DataSheet1_High mechanical property silk produced by transgenic silkworms expressing the Drosophila Dumpy.pdf

Extensive research has been conducted on utilizing transgenic silkworms and their natural spinning apparatus to produce high-performance spider silk fibers. However, research on using non-spider biological proteins to optimize the molecular structure of silk protein and improve the mechanical performance of silk fibers is still relatively scarce. Dumpy, a massive extracellular matrix polypeptide, is essential for preserving the shape and structural integrity of the insect cuticle due to its remarkable tension and elasticity. Here, we constructed two transgenic donor plasmids containing the fusion genes of FibH-Dumpy and FibL-Dumpy. The results indicated the successful integration of two exogenous gene expression cassettes, driven by endogenous promoters, into the silkworm genome using piggyBac-mediated transgenic technology. Secondary structure analysis revealed a 16.7% and 13.6% increase in the β-sheet content of transgenic silks compared to wild-type (WT) silk fibers. Mechanical testing demonstrated that, compared to the WT, HDUY and LDUY transgenic silk fibers exhibited respective increases of 39.54% and 21.45% in maximum stress, 44.43% and 45.02% in toughness, and 24.91% and 28.51% in elastic recovery rate. These findings suggest that Drosophila Dumpy significantly enhanced the mechanical properties of silk, positioning it as an excellent candidate for the development of extraordinary-performance fibers. This study provides rich inspiration for using other biological proteins to construct high-performance silk fibers and expands the possibilities for designing and applying novel biomaterials.</p

Agonist-Activated <i>Bombyx</i> Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway

Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the <i>Bombyx</i> corazonin receptor (BmCrzR) activates both G_αq- and G_αs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a β-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues (⁴¹¹TSS⁴¹³) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of <i>Bombyx</i> corazonin receptors

Viral Infection Affects Sucrose Responsiveness and Homing Ability of Forager Honey Bees<i>, Apis mellifera</i> L.

<div><p>Honey bee health is mainly affected by <i>Varroa destructor</i>, viruses, <i>Nosema</i> spp., pesticide residues and poor nutrition. Interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years. In the present study, the effects of a honey bee virus, Israeli acute paralysis virus (IAPV), on the foraging behaviors and homing ability of European honey bees (<i>Apis mellifera</i> L.) were investigated based on proboscis extension response (PER) assays and radio frequency identification (RFID) systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around 10⁷ copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.</p> </div

The exact number of copies of IAPV in heads of honey bees.

<p>The exact number of copies of IAPV in heads of honey bees collected at day 0, day 1 and day 2 after injections with 1 ul of IAPV (1: 200 dilution) and 1 ul of PBS respectively. Error bars show SD.</p

Comparisons between IAPV-injected honey bees and PBS-injected honey bees in their responsiveness to sucrose.

<p>Sucrose responsiveness of honey bees injected with IAPV with 1: 500 dilution and PBS-injected honey bees were tested at days 0 (A) and 3 (B) after injection. The number of honeybees tested at day 0 post-injection is 60 for each group; the number of honeybees tested at day 3 for IAPV-injected group is 14 and 29 for the PBS-injected group (*=p<0.05, **=p<0.01); sucrose responsiveness of honey bees injected with IAPV with 1: 200 dilution and PBS-injected honey bees tested at days 0 (C) and 2 (D) after injection. The number of honey bees tested at day 0 post-injection is 60 for each group; the number of honey bees tested at day 2 for IAPV-injected group is 22 and 30 for the PBS-injected group (*=p<0.05, **=p<0.01).</p

The RFID system used in homing experiment.

<p>(A) Two RFID readers were placed at the customized tunnel entrance of a nucleus hive with 3 frames. (B) A honey bee with the RFID tag glued to its thorax carrying pollen on its hind legs returns to the hive.</p

Analysis of survival rates.

<p>Comparison of survival rates between honey bees injected with IAPV and honey bees of the control groups at 24, 48 hours after injection. The data are expressed as mean ± SD of four independent experiments. </p

Activation of BNGR-A24 by Direct Interaction with Tachykinin-Related Peptides from the Silkworm <i>Bombyx mori</i> Leads to the G_q- and G_s‑Coupled Signaling Cascades

Tachykinins constitute one of the largest peptide families in the animal kingdom and exert their diverse actions via G protein-coupled receptors (GPCRs). In this study, the <i>Bombyx</i> tachykinin-related peptides (TKRPs) were identified as specific endogenous ligands for the <i>Bombyx</i> neuropeptide GPCR A24 (BNGR-A24) and thus designated BNGR-A24 as BmTKRPR. Using both mammalian cell line HEK293 and insect cell line Sf21, further characterization demonstrated that BmTKRPR was activated, thus resulting in intracellular accumulation of cAMP, Ca²⁺ mobilization, and ERK1/2 phosphorylation in a G_s and G_q inhibitor-sensitive manner. Moreover, quantitative reverse transcriptase polymerase chain reaction analysis and dsRNA-mediated knockdown experiments suggested a possible role for BmTKRPR in the regulation of feeding and growth. Our findings enhance the understanding of the <i>Bombyx</i> TKRP system in the regulation of fundamental physiological processes