Morphological changes of larvae and pupae of Lucilia sericata (Diptera: Calliphoridae) reared at two temperatures and on three food types

Abstract

Determining the minimum postmortem interval (minPMI) from an entomological perspective relies mainly on development data recorded for various species of flies collected from a crime scene or suspicious death. This study focused on the larval and pupal development of Lucilia sericata (Meigen), with an emphasis on the changes of the external morphology of the puparium and its pupal content throughout the duration of metamorphosis. Colonies of L. sericata were reared on 3 types of swine tissue (skeletal muscle, liver tissue, and heart tissue) at 2 different temperature regimes; 24 ± 1 °C and 30 ± 1 °C. The overall developmental time, larval width and length, and inner and outer pupal morphology changes were observed and recorded. The results show that: (i) temperature significantly influenced overall development time, as well as changes in larval width and length, but this effect was not dependent on tissue type; (ii) larval development duration was longest on heart tissue, and shortest on skeletal muscle for both temperatures; and (iii) pupation was longest for larvae reared on skeletal muscle at 24 ± 1 °C, and on liver tissue at 30 ± 1 °C, while those larvae reared on liver tissue at 24 ± 1 °C and heart tissue at 30 ± 1 °C had the shortest pupation period. A seven-character checklist plus 4 landmark stages were developed comprising the external morphology of the puparium and pupal content changes of L. sericata. In conclusion, the study provides larval and pupal development timetables, as well as checklists and photo guides for pupal character development that may be useful for future postmortem determinations.

Introduction

Forensic entomologists use flies (Order Diptera) as the primary source for estimating the minimum postmortem interval (minPMI) or the period of insect activity after a death (Dadour and Morris 2014, Mohr and Tomberlin 2015, Byrd and Tomberlin 2019). Flies are generally the first groups of insects to colonize cadavers, and during the decomposition process many fly species sequentially visit the remains to feed and oviposit (Payne et al. 1968, Voss et al. 2009). Flies leave behind crucial evidence such as egg clusters, dead and living larvae, and full and empty puparia, which can be used to determine an accurate estimate of the minPMI (Voss et al. 2009, Hall et al. 2012, Dadour and Morris 2014). Forensic entomologists utilize fly-related data in 3 distinct ways to determine the minPMI of decomposed remains: via life history tables, matching with species-specific isomegalen/isomorphen curves, and calculating the accumulated degree day/hours (Bambaradeniya et al. 2023a, 2023b, Bugelli et al. 2023). During advanced stages of decomposition, the minPMI can be assessed by the sequential colonization patterns of flies and other arthropods, although this is generally less accurate. This is achieved by referring to the findings of previous carrion successional studies conducted under different death and decomposition scenarios (Voss et al. 2009).

Lucilia sericata is a representative of the carrion entomofauna during the early stages of colonization of animal and human remains, especially in the warmer seasons (Fisher et al. 1998). Researchers have studied the carcass visitation patterns of L. sericata and other blow flies in a variety of decomposition conditions, including those involving carcasses on land, in water, and even those that have been intentionally altered by burning, concealment, or wrapping in cloth (Matuszewski et al. 2020). Due to its frequency on remains, the development times of L. sericata have been published for different regions of the world and it has been used to estimate the time since death for many homicides (Bambaradeniya et al. 2023b).

Studies show that external factors such as temperature, humidity, and tissue type influence the development of egg and larval stages of L. sericata (Bambaradeniya et al. 2023b). In addition, the development of pupae is dependent on the moisture content of the pupation medium (Grassberger and Reiter 2001, Kökdener and Şahin Yurtgan 2022). Karabey and Sert (2018) state that from egg to the beginning of pupariation takes approximately 50% of the total developmental time of L. sericata, and that the residual time can be attributed to the period from pupa to adult emergence (Karabey and Sert 2018). The age of a pupa can be determined by a variety of factors based mainly on color change patterns, and the external morphology of the puparium. The techniques currently include, histology, and sometimes molecular analysis (Davies and Harvey 2013, Brown et al. 2015, Hartmann et al. 2023), hyperspectral imaging (Voss et al. 2017), micro-computed and optical coherence tomography (Brown and Harvey 2014, Martín-Vega et al. 2017). The color changes of the puparium are useful when determining the minPMI during the first 10 h of pupariation (Li et al. 2023). Other external morphological features of pupa have resulted in categorizing the intrapuparial period into different landmark stages. The 4 main stages identified are precryptocephalic pupal stage, cryptocephalic pupal stage, phanerocephalic pupal stage, and pharate adult (Greenberg and Kunich 2002, Li et al. 2023).

