Effect of Seasonal Temperature Variations on the Life Cycle Duration of Forensically Important Calliphorid Fly, Chrysomya saffranea (Bigot, 1877)

The present study deals with the effects of variation in temperature and humidity on the different life cycle stages of Calliphoridae fly Chrysomya saffranea. In India for first time, Chrysomya saffranea was recorded apart from Australia as a native place of this fly. In this study different life cycle stages of Chrysomya saffranea in different seasons were observed. In summer season when the average temperature ranged between 30.5°C and 33.2°C and the average humidity ranged between 12% to 19%, life cycle duration was completed in 220 h (9.17 days) from the depositing of eggs up to the eclosion of adults. But in rainy season when the average temperature ranged between 25.6°C and 28.9°C, and the average humidity ranged between 50% to 65%, life cycle duration was completed in 259 h (10.79 days), while in winter season when the average temperature ranged between 17.8°C and 24.4°C, and the average humidity ranged between 17% to 28%, life cycle duration was completed in 341 h (14.21 days) from eggs up to emergence of the adults. The temperature and humidity are the most important factors playing role in the larval development, decay and degradation of the cadavers. Therefore, the climatic fluctuations and environmental changes play an important role in the life cycle of Chrysomya saffranea. All these factors must be considered in the Post Mortem Interval determination.


Introduction
Forensic entomology is the use of insects and other arthropods as evidence in both civil and criminal investigations [1,2]. It deals with the insects associated with cadavers of human and animal remains to determine the Post-Mortem Interval (PMI) [3]. The wide range of this field can be divided into three subdivisions of urban, stored-product, and medico-legal forensic entomology [4]. Predominantly, blowfly larvae are the most common insects recovered from cadavers of humans; they can provide information on the conditions experienced by a body following death [5].
Chrysomya saffranea (Bigot, 1877), was recorded for first time in India [6], this fly also known as Steelblue fly or Australian blowfly [7]. In this study we used Chrysomya saffranea to study the effect of seasonal temperature on the life cycle duration and the time spent in the development of different stages of this fly and the impact of seasonal temperature on PMI determination. These blowflies of the genera Chrysomya (Diptera: Calliphoridae) are of considerable medical and sanitary importance since it is a myiasis producing agent in animals as well as humans. These flies are also important in forensic entomology since they can be used as biomarkers to estimate the Post Mortem Interval [8][9][10].
There are many factors which play an important role in the growth and development of arthropods, such as temperature, humidity, geographical location, seasons, decomposition time and habitat, which can affect the activity of the forensic flies and their life cycle [11][12][13][14][15][16]. Campobasso et al. [17] proved that the temperature and humidity are the most extrinsic factors playing an important role in the rate of postmortem decay and cadaver degradation.
Clark et al. [18] studied the insect activity at standard condition (21°C temperature and 30% humidity) and observed the insect activity within 96 h after death, while Campobasso et al. [17] reported that the oviposition can occur soon after death at favorable condition or at most within 2 days from the death.
Campobasso et al. [17] observed that the unfavorable temperature and humidity conditions can destroy the eggs easily, and found that in some cases the embryogenesis in eggs was slow down or stop showing a state of quiescence known as diapauses. However, the embryogenesis can resume normally once when the micro environmental condition become more favorable. Nielsen and Nielsen, [19] observed that the eggs of Chrysomya vicina (Diptera: Calliphoridae) did not hatch at temperature below 4°C while hatching of eggs and development of larvae occurred at 6°C to 7°C.
Considerations of critical environmental factors affecting the rate of decomposition are very important during the crime investigations. These factors include location of the body, temperature, general climate, time of year, insect activity, animal activity in the area, and the amount of rainfall [17,20,21].
To study the influence of fluctuating temperatures on insect development, one of five methods must be relied upon [22]. The first method involves transferring the insects from one temperature to another after a period of time has elapsed. The second is to record the temperatures of the natural environment by using a data logger or thermograph and the third method, similar to the first, is to transfer the insects to different temperatures according to a set pattern, The fourth method, which by no means emulates the natural environment, is to set up conditions where the insects develop at temperatures based on a gradient as a result of insect mobility. The fifth and final method is to set the desired conditions for the development and cycle the conditions every 24 h [22].
In this study we used the normal fluctuating temperatures at laboratory condition in different seasons and its effect on the life cycle duration, morphological parameters of different stages and the total life cycle duration from eggs depositing up to the adult eclosion.

