Linking Seasonal Temperature Variations with Laboratory-Derived Development Data for Chrysomya rufifacies (Macquart): A Case for Myiasis

Abstract

Background: The aging of fly larvae is primarily determined by their temperature-dependent growth rates, a concept widely applied in forensic entomology to estimate the minimum postmortem interval using the accumulated degree day/hour (ADD/ADH) method. Method: This study adapted the same approach for veterinary entomology, offering insights into how accumulated degree day (ADD) can be used to estimate both the number and timing of fly generations in nature. This study details a method for identifying the pupation landmarks of Ch. rufifacies (Macquart) by characterising seven distinct pupal stages over time. Following this, ADD values were calculated for each life stage using developmental data collected from two types of ovine muscle: muscle with fat and muscle without fat, at two temperatures: 24 ± 1 °C for autumn and 30 ± 1 °C for summer, reflecting typical seasonal conditions in southwestern Australia. Results: This study also provided a graphical illustration of how to estimate the number of fly generations emerging during a season, based on daily temperature data from autumn and summer in southwestern Australia for the 2023/2024 period. Conclusions: This approach highlights the value of developmental data and ADD methods in veterinary entomology, offering a robust framework for understanding fly population dynamics for effective myiasis control strategies.

1. Introduction

The major Calliphoridae species causing traumatic myiasis in sheep in Australia are Lucilia cuprina (Wiedemann) and Chrysomya rufifacies (Macquart), with the occasional strike by Lucilia sericata (Meigen). Lucilia cuprina and L. sericata are considered primary species, capable of causing myiasis wounds through the feeding activities of larvae on fresh tissues. In contrast, Ch. rufifacies is a secondary species attracted only to necrotic tissues in wounds that are already infested by one or more primary species [1,2,3].

Primarily, fly development data are utilised to estimate the minimum postmortem interval in death investigations and to verify periods of cruelty or neglect in animals and humans afflicted by fly larval infestations in wounds [2,3]. Additionally, previous studies have highlighted that these development data can serve as baseline information for forecasting the number of fly generations that may emerge in a season [4,5]. The accumulated degree day/hour (ADD/ADH) method is the primary technique employed for forensic and veterinary-related temporal predictions involving flies [4,5]. This method is based on the linear relationship between development rates and temperature, assuming a constant increase in development rates with rising temperatures [6]. However, this increase is constrained by species-specific upper and lower threshold temperature limits [6].

A summary of previous laboratory-generated development datasets for Ch. rufifacies by Bambaradeniya et al. (2023) [4] revealed 17 studies conducted across different regions of the world, utilising various tissues and constant temperatures. However, a limitation in all these publications was the absence of any practical application of this data, such as estimating the postmortem interval, assessing neglect periods, or forecasting number of emerging seasonal fly generations. Another significant limitation was that none of the previous studies recorded the pupation landmarks of Ch. rufifacies, which constitutes approximately 50% of the total developmental time of the fly life cycle [7].

The present study addresses these limitations by generating a development dataset for Chrysomya rufifacies including these pupation landmarks at two temperatures: 24 ± 1 °C and 30 ± 1 °C, corresponding to autumn and summer temperatures in southwestern Australia. These baseline data were utilised to forecast emerged fly generation numbers using an ADD model in response to seasonal temperature changes in southwestern Australia for the period of 2023/2024.

2. Materials and Methods

The methodology of this study comprised four key steps: baseline studies, complete development datasets, Degree Day and Accumulated Degree Day calculations, and practical application. The diagram below provides a detailed overview of the methodology (Figure 1).

2.1. Step 1—Baseline Studies

2.1.1. Development Study

The eggs required for the development studies were obtained from laboratory-reared Ch. rufifacies colonies maintained under a 12:12 light–dark photoperiod at approximately 23 °C in an insectary. The emerging adults were housed in insect cages (60 cm × 60 cm × 60 cm) and provided with sugar and milk solutions ad libitum, while the larvae were fed swine muscle pieces. Gravid females were offered swine blood meals to trigger oviposition, followed by swine muscle pieces provided as the oviposition substrate.

