Pseudomonas mosselii: a potential alternative for managing pyrethroid-resistant Aedes aegypti.

BACKGROUND: Aedes aegypti is a widespread mosquito in tropical and subtropical regions that causes significant mortality and morbidity in humans by transmitting diseases, such as dengue fever and Zika virus disease. Synthetic insecticides, such as pyrethroids, have been used to control Ae. aegypti, but these insecticides can also affect nontarget organisms and contaminate soil and water. This study aimed to investigate the mosquitocidal activity of Pseudomonas mosselii isolated from pond sludge against larvae of Ae. aegypti. RESULTS: Based on the initial results, similar time-course profiles were obtained for the mosquitocidal activity of the bacterial culture and its supernatant, and the pellet resuspended in Luria-Bertani (LB) medium also showed delayed toxicity. These results imply that the toxic component can be released into the medium from live bacteria. Further research indicated that the toxic component appeared in the supernatant approximately 4 h after a 3-mL stock was cultured in 200 mL of LB medium. The stabilities of the P. mosselii culture and supernatant stored at different temperatures were also evaluated, and the best culture stability was obtained at 28 °C and supernatant stability at 4 °C. The bacterial culture and supernatant were toxic to larvae and pupae of not only susceptible Ae. aegypti but also pyrethroid-resistant strains. CONCLUSION: This study highlights the value of the mosquitocidal activity of P. mosselii, which has potential as an alternative insecticide to control pyrethroid-resistant Ae. aegypti in the field. © 2024 Society of Chemical Industry.

Efficiency of a Novel Light-Emitting Diode (LED) Trap for Trapping Rhyzopertha dominica (Coleoptera: Bostrichidae) in Paddy Rice Storehouses.

The lesser grain borer Rhyzopertha dominica is the major pest of stored paddy rice globally, including in Taiwan. It has strong phototaxis and is good at flying, suitable for developing a light-trapping method to monitor and control it. In the present study, a wavelength of light-emitting diodes (LEDs), i.e., 373 nm, was determined to be the most efficient to trap R. dominica using a dodecagon maze. Accordingly, an LED trap, named the Taiwan Agricultural Research Institute-LED (TARI-LED) trap, was invented, which comprised LEDs of two distinct wavelengths (373 and 408 nm), a wavelength switch, a suction fan, and an insect collector. The trapping efficiency was assessed in a 4-m3 laboratory arena and two paddy rice storehouses. An initial assessment was performed in the laboratory arena and showed that the TARI-LED trap with 373-nm wavelength for R. dominica rapidly increased in the first 30 min, reaching the highest trapping rate (68.5%) after 3 h. In addition, no significant difference was observed between the suction fan turned on or off. The field tests showed that the 373-nm wavelength had the highest effectiveness for trapping R. dominica in the two paddy rice storehouses, and no significant difference was observed in the number of R. dominica trapped by the 373-nm TARI-LED trap or the CDC-UV light trap. In conclusion, our TARI-LED trap 373 nm exhibited high efficiency in trapping R. dominica in paddy rice storehouses. Moreover, a suction fan-free design should benefit long-term and safe use in paddy rice storehouses trapping R. dominica.

Rapid identification of the invasive fall armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae) using species-specific primers in multiplex PCR.

The fall armyworm (FAW), Spodoptera frugiperda (Smith), is a major pest native to the Americas. A recent invasion of FAWs from Africa eastward to South Asia, the Indochina Peninsula, and mainland China has received much attention due to the considerable economic losses in agriculture. FAWs can rapidly colonise a new area, likely due to the wide range of host plants, good flying capability, and high egg production. Therefore, a convenient, quick, and accurate tool for FAW identification is urgently required to establish a FAW invasion management strategy. In this study, FAW-specific primers were designed to recognise FAWs on the basis of internal transcribed spacer 1 (ITS1). The results revealed the accurate FAW recognition of the three congeneric species and eight common corn lepidopteran pests, especially at their larval stage. Furthermore, species-specific primers have confirmed their efficacy by using 69 FAW specimens from Taiwan, Thailand, and the United States, with a 96% success rate, excluding 3 decayed specimens. By using the simple, reliable, and convenient FAW-specific primers, a pest management programme can be developed not only to reduce sequencing costs and experimental time from 2 days to 4 h, but eradicate the FAW as soon as it enters a new area.

