Establishment of laboratory colony
Sarcophaga dux was obtained from pig carcasses placed in a deserted yard near a reservoir in Changsha city (28º12’N, 112º58’E), Hunan province, China, in March 2018. About 20 adult flies were collected, and species determination was performed by forensic entomologist using the adult identification key [38], and confirmed again by sequencing the COI (Genbank No. MN543093): F: 5-GGTCAACAAATCATAAAGATATTGG-3′ and R: 5-RAAACTTCAGGRTGACCAAAGAATCA-3. These adult S. dux were maintained in an insect-rearing cage of size 35×35×35cm, in forensic entomology laboratory with about 70% humidity, average temperature of 25°C, natural light. The sugar and milk powder (1:1 mixture) and fresh water were provided for adult flies by the dish (12 cm diameter). The fresh pig lung as larviposition medium was placed in the cages, the larvae (between 500 and 1000 individuals) were collected within 4 hours, and reared in a biomimetic appliance of rearing larvae, the sufficient amount of fresh pig lung were provided until pupation. After eclosion, the adult of S. dux were reared once again five generations to obtain a purebred laboratory colony and increase the number of adult flies.
Collection of Developmental Data
The proper amount of fresh pig lung was provided to induce larviposition, about 2,000 to 3,000 larvae were collected within 2h, the larvae was placed in a rearing boxes containing pig lung, and transfer to the artificial climate box (LRH-250-GSI, Taihong Co., Ltd, Shaoguan, China), with the experimental conditions of 75% humidity and photoperiod of 12D:12L, and 16, 19, 22, 25, 28, 31 and 34°C constant temperatures. The fresh pig lung was provided to meet the development needs of the larvae until pupation. Larvae were observed and collected once every 8 h, and 10 larvae were randomly taken from rearing boxes at each examination time, and immediately kill larvae with boiling water for at least 60 s, then stored in 80% ethanol at room temperature [39, 40]. The posterior spiracle slits was observed using a stereoscopic microscope (Motic SMZ-168) to determine the larval instar [41]. After stored 24 h of each larval using tubes, the body length was measured with a digital vernier caliper (Mahr, 16EWRi, Germany), to avoid the variation caused by the preservative solution, due to the increase of the larvae body length of 80% ethanol preserved occurred mainly in the first 12 h, and remained basically constant after 24 h [16]. The time of each developmental stage, including larval, pupation, and eclosion, were recorded. At each constant temperature, five replicates were performed for the study of development parameters.
Data analysis of Developmental
The development data was analyzed by the software Origin Pro 8.6 (SCR: 015636), the one-way ANOVA was used for analyzing the influence of different temperatures on duration of development. The relationship between development time after larviposition and the body length of larval was analyzed by nonlinear regression, to obtain a fitted equation for the PMImin estimation [11]. The Ikemoto and Takai proposed the revised regression model was adopted [42]. The relationship between accumulated degree hours (ADH) and developmental duration was analyzed by the linear regression. The development threshold temperature (D0) and thermal summation constant (K) were determined using the slope of the linear regression equation and the y-intercept, respectively.
Gene expression study
Collection of intrapuparial samples
During the pupae stage at 16, 25 and 34°C constant temperatures, the intrapuparial age of S. dux was set to “zero” when approximately 50% pupae were observed, and sampled firstly from the individuals at this point, then intrapuparial samples were collected once every 24 h at 25 and 34°C, every 48 h intervals at 16°C due to pupae development lasted longer at this temperature, 8-10 intrapuparial were randomly taken at each examination time until 50% adult eclosion [43], and immediately kill with liquid nitrogen, then stored in 5 mL cryovial at -80°C for the subsequent gene expression study. The experiment at each temperature was repeated three times. Pupae development lasted 48 days, 12 days and 7 days at 16°C, 25°C and 34°C, respectively.
Total RNA Isolation and cDNA Synthesis
The TaKaRa MiniBEST Universal RNA Extraction Kit (TAKARA BIO INC) is used to the extraction of each intrapuparial samples RNA according to the instruction. The Evo-M-MLV RT Kit for qPCR with gDNA Clean (Accurate Biotechnology (Hunan) Co., Ltd. China) is used to the synthesis of complementary DNA (cDNA) from Total RNA (10ng-5μg) following the instructions of manufacturer. The reaction condition: 37°C for 15 min, 85°C for 5 s, and -20°C stored for the RT-qPCR.
Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
The candidate reference genes and DEGs involved were selected and downloaded from the NCBI based on the description of relevant literature of diptera insects [24, 25, 26, 27, 44, 45, 46, 47, 48, 49, 50]. These genes of S. dux were amplified and sequenced, and uploading to GenBank BLAST, compared with other insect species, which to verify their identity and confirmed was correctly detected. The RT-qPCR primers of these genes were prepared by the Primer Premier 5, the sensitivity and efficiency were checked by the relative standard curve using the 10-fold concentration dilution method and melting curve [43]. The characteristics of candidate gene and the primer were shown in Table 1. The RT-qPCR reactions were carried out using the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co., Ltd. China), and operated on a 7500 Real-Time PCR System (Applied Biosystems). Amplification conditions: 95◦C 30 s, 40 cycles followed 95◦C 5 s; 60◦C 30 s. All the samples were measured independently three times.
Data Analysis of Reference Genes and DEGs
The raw qPCR data was analyzed using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems), and the cycle threshold value was automatically determined. The reliability and number of candidate reference genes at different temperatures conditions was comprehensively evaluated by geNorm software program [51], which widely used to establish suitable reference genes for qPCR data normalization.
The relative quantification (RQ) of the DEGs was calculated using the 2-△△Ct method and standardized against the reference genes selected [43]. Fold change values of the DEGs (FC i.e. relative expression of gene) was normalized by the log-transform to base 2 [logFC = log2 (RQ)], and the age zero of S. dux intrapuparial was taken as a reference time to calculate the relative change in gene expression of other Intra-puparial at each temperature regime.
The effects of both the temperatures and ageing for the expression level of the Intra-puparial DEGs of S. dux were calculated by a two-way ANOVA. The normal distribution of relative expression of DEGs was conducted by the Kolmogorov–Smirnov and Q–Q plots. The relationship between LogFC values of the DEGs under different temperature conditions and Intra-puparial ageing of S. dux was analyzed through regression to obtain equations for estimating the PMImin. All statistical analysis was conducted by GraphPad Prism 6 (SCR: 002798), and OriginPro version 8.6 (SCR: 015636), SPSS version 22.0 (SCR: 002865).