Approximately 2 kg each of ovine skeletal muscle were obtained from the thigh area of host animals for the study and categorised into muscles with fat (30–50% of total weight) and without fat (all the visible fat tissues were removed). These tissues were cut into 100 g cube-shaped pieces, stored in Ziplock bags, and refrigerated at 2 °C.

Development studies were conducted using eggs from the first three captive generations of Ch. rufifacies to maintain genetic heterogeneity. Egg clusters (50–60 eggs) from this colony were placed on the two tissue types in containers filled with sand and covered with a mesh lid to prevent larvae from escaping. Three replicate studies were conducted for each temperature regime. The autumn temperature was defined as March to May 2023 (average, 24 ± 1 °C), and summer as December 2023 to February 2024 (average 30 ± 1 °C). These are the seasons when fly strike on sheep is most prevalent. These daily temperatures were obtained from the Bureau of Meteorology, Australia website. Relative humidity (RH) and photoperiod were maintained at 70% and 12:12 (L:D) hours within a climatic growth chamber (Fisher & Paykel^® (Waltham, MA, USA) with a purpose-built temperature and humidity regulator).

The initial egg hatch time was recorded, after which the two largest larvae were hot-water-killed and preserved in 70% ethanol every 3 h until larvae reached the post-feeding 3rd instar stage. Subsequently, the same preservation technique was employed every 6 h for post-feeding 3rd instar larvae until pupation, followed by pupae being sampled every 8 h interval until adult emergence. Their developmental stages were then examined microscopically, observing the number of respiratory slits visible in the posterior spiracles. These data were used to develop a detailed developmental timeline for the species across two temperatures and two types of ovine tissue.

2.1.2. Pupation Landmark Study

The development time periods were further expanded to include the pupation landmark stages. However, due to a lack of criteria for determining pupation stages in the previous literature, these stages were instead identified by analysing the character changes within the pupae.

The pupae were extracted and preserved at 8 h intervals under the two temperature regimes. Specifically, one external: puparium, and six internal pupal characteristics: body segments, compound eye, antennae, wings, legs, and abdomen, were analysed. The outer appearance was photographed, and then the pupae were dissected to capture the internal characteristics. The colour changes of each characteristic were assessed over time using the Munsell constant hue system (https://www.andrewwerth.com/aboutmunsell/, accessed on 16 July 2024) under a background light of 400 lux. A character checklist was then generated for each temperature to categorise each pupa into one of four landmark stages: precryptocephalic pupal stage, cryptocephalic pupal stage, phanerocephalic pupal stage, and pharate adult [8,9,10].

The criteria for distinguishing these stages were based on previous studies. The precryptocephalic pupal stage was identified by larval–pupal apolysis, where the prepupa remains tightly attached to the inner surface of the puparium, retaining most larval features, such as body segmentation and the presence of the cephalopharyngeal skeleton. The cryptocephalic pupal stage followed, characterised by the development of legs and wings. This was succeeded by the phanerocephalic pupal stage, where the pupal body is distinctly divided into three segments: head, thorax, and abdomen. Finally, the pharate adult stage represents the adult form within the puparium, with epidermal cells separated from the pupal cuticle [8,9,10].

2.2. Step 2—Complete Development Data Sets

Development datasets at 24 ± 1 °C and 30 ± 1 °C were compiled, encompassing the completion periods for each life stage, including four pupation landmarks. The average duration, calculated for the two tissue types, were converted into days for the purpose of further calculations based on the accumulated degree days.

2.3. Step 3—Degree Day (DD) and Accumulated Degree Day (ADD) Calculations

The total number of fly generations that emerged during the autumn and summer seasons of 2023/2024 was calculated using the Accumulated Degree Day (ADD) method.

The lower developmental threshold temperature (LDT) for Ch. rufifacies was derived from previous studies summarised by Bambaradeniya et al. (2023) [4]. Two values for LDT were identified: 9.50 °C from a study in Thailand [11] and 11.96 ± 0.38 °C from a study in China [12]. Based on the comprehensiveness of the latter study, 11.96 °C was chosen and rounded to 12 °C for simplicity in this study.

