility and good PAA production capacity of Per822 make it a potential candidate in organic synthesis, wastewater treatment, disinfection and biomass pretreatment, etc.

Two new and one little-known damsel bug of the subfamily Prostemmatinae Reuter (Hemiptera, Heteroptera, Nabidae) from China.

Two damsel bugs belonging to two genera of the subfamily Prostemmatinae from China are reported as new to science: Alloeorhynchus (Alloeorhynchus) yunnanensissp. n. and Rhamphocorisguizhouensis sp. n. The little-known species Alloeorhynchus (Alloeorhynchus) reinhardi Kerzhner & Günther, 1999 is redescribed. All species are illustrated in detail. Keys to the Chinese species of Rhamphocoris and Alloeorhynchus are provided aid in identification.

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification.

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/µl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

Chromatographic Fingerprint and Quantitative Analysis of Commercial Pheretima aspergillum (Guang Dilong) and Its Adulterants by UPLC-DAD.

Guang Dilong is a Traditional Chinese Medicine prepared from the dried body of Pheretima aspergillum (E. Perrier), a species of earthworm. However, preparations of Guang Dilong may be adulterated by other species and a method of quality control is needed. A method was developed to analyze and authenticate commercial Guang Dilong, utilizing ultra-high performance liquid chromatography (UHPLC) coupled with diode array detection (DAD). Equipment included an Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm). The mobile phase consisted of acetonitrile and 0.01% formic acid, pumped at 0.3 mL/min. Wavelength detection was at 260 nm. Twenty-two batches of confirmed P. aspergillum samples (reference) from different sources and 20 batches of adulterated samples were analyzed to establish a reference fingerprint for commercial Guang Dilong. Five peaks in the fingerprints of the reference batches were identified as characteristic; six characteristic peaks in the fingerprints of the adulterants were identified by comparing their retention time with those of the references. The total 42 batches of samples were compared with the reference fingerprint, and the fingerprints of the P. aspergillum samples were similar. The UHPLC-DAD method can simultaneously determine the contents of six compounds (hypoxanthine, xanthine, uridine, inosine, guanosine, and adenosine) in the reference and adulterated batches. The six compounds showed good regression (r > 0.9999) within test ranges. The recovery (accuracy) was 98.25 to 101.68%, with relative standard deviation <2.67%. In summary, this UHPLC-DAD method combines chromatographic fingerprint with quantification analysis and could be readily used as an efficient quality control method for Guang Dilong.

Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis.

A new protease gene (pro1437)was separated from an oil-polluted Mud flat metagenomic library. Pro1437 belongs to a peptidase M48 superfamily according to the results of sequence analysis, and it showed very low identities compared to other known proteases or peptidases. The error-prone PCR was used to introduce random mutations and improve the expression of pro1437. After two rounds of mutagenesis and screening, a mutant (Pro2T21) with a 6.6-fold higher activity and a 4.8-fold higher expression level than Pro1437 was obtained. Sequence analysis found three amino acid substitutions (A54V, L192H, F224L) in Pro2T21. 3D structure modelling analysis indicated A54V and L192H probably played a crucial role in the improvement of enzymatic activity and soluble expression level of Pro2T21. Furthermore, Pro2T21opti displayed a 5.8-fold higher expression level than the wild type under optimal pH 8.0 at 50°C after codon-optimization. Also, Pro2T21opti represented robust compatibility with several popular laundry detergents, and blood stains on white cloth pieces were completely washed away when endogenous protease-inactivated Tide and Pro2T21opti were used together. Therefore, Pro2T21opti has great potential for use as an additive in detergents after further study.