Development of Sarcophaga dux (Diptera: Sarcophagidae) at constant temperatures and differential gene expression for age estimation of intrapuparial

Background Sarcophaga dux (Diptera: Sarcophagidae) is a necrophagous esh y with potential forensic value in estimating minimum postmortem interval (PMI min ). The basic developmental data and precise intrapuparial age estimates are signicant for PMI min estimationof application entomological data in legal medicine. The development parameters of S. dux at seven constant temperatures from 16°C to 34°C were investigated by rearing using pig lung in the articial climate box. The appropriate reference genes and intrapuparial differentially expressed genes (DEGs) of S. dux at constant temperatures 34, 25 and 16°C were selected and analyzed using RT-qPCR for more precisely age estimations. The developmental durations of S. dux at 16, 19, 22, 25, 28, 31 and 34°C from larviposition to adult eclosion were 1478.6±18.3, 726.1±15.8, 538.5±0.9, 394.1±9.5, 375.6±10.8, 284.1±7.3, and 252.5±6.1 h, respectively. The thermal summation constant of S. dux was 5341.71±249.29 degree hours, and the developmental threshold temperature was 12.266±0.35°C. The most reliable reference genes under intrapuparial and different temperature in our study were: GST1 and 18S rRNA for 34°C group, GST1 and RPL49 for 25 °C group, and 18S rRNA and 28S rRNA for 16 °C group(cid:0)the four differential expression genes (Hsp60, A-alpha, ARP, RPL8) can be used to more precisely intrapuparial age estimation of S. dux. age estimations of S. dux intrapuparial. The result from this study can make contributes to the use of S.dux for estimating min stability of gene expression normalization at different temperatures and intrapuparial developmental ages. The four DEGs of S. dux including heat shock proteins 60 (Hsp60), sarcocystatin A-alpha (A-alpha), 60S acidic ribosomal protein 8 (RPL8), acidic ribosomal protein (ARP) were used to intrapuparial age estimations. This work provides an important basic developmental data and a more precise intrapuparial stage age estimates way of S. dux, improves the value of this insect of PMI min estimation in forensic investigations. our developmental time of instar of dux is by Kumara et al. (2013) at uctuation temperature 28.9 ± 1.2°C in [55], but faster development at third instar and post-feeding and slower development at pupa stages compared to Kumara et al. (2013) experiments.

(Diptera: Sarcophagidae), a synanthropic esh y species in many parts of the world, is of medical importance as a vector of disease or parasitic, myiasis-producing agent [34], and as forensically important species with the necrophagous habits, it was found to colonize corpses of a male infant in Malaysia [35]. This species widely distributed around the world, such as Oriental region. Previous entomology investigations on S. dux have focused on the description of important morphological features in each developmental stage [34], molecular identi cation analysis by mitochondrial cytochrome oxidase gene subunits I and II (COI and COII) sequences [36], the human death investigations in forensic entomology [34]. However, there are still lacking a detailed investigation of S. dux development. Collecting precise basic data on the growth and development in speci c regions and the analysis of DEGs for a more precise intrapuparial age estimations of insect species has been emphasized as a way to ensure accurate calculation of PMI min [18,24,37].
In the present study, the adult of S. dux were collected from a pig carcasses and established a laboratory colony, the developmental data of S. dux at 16, 19,22,25,28,31 and 34°C from larviposition to adult emergence such as accumulated temperature, larval body length and developmental duration were collected and measured to establish developmental models for estimation the PMI min . In addition, the ten candidate reference genes of S. dux including 60S acidic ribosomal protein P0 (RPLP0), 28S ribosomal RNA (28S rRNA), cAMP-dependent protein kinase (PKA), 18S ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glutathione S-transferases 1 (GST1), α-tubulin (α-TUB), Elongation factor 1-alpha (EF1α), 40S ribosomal protein S3 (RPS3), ribosomal protein L49 (RPL49) were sequenced and rstly evaluated the stability of gene expression normalization at different temperatures and intrapuparial developmental ages. The four DEGs of S. dux including heat shock proteins 60 (Hsp60), sarcocystatin A-alpha (A-alpha), 60S acidic ribosomal protein 8 (RPL8), acidic ribosomal protein (ARP) were used to intrapuparial age estimations. This work provides an important basic developmental data and a more precise intrapuparial stage age estimates way of S. dux, improves the value of this insect of PMI min estimation in forensic investigations.

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 ies were collected, and species determination was performed by forensic entomologist using the adult identi cation key [38], and con rmed 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 ies 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 su cient amount of fresh pig lung were provided until pupation. After eclosion, the adult of S. dux were reared once again ve generations to obtain a purebred laboratory colony and increase the number of adult ies.

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 arti cial 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  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 rst 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, ve 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 in uence 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 tted 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 (D 0 ) 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 rstly 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. 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 ampli ed and sequenced, and uploading to GenBank BLAST, compared with other insect species, which to verify their identity and con rmed was correctly detected. The RT-qPCR primers of these genes were prepared by the Primer Premier 5, the sensitivity and e ciency 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 quanti cation (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 = log 2 (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).

