The Impact of Diabetes on Male Silkworm Reproductive Health.

The increasing prevalence of diabetic reproductive complications has prompted the development of innovative animal models. The use of the silkworm Bombyx mori as a model for diabetic reproductive damage shows potential as a valuable research tool. This study employed silkworms as a novel model to investigate diabetic reproductive damage. The silkworms were fed a high-glucose diet containing 10% glucose to induce a diabetic model. Subsequently, the study concentrated on assessing the influence of diabetes on the reproductive system of male silkworms. The results indicate that diabetes resulted in reduced luteinizing hormone (LH) and testosterone (T) levels, as well as elevated triglyceride (TG) levels in male silkworms. Moreover, diabetes mellitus was associated with pathological testicular damage in male silkworms, accompanied by decreased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels, along with increased malondialdehyde (MDA) levels in the testis. Additionally, diabetes mellitus reduced the expression of siwi1 and siwi2 genes in the testis of male silkworms. Overall, these results support using silkworms as a valuable model for studying diabetic reproductive damage.

The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms.

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.

Safety improvement of the open sun dried Egyptian Siwi dates using closed solar dryer.

Egyptian Siwi dates dried using the open sun drying is exposed to different contaminants. So, the current study aims to use the closed solar dryer to improve Siwi date safety. The impact of washing and closed solar drying on the levels of microbial load, aflatoxins and heavy metals in Egyptian Siwi dates (ESD), in comparison to traditional open sun drying methods were examined. Two different drying techniques were employed to dry 300 kg of ESD. The microbial load was assessed following the two drying procedures. The levels of aflatoxins and heavy metals were analyzed using High-performance liquid chromatography (HPLC) and Inductively Coupled Plasma (ICP) techniques, respectively, after both drying methods. Additionally, the influence of storage time on the microbial load of the ESD was also evaluated using standard methods. The findings of the current study demonstrated that the closed solar drying significantly reduced the total bacterial and fungal counts by 96 % and 93 %, respectively, when compared to open sun-drying. No aflatoxins were detected in both fresh Siwi dates and Siwi dates dried using closed solar drying. However, after open sun drying, two aflatoxins; aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1), were detected in the ESD, with concentrations of 0.95 and 0.23 µg kg-1, respectively. The closed solar drying significantly decreased the levels of lead (Pb), cadmium (Cd), copper (Cu), nickel (Ni), chromium (Cr), zinc (Zn), manganese (Mn), and iron (Fe) in the dried dates by 96 %, 94 %, 48 %, 71 %, 64 %, 4 %, 26 %, and 7 %, respectively, when compared to open sun drying. The stored Siwi dates that was exposed to the open sun drying showed a higher increase in bacterial (4.86 log CFU/g) and fungal (4.46 log CFU/g) counts. However, the stored Siwi dates that was exposed to the closed solar dryer showed a lower increase in bacterial (3.21 log CFU/g) and fungal (2.51 log CFU/g) counts. So, the duration of storage significantly impacted the microbial loads of the closed solar dried dates as compared to open sun drying. Overall, closed solar drying reduced the levels of investigated contaminants and extended the shelf life of ESD, thereby enhancing their safety for human consumption.

The piRNA pathway is required for nucleopolyhedrovirus replication in Lepidoptera.

The Piwi-interacting RNA (piRNA) pathway has been shown to be involved in the antiviral defense against RNA viruses, especially in mosquitoes, but its universality has been questioned. Here, we used the Bombyx mori nucleopolyhedrovirus (BmNPV) -infected silkworm as a model to explore the effects of the key factors of piRNA pathway, BmAgo3 and Siwi, on replication of a large DNA virus (belonging to the family of Baculoviridae). We demonstrated that BmAgo3 and Siwi could promote the replication of BmNPV through both overexpression and knockdown experiments in BmN cell lines and silkworm larvae. In addition, we also studied the effect of PIWI-class genes on Autographa californica nucleopolyhedrovirus (AcMNPV) replication in the Spodoptera frugiperda cell line Sf9. By knocking down the expression of PIWI-class genes in Sf9, we found that Piwi-like-1 and Piwi-like-2-3 could inhibit AcMNPV replication, while Piwi-like-4-5 promoted virus replication. Our study provides compelling evidence that the piRNA pathway affects host infection by exogenous viruses in Lepidoptera. Also, our results reflect the diversity of the roles of PIWI-class genes in virus infection of the host across species. This study is the first to explore the interaction of PIWI-class proteins with DNA viruses, providing new insights into the functional roles of the piRNA pathway.

PIWI Proteins Play an Antiviral Role in Lepidopteran Cell Lines.