The present study recorded the developmental stages of L. sericata on 3 types of swine tissues at 2 constant temperatures corresponding to the average temperature in southwestern Australia during autumn/spring (24 ± 1 °C) and summer (30 ± 1 °C) seasons. As the immature flies developed on each tissue type, larval length and width were measured and both puparium and pupal morphological changes were documented. Although development studies have been conducted on L. sericata, this research is significant as it represents a robust baseline for age estimation of this insect species in cases of forensic interest in the Australian region.

Methodology

Fly Colony

Blow fly larvae were collected from decomposing swine meat pieces placed outside at Murdoch University, Perth, WA (32.0680° S, 115.8352° E). Approximately 50 larvae were subsequently identified as L. sericata after being reared to adults using the key of Williams and Villet (2014). After the adults emerged, they were fed with milk and sugar solution ad libitum. Seven days from the adulthood were fed a blood meal and provided with swine meat to facilitate as an oviposition substrate.

Preparation of Tissue Samples for the Development Studies

Approximately 2 kg each of swine skeletal muscle, liver tissue, and heart tissue was used in this study. Each type of tissue was then cut into small, cube-shaped pieces weighting approximately 100 g, placed inside ziplock polyethylene bags and refrigerated at 2 °C.

Development Study Design

All the development studies were conducted using egg batches harvested from the first 3 captive fly generations to maintain the genetic consistency among replicate studies (Bambaradeniya et al. 2023b). The egg clusters of 50–60 eggs were separated from the oviposition substrate using a fine-tipped artist paint brush and transferred to each tissue type and placed on a small tray inside a plastic container (500 ml) filled to a depth of 4 cm with dry sand. Each container was covered with a paper towel and secured with a rubber band to prevent wandering larvae from the containers. Three replicate studies were conducted for each tissue type at 2 temperature regimes; 24 ± 1 °C and 30 ± 1 °C, while RH and photoperiod were maintained at 70% and 12:12 (L:D) h within a climatic growth chamber (Fisher & Paykel).

Larvae and Pupae Samples

During the development study, 2 larvae were collected every 3 h to record the transition times of each life stage. They were hot water sacrificed and examined under a stereo microscope to determine their life stage. Similarly, 2 of the largest larvae feeding on each tissue type were removed from the colonies every 6 h, hot water sacrificed and preserved in 70% ethanol (Bambaradeniya et al. 2023a). The length and width of each larva were measured using Vernier calipers. In a separate experiment, 150 larvae were reared on skeletal muscle placed in a container with 4 cm of dry sand at the 2 temperatures. After pupariation, when the pupa became static, and every 8 h until adult eclosion, 2 full puparia were collected, pierced, hot water sacrificed, and then preserved in 70% ethanol (Brown et al. 2015). The length and mid width of each puparium were measured using Vernier calipers and photographed using a digital microscope (Dino-Lite edge 3.0 paired with Dino-Capture software). A scalpel was used to dissect each pupa and the dorsal, ventral, and lateral views were photographed to record their morphological characters and color (Voss et al. 2017).

Data Analysis

Development study

The development time of each life stage of L. sericata was tabulated under the 3 tissue types and the 2 temperature regimes. The level of significance of the effect of temperature and tissue type on the development time change, including length and width differences of larvae were determined by a series of analysis of covariance tests (ANCOVA) using the R statistical package. All the recorded development times of larvae and pupae were graphically illustrated using the same statistical package.