Development Duration and Isomorphen Diagram
Between 16 and 34°C, S. dux can complete the development process from larviposition to eclosion, in this range of temperature, the durations of each developmental phase decreases with temperature increasing. The total developmental time was 1478.6 h and 252.5 h at16 and 34°C ( Table 2).
The isomorphen diagram was completed using the development time of each life stages and different temperatures conditions (Fig. 1). The time of all developmental duration gradually shortened with the increasing of growth temperature in 16-34°C.

Thermal Summation Models
The thermal summation models of all developmental process, including total developmental duration, were plotted according to the development time and accumulated degree hours using linear regression analysis (Fig. 2). The thermal summation constants (K) and the developmental threshold temperatures (D 0 ) of S. dux during the whole development process were calculated by the thermal summation model, which were 5341.71±249.29 degree hours and 12.266±0.35°C, respectively (Table 3).

Changes in Larval Body Length and Isomegalen Diagram
The relationship between body length changing of the larva period and development time at different temperatures was shown in Fig. 3. In 16-34°C temperature, with the temperature increasing, the developmental rate of larvae increasing. The equations describing the body length changing of the larva and development time after larviposition were plotted through a regression analysis. The determination coe cient (R 2 ) of the equation at each temperature was greater than 0.95, which indicated that the development data was t well using the regression analysis ( Table 4). The isomegalen diagram show the relationships between the changes of the larval body length after larviposition to peak (3-16cm) and development time under different temperatures (Fig. 4).

Total RNA Quality
The concentration of each intrapuparial samples RNA was quantitated spectrophotometrically (NanoDrop 2000, Thermo, USA). The 1% agarose gel electrophoresis was used to the detection of RNA integrity. The correlation coe cient and PCR e ciency are shown in Table 1.

Stability of Reference Gene Expression
The reliability of reference genes at 34°C, 25°C and 16°C temperatures conditions of different Intra-puparial samples was calculated by geNorm methods, and the results was shown in Supplementary Material 1 and Fig. 5. The lower ranking indicateds more stable expression. The minimum number of candidate reference genes were determined using the pair-wise variation value (V) by the geNorm, V 2 / 3 below 0.15 indicated that two reference genes would be suitable in this experiment condition (Fig. 6), and the most reliable reference genes for this research of S. dux pupae at different constant temperatures were: GST1 and 18S rRNA for 34°C group, GST1 and RPL49 for 25 °C group, and 18S rRNA and 28S rRNA for 16 °C group.

Overall and Detailed Expression Pro les of the DEG
We selected and validated the expression pro les of the four DEGs (Hsp60, A-alpha, ARP, RPL8), which have potential represented developmental-speci c and statistical signi cance for accurately predicting intrapuparial age of S. dux. The overall expression pro les of four DEGs at three constant temperatures were averaged, and plotted against the developmental percentage of S. dux Intra-puparial (Fig. 7). The results shown that the expression pro les of Hsp60 increased at the puparial development process the expression level of A-alpha rst increased steadily until reached its maximum, and then tended to increase steadily until completion puparial development and the expression pro les of ARP has an opposite tendency with A-alpha, the expression level of RPL8 were in a state of uctuation. The simulation equations of fourth-order polynomials were used to describe the relationship between the overall expression pro les of four DEGs at three constant temperatures and developmental percentage of Intra-puparial, which conducted using a nonlinear regression analysis ( Table 5).
The simulation equations (Table 6) and curves (Fig. 8) describing the detailed gene expression and Intra-puparial age at constant 34, 25 and 16°C were obtained using a nonlinear regression analysis, which adopting the differential gene expression levels as the independent variable and Intra-puparial age at each temperature as the dependent variable. The in uence of rearing temperature and Intra-puparial age on the expression of DEGs was tested by a two-way ANOVA analysis (Supplementary Material 2), the result showed that the interaction of age×temperature has a signi cant in uence on the expression pro les of S. dux Intra-puparial DEGs.

