Determination of albendazole and metabolites in silkworm Bombyx mori hemolymph by ultrafast liquid chromatography tandem triple quadrupole mass spectrometry.

Albendazole is a broad-spectrum parasiticide with high effectiveness and low host toxicity. No method is currently available for measuring albendazole and its metabolites in silkworm hemolymph. This study describes a rapid, selective, sensitive, synchronous and reliable detection method for albendazole and its metabolites in silkworm hemolymph using ultrafast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-MS/MS). The method is liquid-liquid extraction followed by UFLC separation and quantification in an MS/MS system with positive electrospray ionization in multiple reaction monitoring mode. Precursor-to-product ion transitions were monitored at 266.100 to 234.100 for albendazole (ABZ), 282.200 to 208.100 for albendazole sulfoxide (ABZSO), 298.200 to 159.100 for albendazole sulfone (ABZSO2) and 240.200 to 133.100 for albendazole amino sulfone (ABZSO2-NH2). Calibration curves had good linearities with R2 of 0.9905-0.9972. Limits of quantitation (LOQs) were 1.32 ng/mL for ABZ, 16.67 ng/mL for ABZSO, 0.76 ng/mL for ABZSO2 and 5.94 ng/mL for ABZSO2-NH2. Recoveries were 93.12%-103.83% for ABZ, 66.51%-108.51% for ABZSO, 96.85%-105.6% for ABZSO2 and 96.46%-106.14% for ABZSO2-NH2, (RSDs <8%). Accuracy, precision and stability tests showed acceptable variation in quality control (QC) samples. This analytical method successfully determined albendazole and its metabolites in silkworm hemolymph in a pharmacokinetic study. The results of single-dose treatment suggested that the concentrations of ABZ, ABZSO and ABZSO2 increased and then fell, while ABZSO2-NH2 level was low without obvious change. Different trends were observed for multi-dose treatment, with concentrations of ABZSO and ABZSO2 rising over time.

Analysis of midgut gene expression profiles from different silkworm varieties after exposure to high temperature.

The silkworm is a poikilothermic animal, whose growth and development is significantly influenced by environmental temperature. To identify genes and metabolic pathways involved in the heat-stress response, digital gene expression analysis was performed on the midgut of the thermotolerant silkworm variety '932' and thermosensitive variety 'HY' after exposure to high temperature (932T and HYT). Deep sequencing yielded 6,211,484, 5,898,028, 5,870,395 and 6,088,303 reads for the 932, 932T, HY and HYT samples, respectively. The annotated genes associated with these tags numbered 4357, 4378, 4296 and 4658 for the 932, 932T, HY and HYT samples, respectively. In the HY-vs-932, 932-vs-932T, and HY-vs-HYT comparisons, 561, 316 and 281 differentially expressed genes were identified, which could be assigned to 179, 140 and 123 biological pathways, respectively. It was found that some of the biological pathways, which included oxidative phosphorylation, related to glucose and lipid metabolism, are greatly affected by high temperature and may lead to a decrease in the ingestion of fresh mulberry. When subjected to an early period of continuous heat stress, HSP genes, such as HSP19.9, HSP23.7, HSP40-3, HSP70, HSP90 and HSP70 binding protein, are up-regulated but then reduced after 24h and the thermotolerant '932' strain has higher levels of mRNA of some HSPs, except HSP70, than the thermosensitive variety during continuous high temperature treatment. It is suggested that HSPs and the levels of their expression may play important roles in the resistance to high temperature stress among silkworm varieties. This study has generated important reference tools that can be used to further analyze the mechanisms that underlie thermotolerance differences among silkworm varieties.