The degree day and accumulated degree day calculations were based on following two steps (Figure 2).

2.4. Step 4—Practical Application

Two graphs were developed to track the progression of the developmental stages across seasons by correlating the degree days calculated based on daily temperatures of each season with the required degree days for the emergence of successive fly generations.

3. Results

3.1. Development Study

The development time periods of Ch. rufifacies at the two temperatures across the two tissue types are presented in

Table 1. Time (mean days ± SE, n = 3) needed by Ch. rufifacies to complete each developmental stage when reared on different tissues: ovine skeletal muscle—without fat, and ovine skeletal muscle—with fat, at two temperatures: 24 ± 1 °C and 30 ± 1 °C. The average development time (days) for both tissue types also are given.

Temperature	Tissue Type	Eggs	1st Instar	2nd Instar	3rd Instar	3rd Instar Post Feeding	Pupa
24 ± 1 °C	Ovine skeletal muscle (without fat)	0.57 ± 0.06	1.42 ± 0.08	1.39 ± 0.40	1.34 ± 0.08	2.85 ± 0.20	6.47 ± 0.07
	Ovine skeletal muscle (with fat)	0.49 ± 0.10	1.39 ± 0.09	1.05 ± 0.40	1.41 ± 0.08	2.84 ± 0.48	6.46 ± 0.13
Ave. development times in days		0.53	1.40	1.21	1.39	2.84	6.46
30 ± 1 °C	Ovine skeletal muscle (without fat)	0.44 ± 0.18	0.67 ± 0.23	0.63 ± 0.19	0.24 ± 0.04	2.04 ± 0.35	3.59 ± 0.25
	Ovine skeletal muscle (with fat)	0.41 ± 0.11	0.60 ± 0.15	0.53 ± 0.20	0.28 ± 0.11	1.31 ± 0.24	3.89 ± 0.18
Ave. development times in days		0.42	0.63	0.58	0.25	1.67	3.74

. Overall, at 24 ± 1 °C, the total development time was longer for both tissue types compared to 30 ± 1 °C. To calculate degree days (DD), the time values were averaged to obtain an overall value for ovine muscle collectively (Table 1).

3.2. Pupation Landmarks

The four pupation landmarks were identified based on the morphological changes observed using the seven distinct characters: puparium, body segments, compound eye, antennae, wings, legs, and abdomen. These changes are illustrated with corresponding photographs (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9). Additionally, the time required to reach each landmark at two different temperatures was determined by summarising these morphological changes, as outlined in the checklist provided in Table 2 and Table 3 for 24 ± 1 °C and 30 ± 1 °C, respectively. Similar to the previous study of the same research group, Table 4 presented the timing for each pupation landmark at two temperatures, based on the characterisation of the stages [10].

• Puparium (Figure 3: Character 1)

Figure 3. Character 1: Development of puparium (identified stages: P1–P4).

Figure 3. Character 1: Development of puparium (identified stages: P1–P4).

The external appearance of the puparium maintained a barrel shape throughout the pupation period with clearly visible segmentation on its surface (P1, P2, P3, and P4). The colour of the puparium did not change notably, remaining consistently brown (7.5R 5/16). However, during the later part of pupation, specifically in the pharate adult stage, the puparium exhibited a shrivelled (P4) and sunken appearance compared to the earlier stages (P1, P2, and P3).

• Body Segments (Figure 4: Character 2)

Figure 4. Character 2: Development of the body segments (Identified stages: B1–B6).

Figure 4. Character 2: Development of the body segments (Identified stages: B1–B6).

Internally, the puparium initially had a 12-segmented tapered body with several outer, brown-coloured spines on top (B1, B2). As the body separated into three segments—head, thorax, and abdomen—these spines began to disappear (B3). Gradually, the white-coloured body segments (B3, B4) turned light brown (B5) (2.5YR 5/10) and finally appeared black (B6) (N1), with the setae becoming more visible and exhibiting robust patterns.