PCR-RFLP of mitochondrial DNA reveals two origins of Apis mellifera in Taiwan.

Beekeeping has been a highly valued industry in Taiwan. As a result, many subspecies of Apis mellifera have been introduced to Taiwan since 1911, leading to the hybridization of different subspecies. In order to know the matrilineal origins of Taiwan A. mellifera, a total of 280 samples collected from 33 apiaries throughout the island were examined. Using PCR-RFLP of four mitochondrial gene fragments, i.e., the non-coding region between tRNAleu and cytochrome c oxidase subunit II (intergenic tRNAleu-COII), cytochrome b (Cyt b), large subunit rRNA (Ls rRNA) and cytochrome c oxidase subunit I (COI), we only found two haplotypes exist in 280 samples. Haplotypes ababa and bbbaa account for 87% of these Western bees belonged to the Eastern European (C) lineage and 13% belonged to the Middle East (Z) lineage, respectively, with the latter being totally absent in northern Taiwan. African (A) and Mellifera (M) lineages, officially imported once in 1990s and 1930s respectively, were not detected. The identification of subspecies of A. mellifera and survey of their distribution on the island are expected to facilitate efficient breeding programs and establish a more booming beekeeping industry.

Gene expression changes in honey bees induced by sublethal imidacloprid exposure during the larval stage.

Honey bee larvae exposed to sublethal doses of imidacloprid show behavioural abnormalities as adult insects. Previous studies have demonstrated that this phenomenon originates from abnormal neural development in response to imidacloprid exposure. Here, we further investigated the global gene expression changes in the heads of newly emerged adults and observed that 578 genes showed more than 2-fold changes in gene expression after imidacloprid exposure. This information might aid in understanding the effects of pesticides on the health of pollinators. For example, the genes encoding major royal jelly proteins (MRJPs), a group of multifunctional proteins with significant roles in the sustainable development of bee colonies, were strongly downregulated. These downregulation patterns were further confirmed through analyses using quantitative reverse transcription-polymerase chain reaction on the heads of 6-day-old nurse bees. To our knowledge, this study is the first to demonstrate that sublethal doses of imidacloprid affect mrjp expression and likely weaken bee colonies.

Using DNA chips for identification of tephritid pest species.

BACKGROUND: The ability correctly to identify species in a rapid and reliable manner is critical in many situations. For insects in particular, the primary tools for such identification rely on adult-stage morphological characters. For a number of reasons, however, there is a clear need for alternatives. This paper reports on the development of a new method employing DNA biochip technology for the identification of pest species within the family Tephritidae. RESULTS: The DNA biochip developed and tested here quickly and efficiently identifies and discriminates between several tephritid species, except for some that are members of a complex of closely related taxa and that may in fact not represent distinct biological species. The use of these chips offers a number of potential advantages over current methods. Results can be obtained in less than 5 h using material from any stage of the life cycle and with greater sensitivity than other methods currently available. CONCLUSIONS: This technology provides a novel tool for the rapid and reliable identification of several major pest species that may be intercepted in imported fruits or other commodities. The existing chips can also easily be expanded to incorporate additional markers and species as needed.

A SCAR-based method for rapid identification of four major lepidopterous stored-product pests.

Since Taiwan became a World Trade Organization member in 2002, large quantities of grain have been imported from different countries, and insect pests are frequently intercepted from these imported commodities in quarantine inspection. Because most insects are intercepted as immature forms, morphological identification is problematic; therefore, we developed a DNA identification method based on a sequence-characterized amplified region- polymerase chain reaction (SCAR-PCR). Three sets of multiplex SCAR-PCR mixtures, namely SCAR-I, -II, and -III, were developed with each set composed of four species-specific primer pairs derived from the genomic DNA of four major lepidopterous stored-product pests: Corcyra cephalonica (Stainton), Cadra cautella (Walker), Sitotroga cerealella Oliver, and Plodia interpunctella (Hübner). The SCAR-I amplicons of C. cephalonica, C. cautella, S. cerealella, and P. interpunctella were 205, 550, 324, 382 bp, respectively, while those of SCAR-II were 341, 565, 261, and 170 bp, and those of SCAR-III were 514, 555, 445, and 299 bp. These multiplex PCR mixtures could sensitively and unambiguously detect and identify in approximately 5 h individuals among the four lepidopterous pests intercepted in imported stored-products. In summary, the SCAR-PCR method we developed represents a rapid, sensitive and accurate technique for identifying insect species of stored products in plant quarantine operation.

