The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

Effects of Acute Temperature Stress on mRNA Expression of Transferrin in the Yellow Pond Turtle Mauremys mutica

The yellow pond turtle Mauremys mutica is widely cultured using both greenhouse-reared and outdoor pond-reared models.Individuals from the two models often show different tolerances to dramatic temperature changes caused by extreme weather events.However,the mechanism underlying the difference is unclear.In this study,we found that for greenhouse-reared turtles(GRTs),the expression levels of an immune-related gene for transferrin were significantly different(P<0.05)between the control group and the acute cold stress(ACS)group for most time points(3 h,6 h and 48 h),while at two time points(6 h and 12 h)there was a significant difference(P<0.05)between the control group and the acute heat stress(AHS)group.However,for the outdoor pond-reared turtles(OPTs),we found the opposite pattern:the ACS group showed no significant difference(P>0.05)from the control group for all time points(3 h,6 h,12 h,24 h and 48 h),whereas two time points(12 h and 24 h)were significantly different(P<0.05)for the AHS group.Our results indicate that ACS may influence the immunity of GRTs and have no influence on OPTs,whereas AHS may largely affect the immunity of OPTs and have little influence on GRTs.The findings provide insights into the mechanism underlying the different morbidity and mortality rates of turtles from different culture models after extreme weather events.

Matrix metalloproteinase 2 contributes to adult eclosion and immune response in the small hive beetle,Aethina tumida

During the pupal-adult eclosion process of holometabolous insects,the old cuticle is shed and replaced by a completely different new cuticle that requires tanning and expansion,along with extensive extracellular matrix(ECM)remodeling.In vertebrates,matrix metalloproteinases(MMPs),a class of zinc-dependent endopeptidases,play key roles in regulating the ECM that surrounds cells.However,little is known about these extracellular proteinases available in insects.The small hive beetle(SHB),Aethina tumida,is a widespread invasive parasite of honey bees.In this study,6 MMP homologs were identified in the SHB genome.RNA interference experiments showed that all 6 AtMmps are not required for the larval-pupal transition,only AtMmp2 was essential for pupal-adult eclosion in SHB.Knockdown of AtMmp2 resulted in eclosion defects and wing expansion failure,as well as mortality within 3 d of adult eclosion.Transcriptomic analysis revealed that knockdown of AtMmp2 significantly increased expression of the Toll and Imd pathways,chitin metabolism,and cross-linking(such as the pro-phenoloxidase activating cascade pathway and the tyrosine-mediated cuticle sclerotization and pigmentation pathway).These data revealed evolutionarily conserved functions of Mmp2 in controlling adult eclosion and wing expansion,also provided a preliminary exploration of the novel function of regulating Toll and Imd pathways,as well as new insights into how MMPs regulate insect development and defense barriers.