Recording of internal and external morphological characters of pupae

The puparia development of preserved specimens was assessed under 1 external and 6 internal pupal characteristics. The changes that occurred in the puparium were recorded in relation to their shape and color change and they were labeled sequentially (pupa: P1–P4, body segment: B1–B6, compound eye: E1–E8, antennae: A1–A8, wings: W1–W6, legs: L1–L8, and abdomen: AB1–AB7). All the color differentiations were recorded using a Munsell constant hue system (https://www.andrewwerth.com/aboutmunsell/) under a background light of 400 lux. The Muncell notations for each color of a specific body location in a photograph were recorded by carefully considering and referencing the hues and chromas that given in the corresponding chart. In addition, the development timeline of body segmentation, and the formation of the compound eye, antennae, wings, legs, and abdomen were recorded and photographed under the 2 temperatures regimes. Based on overall changes of these selected characteristics, the full length of pupation for the 2 temperature regimes were grouped into the 4 landmark stages: precryptocephalic pupal stage, cryptocephalic pupal stage, phanerocephalic pupal stage, and pharate adult. The precryptocephalic pupal stage was characterized by larval–pupal apolysis with the prepupa tightly attached to the inner surface of the puparium while retaining most of the larval features such as body segmentation, and the presence of the cephalopharyngeal skeleton. The next or cryptocephalic pupal stage was marked by the formation of legs, and wings. This was followed by the phanerocephalic pupal stage denoted by defining the pupal body into 3 segments: head, thorax, and abdomen. Finally, the pharate adult was the form of adult within the puparium with their epidermal cells separated from pupal cuticle (Greenberg and Kunich 2002, Li et al. 2023).

Results

Overall Development

The total time required for the life cycle to complete varied depending on the temperature and tissue type. Statistical analysis demonstrated that there was a significant difference between temperature and the total development time (df = 1, F = 432.26, P ≤ 0.01). However, there was no significant difference between tissue type and development time (df = 2, F = 0.04, P = 0.96). Closer scrutiny of the tissue types showed that, at 24 ± 1 °C, liver tissue yields the shortest completion time, followed by heart tissue and skeletal muscle (Table 1, Fig. 1). Conversely, at 30 ± 1 °C, heart tissue yields the shortest completion time, followed by liver tissue and skeletal muscle.

Larval Length and Width

The temperature had a significant impact on larval length change (df = 1, F = 8.45, P < 0.01), with the highest and lowest average larval lengths recorded for larvae reared on heart tissue at 24 ± 1 °C (1.36 mm, 16.10 mm) and 30 ± 1 °C (1.80 mm, 15.62 mm), respectively. However, the tissue type did not appear to have any significant effect on larval length change (df = 2, F = 0.74, P < 0.01).

The temperature had a significant impact on the larval width change (df = 1, F = 29.12, P ≤ 0.01), with both maximum and minimum ranges of average cross width of larvae recorded on skeletal muscle (0.57 mm, 2.62 mm) at 30 ± 1 °C. At 24 °C, the highest average larval width was recorded for larvae reared on liver tissue (2.95 mm), while the lowest was on skeletal muscle (0.21 mm). Tissue type did not have a significant effect on larval width change (df = 2, F = 1.99, P = 0.14).

Inner and Outer Morphological Character Changes of L. sericata Pupa

This study examined the external morphological changes in puparium development, as well as the formation of body segments of the pupa. These included the compound eyes, antennae, wings, and legs of L. sericata in the head and thorax regions. The whole abdomen was considered as a single unit during this analysis. The time scale for tracking changes in character descriptions are provided in Table 2.

During the onset of pupariation, the puparium maintained a similar shape to that of the larvae (P1). Over time, after 8–16 h from pupation, it developed into a barrel shape that persisted throughout the entire process (P2, P3, P4). However, prior to emergence, a small groove appeared near the front end demarcating the operculum, that signifying the separation point of the puparium to emerge as an adult (P3, P4). The exterior color of the puparium changed from light brown (P2) (2.5YR 5/10) to brown (P3) and ultimately black (P4) (N1). Throughout the entire pupation period, the surface of the puparium displayed segmentation (P1, P2, P3, P4) (Fig. 2).