cDNA isolation, expression, and hormonal regulation of yolk protein genes in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

Yolk protein (YP) or vitellogenin (Vg), the main component of yolk, is the key nutrient for embryonic development. YPs, encoded from uncleaved genes existing mainly in cyclorraphan flies, are different from VGs that are present in most non-cyclorraphan dipterans and other insects. In this study, cDNAs of two YPs, namely Bdyp1 and Bdyp2 (GenBank accession Nos. AF368053 and AF368054), were isolated in the oriental fruit fly, Bactrocera dorsalis (Hendel). RT-PCR analysis revealed that Bdyp1 and 2 are expressed in the fat body and ovary during egg development. However, the expression profiles of Bdyp1 and 2 in the fat body are different, indicating that divergent mechanisms might exist in the regulation of these two genes. Twenty-hydroxyecdysone (20E) plays a major role in promoting Bdyp1 expression, yet the expression of Bdyp2 exhibits a greater response to juvenile hormone (JH) in fat body in vitro. Unexpectedly, 20E-induced expression of both Bdyp1 and 2 is suppressed by JH prior to 20E treatment of in vitro fat body; conversely, it is enhanced by the addition of JH following 20E treatment.

CDNA cloning and characterization of S6 kinase and its effect on yolk protein gene expression in the oriental fruit fly Bactrocera dorsalis (Hendel).

p70 S6 kinase (S6K), a serine/threonine protein kinase, is a downstream target of target of rapamycin (TOR) gene and an important regulator of protein synthesis responsible for cell growth and reproduction. In this study, a S6K gene, named BdS6K (GenBank Accession No. GQ203802), was isolated from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed that BdS6K mRNA is expressed at a higher level in egg than in other developmental stages, as well as in ovary than in fat body. Downregulation of BdS6K activity by rapamycin treatment in larval stage resulted in the developmental defects of larvae, pupae, and adults, with a reduced yolk protein (YP) expression in the fat body throughout the first reproductive cycle with a substantial reduction in ovary size, and also repressed the egg development in female fruit fly. Knockdown of BdS6K gene by RNA interference in the adult significantly decreased the YP expression. These observations support the involvement of BdS6K signaling in the regulation of the YP synthesis and egg development in B. dorsalis.

Evaluating antioxidative activities of amino acid substitutions on mastoparan-B.

Mastoparan-B is a peptide toxin isolated from the venom of Vespa basalis, the most dangerous hornet found in Taiwan. This study is aimed to evaluate the antioxidative activities of several amino acid substitutions on MP-B, and examined the influences of mast cell degranulation and hemolytic activities in parallel with antioxidative activities. The correlations between the biological function and amino acid sequence were assessed. Our study shows original MP-B is a valuable antioxidant at low concentration in competing with nitric-oxide for oxygen molecules and possesses good antioxidative enzyme activities resembled to superoxidase dismutase and glutathione peroxidase. And there are no predominant rates of mast cell degranulation and hemolytic effects in such condition. With proper substitutions, the reducing power, DPPH scavenging activity and glutathione reductase-like enzyme activity of MP-B can increase clearly. The results demonstrate that MP-B analogs are very potential to be applicable antioxidants for other antioxidative usages.

Display female-specific doublesex RNA interference in early generations of transformed oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND: The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most destructive pests in many Asian countries. An effective strategy to reduce fly density in the field is urgently required. Recently, the doublesex of B. dorsalis (Bddsx(f) ) has been cloned, and RNA interference (RNAi) indicates that it can reduce the offspring in vitro. In this study, a piggyBac-based construct that generates short hairpin RNA (shRNA) against the female-specific region of Bddsx was introduced into the pest to test the RNAi effects on reproductive functions in vivo. RESULTS: After embryonic injection and backcross, 21 transgenic lines with germline transformation were identified. Genomic DNA analysis showed that the exogenous transgene including short hairpin Bddsx(f) and a DsRed marker had integrated into the genomes of 11 transformed lines. Northern blot analysis indicated the presence of Bddsx(f) short interfering RNA (siRNA) under the control of a U6 promoter in transformed flies. As expected, the specific effects of RNAi led to the delay of egg maturation, and the offspring was significantly reduced. Reverse transcription real-time PCR further demonstrated that in vivo interference not only specifically inhibited the Bddsx(f) transcript but also repressed expression of the downstream yolk protein gene (Bdyp1). CONCLUSION: The results clearly indicate that RNAi is heritable through the expression of specific siRNA in early generations of transformed oriental fruit fly. These results can broaden the understanding of sex-related developmental mechanisms in the fly, and also offer a possible molecular approach for pest control in the future.