Materials and Methods
Larvae of Calliphoridae species Chrysomya saffranea were collected from the dead body of cat at the garden of the Zoology Department at Dr Ambedkar Marathwada University Reserve (19° 522 483 N, 75° 192 123 E), Aurangabad City, Maharashtra State in India. Posteriorly, these larvae were reared in the laboratory by providing daily feed of buffalo`s fresh liver. Maggot culture was provided with fresh liver as a food till the prepupae stage. Prepupae were kept in 500 ml beakers containing dry soil which is required for the pupation. Adult flies which emerged out from pupae were reared in rearing boxes of size 22 × 12 × 10 inches in dimensions (length × width × depth respectively). Purity of culture was maintained by separating the eggs immediately after depositing from the female. Larvae and adult were dissected from the pure culture with the help of stereo-zoom microscope (ERMA Optical works, Tokyo, No. 44883) and light microscope (Magnus Trinocular Microscope MLX-DX, Olympus -India PVT. LTD. No. 4B525145) and identified according to the identification keys in [7,6,23].

Experimental Design
In these experiments eggs were collected with the help of fine brush directly after laying and reared in summer, and rainy winter seasons at laboratory condition. After maintaining the pure culture of Chrysomya saffranea, to insure the same environmental condition at laboratory three experiments were conducted at the same time. Three groups of 80 larvae separately transferred into three glass beakers 500 ml capacity; each group of larvae were daily fed on 50 gm of buffalo`s fresh liver till pupation. At the pupation time larvae were placed in small plastic tub 2 L capacity with 200 gm of dried soil which is necessary for the pupation. Life cycle duration of different stages and the morphological parameters were recorded daily. The temperature and the relative humidity were also recorded by using Hygrothermometer clock. The experiment was repeated three times seasonably.

Statistical Analysis
Statistical analysis was performed using the excel sheet, data were analyzed statistically by using Two-ways analysis of variance (ANOVA) and significance level at P ≤ 0.05 was used.

Life cycle in summer season
The life cycle duration of Chrysomya saffranea has been studied when the average temperature ranged between 30.5°C and 33.2°C and the average humidity ranged between 12% to 19% (Table 1)

Life cycle in rainy season
Life cycle duration of Chrysomya saffranea has been studied in rainy season when the average temperature and relative humidity were ranged between 25.6°C to 28.9°C, and 50% to 65% respectively (Table  1)