• Compound Eye (Figure 5: Character 3)

Figure 5. Character 3: Development of compound eyes (Identified stages: E1–E7).

Figure 5. Character 3: Development of compound eyes (Identified stages: E1–E7).

The separation of eye margins from the rest of the head appeared without a colour change in the head (E1, E2). However, later, the eye colour changed, from creamy white (E2), to red brown (E3, E4, E5), (7.5R 4/12), and finally to red (E6, E7), (5R 5/18). Ch. rufifacies eyes were not as distinctly separated from the rest of the head region (E7).

• Antennae (Figure 6: Character 4)

Figure 6. Character 4: Development of antennae (Identified stages: A1–A7).

Figure 6. Character 4: Development of antennae (Identified stages: A1–A7).

As the cephalopharyngeal skeleton disappeared, the anterior end of the head became completely sealed before the antennae appeared (A1, A2). The maturation of antennae underwent colour changes from brown (7.5R 5/16) to black (A4, A5, A6, A7) (N1).

• Wings (Figure 7: Character 5)

Figure 7. Character 5: Development of wings (identified stages: W1–W6).

Figure 7. Character 5: Development of wings (identified stages: W1–W6).

Wings appeared with the formation of the three body segments after the legs emerged (W1, W2). The colour of the wings changed from white to light brown (2.5YR 5/10) (W3, W4), then to brown (W5), and finally to black (N1), (W6), with intricate veins becoming visible.

• Legs (Figure 8: Character 6)

Figure 8. Character 6: Development of legs (Identified stages: L1–L6).

Figure 8. Character 6: Development of legs (Identified stages: L1–L6).

The leg formation initiated as an inflated tube-like mass from the head and thorax separating groove (L1, L2). The overall body length eventually extended to about two-thirds of the pupal length (L3). The colour of the legs changed from creamy white (L1, L2) to light brown (L3), then to dark brown (L4), and finally to black (L5, L6). The well-defined legs consisted of the femur, tibia, and tarsi, with dark bristles (L6).

• Abdomen (Figure 9: Character 7)

Figure 9. Character 7: Development of abdomen characters (Identified stages: AB1–AB6).

Figure 9. Character 7: Development of abdomen characters (Identified stages: AB1–AB6).

Three lines of bristle points appeared on the abdomen after segmentation (AB4), and the colour changed from creamy white (AB3) to brown AB4). Finally, the colour of the abdomen turned black (AB5, AB6).

Table 2. The checklist of identified characters over time within four pupation landmark phases of Ch. rufifacies at 24 ± 1 °C.

Age (D)	24 ± 1 °C
	Puparium (Figure 3)	Body Segmentation (Figure 4)	Compound Eye (Figure 5)	Antennae (Figure 6)	Wings (Figure 7)	Legs (Figure 8)	Abdomen (Figure 9)
0.00	P1	B1	E1	A1	W1	L1	AB1
0.33	P2	B2	E2	A2	W2	L2	AB2
0.66	P2	B3	E2	A3	W2	L2	AB2
1.00	P3	B4	E3	A4	W3	L3	AB3
1.33	P3	B4	E3	A4	W3	L3	AB3
1.66	P3	B4	E3	A4	W3	L3	AB3
2.00	P3	B4	E3	A4	W3	L3	AB3
2.33	P3	B4	E3	A4	W3	L3	AB3
2.66	P3	B4	E4	A5	W3	L4	AB3
3.00	P3	B5	E4	A5	W4	L4	AB5
3.33	P3	B5	E4	A5	W4	L4	AB5
3.66	P3	B5	E5	A5	W5	L4	AB4
4.00	P4	B6	E6	A6	W5	L5	AB5
4.33	P4	B6	E6	A6	W6	L6	AB5
4.66	P4	B6	E7	A7	W6	L6	AB6

Table 2. The checklist of identified characters over time within four pupation landmark phases of Ch. rufifacies at 24 ± 1 °C.