Target of rapamycin in the oriental fruit fly Bactrocera dorsalis (Hendel): its cloning and effect on yolk protein expression.

Target of rapamycin (TOR), a serine/threonine protein kinase, is involved in regulating a number of growth and developmental processes of an organism, including yolk protein synthesis in insects. In this study, TOR gene was isolated, designated BdTOR (GenBank accession no. FJ167395), from the oriental fruit fly Bactrocera dorsalis (Hendel). Quantitative RT-PCR showed a higher expression of BdTOR in the pupa than in other developmental stages, as well as in ovary than in the fat body. Downregulation of BdTOR activity by rapamycin treatment and RNA interference (RNAi) in vivo resulted in a significant reduction in yolk protein transcripts in both fat body and ovary, with a substantial reduction in ovary size. However, an unexpected increase in the expression of yolk protein gene was observed in adult ovary 9 days after rapamycin treatment. Taken together, the results suggest the involvement of BdTOR in the regulation of yolk protein synthesis in B. dorsalis.

Effects of irradiation on Planococcus minor (Hemiptera: Pseudococcidae).

Irradiation has been recognized and endorsed as a potential phytosanitary measure that could be an alternative to current quarantine treatments. Dosages of 50, 100, 150, 200, and 250 Gy were used to irradiate three different life stages (eggs, immatures, and adults) of Planococcus minor (Maskell) (Hemiptera: Pseudococcidae), focusing on females due to its parthenogenesis ability, with an aim to find the most tolerant stage and the most optimal dose to control P. minor. Cobalt 60 was the source of irradiation used. Irradiation of 150-250 Gy has a significant effect on all life stages of P. minor, decreasing its survival rate, percentage of adult reproduction, oviposition, and fertility rate. The adult was the most tolerant life stage in both mortality and fertility rate. All the different irradiated target life stage groups oviposited eggs, but none of the F2 eggs hatched at the most optimal dosage of 150-250 Gy.

Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone.

A signature of eclosion hormone (EH) action in insect ecdysis is elevation of cGMP in Inka cells, leading to massive release of ecdysis triggering hormone (ETH) and ecdysis initiation. Although this aspect of EH-induced signal transduction is well known, the receptor mediating this process has not been identified. Here, we describe a receptor guanylyl cyclase BdmGC-1 and its isoform BdmGC-1B in the Oriental fruit fly Bactrocera dorsalis that are activated by EH. The B form exhibits the conserved domains and putative N-glycosylation sites found in BdmGC-1, but possesses an additional 46-amino acid insertion in the extracellular domain and lacks the C-terminal tail of BdmGC-1. Combined immunolabeling and in situ hybridization reveal that BdmGC-1 is expressed in Inka cells. Heterologous expression of BdmGC-1 in HEK cells leads to robust increases in cGMP following exposure to low picomolar concentrations of EH. The B-isoform responds only to higher EH concentrations, suggesting different physiological roles of these cyclases. We propose that BdmGC-1 and BdmGC-1B are high- and low-affinity EH receptors, respectively.

Juvenile hormone induction of glutathione S-transferase activity in the larval fat body of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

The effect of pyriproxyfen, a juvenile hormone analog (JHA), on the pupation of S. litura was examined. A topical application of 100 mug JHA/larva on the newly ecdysed (0-day) sixth instar larvae resulted in more than 80% pupation, while most of the 1- or 2-day-old larvae similarly treated developed into supernumerary larvae. Glutathione S-transferse (GST) activity in the fat body of 0-day-old sixth instar larvae was significantly induced within 12 h of JHA (100 microg/larva) treatment. In contrast, no such induction was found when 1- and 2-day-old sixth instar larvae were similarly treated. This induction phenomenon was also observed when 0-day-old sixth instar larvae were treated with the natural JH III. The suppressive effects of alpha-amanitin and cycloheximide suggest that JHA induction of GST activity in these cutworm larvae presumably occurred at the gene transcription level.