Insect antiviral immunity primarily relies on RNAi mechanisms. While a key role of small interfering (si)RNAs and AGO proteins has been well established in this regard, the situation for PIWI proteins and PIWI-interacting (pi)RNAs is not as clear. In the present study, we investigate whether PIWI proteins and viral piRNAs are involved in the immunity against single-stranded RNA viruses in lepidopteran cells, where two PIWIs are identified (Siwi and Ago3). Via loss- and gain-of-function studies in Bombyx mori BmN4 cells and in Trichoplusia ni High Five cells, we demonstrated an antiviral role of Siwi and Ago3. However, small RNA analysis suggests that viral piRNAs can be absent in these lepidopteran cells. Together with the current literature, our results support a functional diversification of PIWI proteins in insects.

Functional Characterization of Silkworm PIWI Proteins by Embryonic RNAi.

The molecular mechanisms of sex-determination systems among insect orders and species are diverse. Therefore, genes involved in sex determination are strong candidates for insect pest management. Even though lepidopterans are major agricultural insect pests that cause widespread economic damage to various crops, their sex-determination systems have not been fully elucidated, even in the silkworm (Bombyx mori), a model lepidopteran insect. In 2014, we found that a female-specific W chromosome-derived PIWI-interacting RNA (piRNA) determines femaleness in silkworms. To analyze the function of two core silkworm piRNA biogenesis pathway genes, Siwi and BmAgo3, in the sex-determination system, we developed a genomic DNA and total RNA extraction strategy for a siRNA-injected single embryo. The siRNA-injected embryo can be molecularly sexed by W chromosome-specific DNA markers. Using complementary DNA (cDNA) reverse transcribed from the sexed RNA, we evaluated the knockdown effect of the PIWI protein-coding genes on a sexual development-related gene, Bombyx mori doublesex.

Siwi levels reversibly regulate secondary piRISC biogenesis by affecting Ago3 body morphology in Bombyx mori.

Silkworm ovarian germ cells produce the Siwi-piRNA-induced silencing complex (piRISC) through two consecutive mechanisms, the primary pathway and the secondary ping-pong cycle. Primary Siwi-piRISC production occurs on the outer mitochondrial membrane in an Ago3-independent manner, where Tudor domain-containing Papi binds unloaded Siwi via its symmetrical dimethylarginines (sDMAs). Here, we now show that secondary Siwi-piRISC production occurs at the Ago3-positive nuage Ago3 bodies, in an Ago3-dependent manner, where Vreteno (Vret), another Tudor protein, interconnects unloaded Siwi and Ago3-piRISC through their sDMAs. Upon Siwi depletion, Ago3 is phosphorylated and insolubilized in its piRISC form with cleaved RNAs and Vret, suggesting that the complex is stalled in the intermediate state. The Ago3 bodies are also enlarged. The aberrant morphology is restored upon Siwi re-expression without Ago3-piRISC supply. Thus, Siwi depletion aggregates the Ago3 bodies to protect the piRNA intermediates from degradation until the normal cellular environment returns to re-initiate the ping-pong cycle. Overall, these findings reveal a unique regulatory mechanism controlling piRNA biogenesis.

Impact of Pretreatments and Drying Temperatures on Quality of Siwi and Sakkoti Dates.

BACKGROUND AND OBJECTIVE: Drying of date helps in preserving it to be consumed outside the harvest season and removes some moisture from dates and also slows down the action of date endogenous enzymes. This study was carried out to investigate pretreatments and drying temperature on the physical and chemical properties of 2 date varieties (Siwi and Sakkoti) at the khalal stage. MATERIALS AND METHODS: The date fruits at khalal stage were dipped in ascorbic acid solution, sodium metabisulfite solution and sulfur dioxide before cut into pieces, halves and as whole. Then dates were dried at 50, 55, 60 and 65°C, respectively till ~20% moisture content and examined the physical and chemical properties of dried dates. RESULTS: Moisture content of Siwi and Sakkoti at the khalal stage was 56.90 and 51.72%, respectively, while total sugars were 79.76 and 75.74%, respectively on dry weight bases. The color of dates Hunter (L and b) were the highest of treated with meta-bisulfate solution or sulfur dioxide and the lowest of color date observed (Hunter, a) comparing with control and ascorbic acid. CONCLUSION: The pretreatments indicated that the dipping dates in sodium meta-bisulfate solution or sulfur dioxide then, dried at 60˚C produce high quality parameters of semi-dry dates comparing control and ascorbic acid.

Crystal Structure of Silkworm PIWI-Clade Argonaute Siwi Bound to piRNA.

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.