Age (D)	24 ± 1 °C
	Puparium (Figure 3)	Body Segmentation (Figure 4)	Compound Eye (Figure 5)	Antennae (Figure 6)	Wings (Figure 7)	Legs (Figure 8)	Abdomen (Figure 9)
0.00	P1	B1	E1	A1	W1	L1	AB1
0.33	P2	B2	E2	A2	W2	L2	AB2
0.66	P2	B3	E2	A3	W2	L2	AB2
1.00	P3	B4	E3	A4	W3	L3	AB3
1.33	P3	B4	E3	A4	W3	L3	AB3
1.66	P3	B4	E3	A4	W3	L3	AB3
2.00	P3	B4	E3	A4	W3	L3	AB3
2.33	P3	B4	E3	A4	W3	L3	AB3
2.66	P3	B4	E4	A5	W3	L4	AB3
3.00	P3	B5	E4	A5	W4	L4	AB5
3.33	P3	B5	E4	A5	W4	L4	AB5
3.66	P3	B5	E5	A5	W5	L4	AB4
4.00	P4	B6	E6	A6	W5	L5	AB5
4.33	P4	B6	E6	A6	W6	L6	AB5
4.66	P4	B6	E7	A7	W6	L6	AB6

Table 3. The checklist of identified characters over time within four pupation landmark phases of Ch. rufifacies at 30 ± 1 °C.

Age (D)	30 ± 1 °C
	Puparium (Figure 3)	Body Segmentation (Figure 4)	Compound Eye (Figure 5)	Antennae (Figure 6)	Wings (Figure 7)	Legs (Figure 8)	Abdomen (Figure 9)
0.00	P1	B1	E1	A1	W1	L1	AB1
0.33	P2	B2	E2	A2	W2	L2	AB2
0.66	P2	B3	E2	A3	W2	L2	AB2
1.00	P2	B3	E2	A3	W3	L3	AB2
1.33	P3	B4	E3	A4	W3	L3	AB3
1.66	P3	B4	E3	A4	W3	L3	AB3
2.00	P3	B4	E3	A4	W3	L3	AB3
2.33	P3	B5	E4	A5	W4	L4	AB4
2.66	P3	B5	E5	A5	W4	L4	AB4
3.00	P4	B6	E6	A6	W5	L5	AB5
3.33	P4	B6	E7	A7	W6	L6	AB6

Table 3. The checklist of identified characters over time within four pupation landmark phases of Ch. rufifacies at 30 ± 1 °C.

Age (D)	30 ± 1 °C
	Puparium (Figure 3)	Body Segmentation (Figure 4)	Compound Eye (Figure 5)	Antennae (Figure 6)	Wings (Figure 7)	Legs (Figure 8)	Abdomen (Figure 9)
0.00	P1	B1	E1	A1	W1	L1	AB1
0.33	P2	B2	E2	A2	W2	L2	AB2
0.66	P2	B3	E2	A3	W2	L2	AB2
1.00	P2	B3	E2	A3	W3	L3	AB2
1.33	P3	B4	E3	A4	W3	L3	AB3
1.66	P3	B4	E3	A4	W3	L3	AB3
2.00	P3	B4	E3	A4	W3	L3	AB3
2.33	P3	B5	E4	A5	W4	L4	AB4
2.66	P3	B5	E5	A5	W4	L4	AB4
3.00	P4	B6	E6	A6	W5	L5	AB5
3.33	P4	B6	E7	A7	W6	L6	AB6

Table 4. Time (days) attained by Ch. rufifacies at 24 ± 1 °C and 30 ± 1 °C to complete each landmark stages of pupation [10].