Female-specific doublesex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel).

A homologue of the doublesex gene (Bddsx) has been cloned from the oriental fruit fly, Bactrocera dorsalis (Hendel). Northern analysis indicates a differential expression of Bddsx in male and female flies, as reported for other dsx genes. A structural conservation of DNA binding domain/oligomerization domain 1 and oligomerization domain 2 suggests that the doublesex protein (BdDSX) of this fruit fly serves as a transcriptional factor for downstream sex-specific gene expression. The putative transformer/transformer-2 protein binding sequence in female-specific transcript suggests that a preserved alternative splicing process found in other flies mediates the synthesis of Bddsx transcript. RNA interference (RNAi) data from adult abdominal dsRNA injection assays indicate that female-specific dsx dsRNA reduces specifically its own transcript, inhibits selectively expression of the yolk protein gene (Bdyp1), and delays ovary development. The number of matured eggs is significant reduced after RNAi treatment, but the sex ratio of offspring is not biased. Moreover, 27% of female progeny with RNAi show deformed ovipositor, but male flies are not affected. Although this is a transient treatment, the specific Bddsx(f) interference offers a promising and novel approach to oriental fruit fly control in the future.

Generation of insulin-producing islet-like clusters from human embryonic stem cells.

Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to beta cells. Generation of glucose-responsive, insulin-producing beta cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Ptf1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mug of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner. Disclosure of potential conflicts of interest is found at the end of this article.

Rapid and efficient in vitro generation of pancreatic islet progenitor cells from nonendocrine epithelial cells in the adult human pancreas.

The absence of efficient and directed methods for the differentiation of adult pancreatic progenitor cell populations to pancreatic islet cells has raised doubts concerning the regeneration potential inherent in the adult pancreas. Relatively low levels of islet cell differentiation have been reported using adult pancreatic cells in vivo and in vitro. In the present study, we initially enriched for a nonendocrine epithelial component of the adult human pancreas and defined conditions that are permissive to islet cell differentiation in vitro. Sequential progression of cell differentiation in the permissive conditions allowed for incremental evaluation of changes occurring in the cell population. Optimization of the differentiation process, for the efficient production of islet endocrine cells, was accomplished by identifying specific factors and culture conditions that increased islet progenitor production 250-fold. Ultimately, 85% percent of the nonendocrine epithelial cells isolated from human pancreatic tissue and cultured in the optimized conditions for 8 days, readily re-expressed pancreatic duodenal homeobox-1 (Pdx1). Sixty-five percent of these Pdx1-expressing cells were capable of additional islet endocrine cell differentiation. This represents a significant advancement in the differentiation of an adult pancreatic progenitor cell population in vitro and suggests that the nonendocrine compartment of the human pancreas remains an important cell resource for the generation of transplantable islets to treat diabetes.

Association of matrix metalloproteinase-9 and Purkinje cell degeneration in mouse cerebellum caused by Angiostrongylus cantonensis.

Angiostrongylosis is a neurological disorder caused by invasion of the central nervous system by developing larvae of Angiostrongylus cantonensis. Purkinje cells in infected mouse cerebellums are small and irregular with degenerative atrophy or partial loss. Ultrastructural changes in degenerative cells included enlarged vacuolar structures and swollen mitochondria within the cytoplasm. The matrix metalloproteinase-9 mRNA which is low in normal cerebellums was expressed in A. cantonensis-infected mice cerebellum prior to Purkinje cell degeneration. Matrix metalloproteinase-9 protein level and enzyme activity increased when the Purkinje cells appeared degenerated. Using immunohistochemistry, matrix metalloproteinase-9 was localised within degenerative Purkinje cells. In addition, when the specific matrix metalloproteinase inhibitor, GM6001, was added, matrix metalloproteinase-9 enzyme activity was reduced by 41.6%. The numbers of degenerative Purkinje cells increased significantly upon establishment of infection but subsided upon inhibition. These results suggested that the expression of matrix metalloproteinase-9 may be associated with degeneration of Purkinje cells in mouse cerebellum infected by A. cantonensis.