Discussion
Although many studies have emphasized the importance of S. dux in forensic science, the development durationre data available is limited. In this study, the basic developmental data of S. dux at seven constant temperatures from 16°C to 34°C was reported, and the age indication index to use S. dux for estimation PMImin including body length changing of the larva period, accumulated degree hours and duration of development was provided. The duration of development of S. dux from larviposition to adult eclosion at 16, 19 [34]. In addition, the developmental durations of this species from the larvae to pupariation were 72-96h in ranging between 27.1-29.8°C in Thailand [52], 168h at 29.5°C in Guam [53], 196-244h at 25°C in South Africa [54], and 114, 108, 86h at 25, 28, 31°C in our study. The developmental time of second instar of S. dux that we measured (19h) at constant temperatures 28°C is similar with the reported (19h) by Kumara  The three development models of isomegalen diagram, isomorphen diagram, and thermal summation model were established basis on the developmental data obtained for facilitating the estimation of PMImin. The application range of isomorphen diagram widespread, and can calculate the time of different stages of development at various temperatures, but does not include the variability between events [7]. The isomegalen diagram can clearly to calculate the relationships between the changes of the body length of larval after larviposition to peak and development time under different temperatures [11,12,13]. The thermal summation model is more applicable to uctuating temperatures environments [12,14]. The developmental threshold temperatures (D 0 ) of S. dux at total developmental process in our study were calculated were 12.266±0.35°C using thermal summation model, and this species has been recorded in Nepal at altitudes of 2,000 m, indicated they are well adapted to low temperatures and high altitudes [56].
The biological and abiotic factors can affect the developmental process of necrophagous insects, such as, Maggots hardly reared individually because of the gregarious behavior [57]. Too much food will lead to delayed development and even larvae death [58], and starvation can result adults smaller and early larvae pupariation [59]. The different laboratory equipment and conditions, including photoperiod and humidity impact the development process, but temperature is still main factor affecting growth and development of insect [12,16,17], thus, evaluated the above factors and detailed data recording and careful experimental design to establish a development duration of fly should be emphasized.
RT-qPCR is a reliable and sensitive technology to explore the DEGs in many complex biological progresses [60]. However, the key to obtain accurate target gene results with stable standard gene is normalization, and the normalization of qPCR data requires the appropriate reference genes [61]. The studies have suggests that in different environmental conditions or developmental stages, the stably reference genes are differently [62]. Moreover, standardizing experimental shown that two or more reference genes will be su cient than single reference gene [63]. In this study, the stability of ten candidate reference genes at different pupae developmental stages and constant temperatures of S. dux was rstly evaluated for gene expression research.
During y metamorphosis, the analysis of differentially expressed genes (DEGs) may provide a way to estimate age of forensically important y in death investigations [64]. DEGs have the advantage in objective data. Importantly, its method is easier to master than morphology. Therefore, it would be bene cial to have an alternative way which could be carried out by a standard forensic laboratory, leading to produce more standardized results. Until now, the forensically relevant species involved in DEGs studies including C. vicina, L. sericata, and S. peregrine, the DEGs including bcd, sll, cs, ace, hsp90, hsp 60, usp, ecr, rop-1, w, 15_2, 2014192, actin, arylphorin receptor [24,25,26,27,28,29,43]. There are lots of other forensically important species need to be investigated.
In this study, the expression level of S. dux Intra-puparial four DEGs, including Hsp60, RPL8, A-alpha, ARP were rstly evaluated at 34°C, 25°C and 16°C constant temperatures for Intra-puparial age estimation. The results indicated that for the same DEGs, the expression pro les at three temperature conditions were similar, and with the increase of temperature, puparial development time was shortened, which led to the faster change of gene expression. The research show that in the early embryonic and pupa stage, the sarcocystatin A (A-alpha) gene was activated, indicating that sarcocystatin A was involved in the morphogenesis of the Sarcophaga larvae and adult structures [65], and the expression pro les of A-alpha has the potentially used to more precisely age estimation of S. dux intra-puparial in our study. The potential of hsp 60 gene used to estimate the puparia age of y has been proved by  and this study [27]. In the ideal of circumstances, the bestsuited gene for indicating insect age ought to exhibit the continuous increase or decrease trend in expression level and development time, resulting in the expression pro les would match with the sole corresponding development time. But in fact, it is hard to nd out the genes that accord with this criterion because the insects change dramatically during pupal stage. Therefore, combining DEGs is an effective way for precisely age estimation of intra-puparial.
Recent studies have shown that geographically variation can affect the rates of insect developmental, such as C. vicina and L. sericata [66,67,68]. The geographically separate populations may result a different discoveries of the same species. Therefore, the precisely PMImin estimation at speci c crime scene by necrophagous sarcophagids requires collection the development data from different geographical regions in the world. Furthermore, the effect of geographic variation on DEGs expression pro le is still unclear and needs further study.

Conclusions
The basic developmental data and precise intrapuparial age estimates are signi cant for PMI min estimation of application S. dux in crime investigations. In present study, the development parameters of S. dux at seven constant temperatures from 16°C to 34°C in body length changing of the larva period, accumulated degree hours and duration of development were investigated, the appropriate reference genes in intrapuparial and different temperature of S. dux were selected for the accuracy of RT-qPCR, the four differential expression genes (Hsp60, Aalpha, ARP, RPL8) has the potentially used to more precisely age estimation of S. dux intra-puparial.     Figure 1 Isomorphen diagram of Sarcophaga dux. The duration of each development milestone (first-ecdysis, second-ecdysis, wandering, pupation, and eclosion) was plotted with the time from larviposition to the onset of each milestone. Each curve corresponds to a developmental milestone, and the error bar is the standard deviation of each milestone.

Figure 2
Thermal summation models of five developmental stages and total development duration of Sarcophaga dux, the solid line represents the regression line. Dashed line represents 95% confidence interval.