Overall, the puparium had a tapered body with 12 segments similar to the larval stage (B1). However, over time, after completion of the first 20% of the pupal period, their shape changed to become more barrel-like (B2). Also the cephalopharyngeal skeleton is visible (B1, B2), but this progressively detached from the pupa and later can be seen at the anterior end of the puparium, stuck to the inner surface (B3). At the same stage the legs form and the head became distinctly visible from the rest of the body. Once the pupae had fully formed the 3 body segments (head, thorax, and abdomen), their size almost remained the same, but their color changed. At the same time the setae began to form (B4, B5, B6) (Fig. 3).

Antennae emerged after completing the prepupa transformation in the anterior end, with the disappearance of the cephalopharyngeal skeleton (A1, A2). Two circular buds surfaced on either side of the pupa and gradually matured into antennae (A3, A4). It is noteworthy that the hue of the antennae underwent a transformation while growing, initially beginning as white (A3, A4), then shifting to brown (A5, A6) (7.5R 5/16), and ultimately tanning into black (A7, A8) (N1) (arrowed) (Fig. 5).

It is important to note that the compound eyes appear as protuberances on both sides of the head, with the head region distinguishable from the rest of the body (E2, E3). Additionally, the color of the eye develops over time, starting from creamy white (E2, E3) and eventually becoming red-brown (E8) (7.5R 4/12), passing through different shades like brown (E4) (7.5R 5/16), pale pink (E5) (10RP 6/12), pink (E6) (10RR 6/16), and red (E7) (5R 5/18) (Fig. 4).

The legs of L. sericata pupa begin as a single inflated tube-like mass attached to the body (L, L2), but as they elongate and become segmented (L3, L4, L5), they detach from the body and grow to be about two-thirds of the pupal length (L6, L7, L8). The legs change color from creamy white (L3) to light (L4, L5) and dark brown (L6, L7) (7.5R 5/16), eventually darkening to black (L8) (N1) (arrowed). They finally become well-defined as the femur, tibia, and tarsi, with dark bristles (L8) (Fig. 7).

Once the legs had emerged (W1, W2), wings appeared in the thoracic area (W3). Initially, the wings are tightly folded (W3, W4) but eventually unfurl (W5), revealing their intricate veins (W6). The hue of wings transitions from white (W3) (N9) to brown (W4, W5) (7.5R 5/16) and finally to translucent (W6) (arrowed) (Fig. 6).

Before segmentation (B3), bristle points appear on the abdomen (AB1, AB2). At first, they are brownish (AB4, AB5) (7.5R 5/16), but later they turn tan to black (AB6, AB7) (N1) (Fig. 8).

Internal and External Morphological Changes of L. sericata Pupa at 24 ± 1 °C and 30 ± 1 °C

The pupation phase of L. sericata was 184 h when raised on skeletal muscle at 24 ± 1 °C. The puparium had a minimum length of 5.75 mm and a maximum length of 8.27 mm. The cross width parameters of the puparium varied from 2.36 mm to 3.21 mm.

In contrast, the pupation phase of L. sericata was 136 h when fed on swine skeletal muscle at 30 ± 1 °C. The size of the puparium varied from 5.75 mm to 8.24 mm in length and 1.97 mm to 3.12 mm in width.

Figures 9 and 10 illustrate the characteristics of the morphological changes of the puparium and the pupal changes [dorsal (D), lateral (L), and ventral (V) view] over time.

Observations on 4 Landmark Phases of L. sericata Pupation

The 4 apolysis phases of the intrapuparial process; precryptocephalic pupal stage, cryptocephalic pupal stage, phanerocephalic pupal stage, and pharate stage of L. sericata were distinguished using a photo series that indicated the development of characteristics and the pupation character checklist at each temperature (Figs. 9 and 10). Specific baseline characters were determined to define each phase based on previous publications (Greenberg and Kunich 2002, Karabey and Sert 2018).