Life cycle in winter season
Life cycle duration of Chrysomya saffranea has been studied in winter season when the average temperature ranged between 17.8°C and 24.4°C, and 17% to 28% average humidity (Table 1), different life cycle stages and morphological parameters were recorded as below: Eggs: After depositing, the eggs were immediately collected; the average length width and weight of the eggs were 1.13 mm, 0.4 mm and 0.20 mg respectively. After 27 h of development the eggs hatched into 1st instar larvae.
Larvae (Maggot): The 1 st instar larvae took 30 h to develop, the average length, width and weight of the 1 st instar larvae were 4 mm, 1.3 mm and 7.6 mg respectively. The duration spent for 2 nd instar to reach the 3 rd instar was 30 h, and the average length, width and weight of the 2 nd instar larvae were 6 mm, 1.8 mm and 20.1 mg respectively. The 3 rd instar larvae took 42 h, and the average length, width and weight of 3 rd instar larvae were 9. The total life cycle duration of Chrysomya saffranea in summer was completed in 220 hrs (9.17 days) when the average temperature ranged from 30.5°C to 33.2°C and the average humidity ranged from 12% to 19%, but in rainy season it was completed in 259 h (10.79 days) when the average temperature ranged from 25.6°C to 28.9°C and the average humidity ranged from 50% to 65%, while in winter it was completed in 341 h (14.21 days) when the average temperature and humidity ranged from 17.8°C to 24.4°C and 17% to 28% respectively. Likewise, the shortest life cycle duration was recorded in summer, then followed by the rainy season and the longest life cycle duration was in winter season.
This result supported the previous study on Lucilia sericata (Diptera: Calliphoridae), when it was reared in different temperatures of 15°C, 17°C, 19°C, 20°C, 21°C, 22°C, 25°C, 28°C, 30°C and 34°C, and the shortest life cycle duration of 259 h was observed in 34°C, while the longest duration of 842 h was observed in 17°C but in 15°C, the period recorded up to post feeding was 340 h and there are no records of emergence of the adult in this lowest temperature [24].
Similarly in the previous study on the developmental duration of Chrysomya megacephala (Diptera: Calliphoridae), in rainy season and low constant temperature of 10°C and humidity 19%, reported that the total life cycle duration in rainy season was completed in 265 h ± 2 h (11.04 days ± 0.08 days) when the temperature ranged between 26°C and 29°C and humidity ranged between 35% and 50%, while in the low constant temperature 10°C ± 0.5°C the life cycle was completed in 609 h ± 4 h (25.38 days ± 0.16 days) indicating a delay in the life cycle by 14.37 days ± 0.13 days [25].
These results are in agreement with the data obtained in the present study. Similarly our current results are in agreement with another study on the effect of different temperature and humidity on the life cycle duration and morphological parameters of Chrysomya rufifacies (Diptera: Calliphoridae) in different seasons, it was reported that the life cycle of Chrysomya rufifacies in summer was completed in 241 ± 2.17 h (10.04 days ± 0.12 days) when the temperature ranged between 30.1°C and 37.2°C, but in the rainy season it was completed in 275 h ± 2.27 h (11.46 days ± 0.45 days), when the temperature ranged between 26.2°C and 30.1°C, while in winter the life cycle was completed in 318 h ± 2.45 h (13.25 days ± 0.25 days) when the temperatures ranged between 26.4°C and 18.2°C respectively [26]. Similar study on the developmental time of forensically important blow fly species (Diptera: Calliphoridae) Phormia regina (Meigen, 1826) at different constant minimum and maximum threshold temperatures (8°C to 32°C) reported that no development occurred at 12°C and below but in 14°C the total life cycle was of 45 days and at 26°C the total life duration was 13 days, while at 32°C the total life cycle was of 11.3 days [27].
Warren and Anderson, [28] compared the immature development of (Diptera: Calliphoridae) Protophormia terraenovae (Robineau-Desvoidy, 1830) at fluctuating temperatures of 4-28 and 9°C to 23°C and at their mean constant temperature 16°C, they reported that the development was fastest at the greater fluctuation and slowest at the constant temperature and showed similar percentages of development time in each stage. They conclude that the fluctuations above the mean enhance the developmental rate relatively more than temperatures below the mean can reduce the developmental rate.
Another study on the developmental rate of forensically important (Diptera: Calliphoridae) species Chrysomya albiceps at different   Higher temperatures generally support egg hatching and accelerate maturation of larvae which can double their size in few h and reduce the development time of Diptera. Once the Calliphoridae larvae have reached maximum length at the peak of feeding, they tend to decline progressively to about 75% of the length in the Calliphorid pre-adult cycle spent in post feeding and pupation [30,17].
The data obtained on PMI, length, width and weight of (Diptera: Calliphoridae) Chrysomya saffranea in the present study during three seasons (summer, Rainy and winter) were statistically analyzed for analysis of variance (ANOVA) and the P-value obtained are shown in Tables 2-5.   On the basis of the P-values, it can be stated that there was significant variation in PMI, length, width and weight of Chrysomya saffranea (Diptera: Calliphoridae) species among the various growth stage as well as among the three seasons.

Summer Rainy Winter
Thus, it can be concluded that different growth stages as well as different seasons were responsible for the variation in the morphological parameter of this species. The values obtained during ANOVA analysis clearly indicated significant variation among duration of PMI of different stages, length, width and weight of all the three species.