Pupation Landmark	Characteristics	Ch. rufifacies
		24 ± 1 °C	30 ± 1 °C
Pre-cryptocephalic pupal stage	The larval hypodermis remains attached to the puparium, while the cephalopharyngeal skeleton is firmly connected to the prepupa at the anterior end. Notable differences in the shape and color of the puparium were observed compared to later stages, as it retained the larval form and exhibited a lighter brown hue (Figure 10).	0.33	0.33
Cryptocephalic pupal stage	The initial formation of legs appeared as a swollen, tube-like structure in the midsection of the body at this stage. As development advances, a distinct groove emerges, separating the head and thorax regions. The cephalopharyngeal skeleton is still visible, but it is less firmly attached to the puparium compared to the preceding stage (Figure 10).	0.66	0.66
Phanerocephalic pupal stage	The respiratory horns, situated within the groove between the head and thorax regions, become visible. As development advances, the compound eyes and antennae emerged on the head, wings form on the thorax, and setae develop on the abdomen (Figure 10).	3.00	2.00
Pharate adult	In this final phase, the insect matures into an adult, undergoing body tanning and the hardening of visible setae and bristles. The pupal cuticle, which encases the developing adult, begins to shed as the maturation process progresses (Figure 10).	1.00	0.66

Table 4. Time (days) attained by Ch. rufifacies at 24 ± 1 °C and 30 ± 1 °C to complete each landmark stages of pupation [10].

Pupation Landmark	Characteristics	Ch. rufifacies
		24 ± 1 °C	30 ± 1 °C
Pre-cryptocephalic pupal stage	The larval hypodermis remains attached to the puparium, while the cephalopharyngeal skeleton is firmly connected to the prepupa at the anterior end. Notable differences in the shape and color of the puparium were observed compared to later stages, as it retained the larval form and exhibited a lighter brown hue (Figure 10).	0.33	0.33
Cryptocephalic pupal stage	The initial formation of legs appeared as a swollen, tube-like structure in the midsection of the body at this stage. As development advances, a distinct groove emerges, separating the head and thorax regions. The cephalopharyngeal skeleton is still visible, but it is less firmly attached to the puparium compared to the preceding stage (Figure 10).	0.66	0.66
Phanerocephalic pupal stage	The respiratory horns, situated within the groove between the head and thorax regions, become visible. As development advances, the compound eyes and antennae emerged on the head, wings form on the thorax, and setae develop on the abdomen (Figure 10).	3.00	2.00
Pharate adult	In this final phase, the insect matures into an adult, undergoing body tanning and the hardening of visible setae and bristles. The pupal cuticle, which encases the developing adult, begins to shed as the maturation process progresses (Figure 10).	1.00	0.66

Figure 10. Inner and outer morphological changes of Ch. rufifacies at 24 ± 1 °C (a) and 30 ± 1 °C (b) (P = puparium, D = dorsal, L = lateral, V = ventral).

Figure 10. Inner and outer morphological changes of Ch. rufifacies at 24 ± 1 °C (a) and 30 ± 1 °C (b) (P = puparium, D = dorsal, L = lateral, V = ventral).

3.3. Development Data and Required Degree Days for Each Life Stage at Two Temperatures

The pupation landmark data, combined with the development data from eggs to the third instar post-feeding stage, produced the following development dataset (

Table 5. Complete development dataset for Ch. rufifacies at the two temperatures in ovine muscle along with the degree days (DD) and accumulated degree days (ADD).

Temperature	Stage	Time (Days)	DD	ADD
24 ± 1 °C	Eggs	0.53	6.36	6.36
	1st instar	1.40	16.80	23.16
	2nd instar	1.21	14.52	37.68
	3rd instar: pre feeding	1.39	16.68	54.36
	3re instar: post feeding	2.84	34.08	88.44
	Pre-cryptocephalic pupal stage	0.33	3.96	92.40
	Cryptocephalic pupal stage	0.66	7.96	100.32
	Phanerocephalic pupal stage	3.00	36.00	136.32
	Pharate adult	1.00	12.00	148.32
30 ± 1 °C	Eggs	0.42	7.56	7.56
	1st instar	0.63	11.34	18.90
	2nd instar	0.58	10.44	29.34
	3rd instar: pre feeding	0.25	4.50	33.84
	3re instar: post feeding	1.67	30.06	63.90
	Pre-cryptocephalic pupal stage	0.33	5.94	69.84
	Cryptocephalic pupal stage	0.66	11.88	81.72
	Phanerocephalic pupal stage	2.00	36.00	117.72
	Pharate adult	0.66	11.88	129.60

) for Ch rufifacies at the two temperatures. Additionally, the table also presents the calculated degree days (DD) and accumulated degree days (ADD) for each stage.

3.4. Seasonal Emergence of Fly Generations

Figure 11 illustrates that a total of eight fly generations emerged during the autumn season, spanning from March to May 2023 in southwestern Australia. On average, 3.25 generations emerged in both March and April, while only one generation was recorded in May. In contrast, during the summer season from December 2023 to February 2024 (Figure 12), 14 generations were estimated to emerge. Within this period, four generations emerged in December, followed by five in both January and February.

4. Discussion

The primary aim of this study was to determine the developmental periods of distinct life stages, from eggs to adult, in Ch. rufifacies reared on two types of ovine tissue at two temperatures: 24 ± 1 °C and 30 ± 1 °C. Additionally, this study explored the practical application of these laboratory-generated developmental datasets for predicting the emerged number of fly generations under field conditions.

Previous studies on the development of Ch. rufifacies have primarily used swine muscle tissue, which is the main proxy for human tissue in forensic entomology field studies [4]. This study attempts to address this gap by using ovine muscle tissues as the larval feeding medium, which is one of the primary hosts for parasitic fly infestations [13]. This study also compared the possible variations in fat composition within ovine skeletal muscle tissue, emphasising that different muscle and adipose tissue concentrations may influence the severity of symptoms, complications, and overall health outcomes of the sheep [4]. Notably, there were differences in the development of flies reared on the two muscle types. This may be due to fat-containing tissues providing more calories per gram, making them a higher energy nutrient source for larvae. Furthermore, fat-rich tissues are softer and easier to digest, allowing larvae to obtain energy more efficiently for their growth and development.

The development periods of pupal life stages of Ch. rufifacies differed between the two temperatures [3]. These findings suggest that temperature is critical when predicting the number of emerging fly generations in each season. By incorporating larval and pupal developmental landmarks into Accumulated Degree Day/Hour models, it becomes possible to predict the emergence of distinct, temperature-dependent fly waves [5]. Although, the larval feeding medium and ambient temperature are important when developing a predictive model, it is essential to consider additional factors such as the type, texture, and moisture content of the pupation medium, which have been shown to impact the development periods of pupation landmarks in Ch. rufifacies [14].

This study presented a clear methodology using pupation landmarks through the character changes as the pupae matured to calculate the accumulated degree days (ADD) and correlate it with the environmental temperature data to estimate the timing of fly emergence. These data are the first step to assess the population dynamics of flies in relation to temperature changes. Additionally, if humidity was incorporated, then these data could serve as an even more robust indicator for evaluating the effectiveness of specific fly mitigation practices.

A study conducted by Wall et al. (1992) [5] used a simulation method to predict the emergence numbers of Lucilia sericata based on laboratory development data and observations of the timing of fly wave appearances in the field. The current study focuses on predicting fly generations rather than emergence numbers predicted by Wall et al. (1992). Both studies utilised the accumulated degree days (ADD) method. It seems that the 1992 study was never adopted into a model to predict myiasis. The current study, however, may be a more useful tool for predicting fly strike as it informs farmers on when populations of flies increase throughout a season. To achieve this, a further step would be to conduct a field trial to compare the development data presented in this study by correlating it with actual environmental conditions. This could be accomplished by recording daily temperatures in the field and correlating them with both the pupation stages, as well as the exact timing of oviposition. Additionally, a population study using fly traps would refine this validation process by identifying the precise timing of fly emergence in each season, rather than assuming it to begin at the onset of summer.

In addition, the present study was conducted at only two temperatures, which presents a limitation when calculating the minimum threshold temperature. Determining the minimum threshold requires regression analysis, where the development rate is plotted against temperature, and the threshold is identified as the point of intersection with the x-axis. It is crucial to establish this value for the study area, as the minimum threshold can vary depending on location-specific genetic variations within the same species (Higley and Haskell. 2009).