Precryptocephalic pupal stage

Based on observation, it was found that the precryptocephalic pupal stage lasted approximately 16 h at 24 ± 1 °C and 8 h at 30 ± 1 °C (Figs. 9 and 10, Table 2). During this stage, it was difficult to separate the hypodermis of the larvae from the puparium as they adhered to each other. The cephalopharyngeal skeleton was also closely attached to the prepupa at the anterior end. At this stage, slight differences in the shape and color of the puparium compared to the other stages were noticeable, as it still retained the shape of the larvae and had a lighter brown color (7.5R 5/16).

Cryptocephalic pupal stage

The cryptocephalic pupal stage at 30 ± 1 °C required 8 h to complete, whereas 16 h at 24 ± 1 °C (Figs. 9 and 10, Table 2). During this stage the formation of legs was observed in the middle of the body, appearing as an inflated tube-like mass. As this stage progressed, a groove became visible between the head and thorax regions. The cephalopharyngeal skeleton was noticeable, but not tightly bound to the puparium as in the previous stage.

Phanerocephalic pupal stage

During the phanerocephalic pupal stage, the 3 body segments of head, thorax, and abdomen can be observed. This stage can range from 40 to 160 h at 24 ± 1 °C and 24–112 h at 30 ± 1 °C (Figs. 9 and 10, Table 2). The respiratory horns located on the groove between the head and thorax regions become visible. It is imperative to note that as development progresses throughout the stage, compound eyes and antennae appear on the head, wings on the thorax, and setae on the abdomen.

Pharate adult

This phase involves insect maturation to adult which includes the tanning of the body and hardening of visible setae and bristles. The pupal cuticle that covers the body of the adult begins to shed. This process required between 168 and 184 h at 24 ± 1 °C and 120 and 136 h at 30 ± 1 °C (Figs. 9 and 10, Table 2) to complete.

Discussion

The purpose of this study was to gather information about the growth stages of L. sericata larvae and pupae reared on swine skeletal, liver, and heart tissue at 2 different temperatures (autumn/spring and summer) in Australia. Previous studies on the larval and pupal development of L. sericata have been conducted separately (see Bambaradeniya et al 2023b for review) (Grassberger and Reiter 2001), but this is the first study to detail the total developmental time of the immature life history stages, with a specific emphasis on pupal development during metamorphosis.

Bambaradeniya et al. (2023b) compiled a list of 15 studies that documented the development of L. sericata under different temperatures and tissue types obtained from various host animals and found that 11 studies used bovine tissue, 2 used lamb meat, 1 used chicken liver, and 1 used swine tissue as the larval food. These studies were conducted in growth chambers with constant temperatures ranging from 10 to 36 °C. A single study conducted by Hans and Vanlaerhoven (2021) in Canada used swine tissue and similar temperature parameters to the present study, but they were unable to record a complete development period for L. sericata. A much earlier study, however, by Grassberger and Reiter (2001) again using similar temperatures but on bovine muscle, showed a contrary result, indicating that total development took 297 h at 25 °C and 267 h at 30 °C. In the present study, total development time was between 401 and 413 h at 24 °C and 241 and 247 h at 30 °C for all tissue types. This discrepancy may be attributed to a variety of factors such as the food medium, the genetic homogeneity of the colony, the geographical location of the source specimens collected for the colonies, and the variations in methodology, such as the sampling frequency (Bambaradeniya et al. 2023b).

To date, there have been no studies conducted on the impact of tissue types obtained from different locations of the same host animal, in this case swine, on the development of L. sericata. In this study only fresh tissues were presented to flies for oviposition. There are limitations concerning this method especially when presenting organs as a food substrate for development. The current design attempted to simulate fly oviposition and subsequent larval feeding on a carcass, mirroring the natural process whereby a female fly lays eggs on fresh meat such as skeletal muscle, and larvae emerge to feed on the decaying tissue. However, female flies are unlikely to lay eggs directly on internal organs of a fresh carcass without an entry point, such as an orifice, but more likely via a deep cut or bullet wound. Therefore, if no entry point is present, then consideration in future studies should be given to feeding larvae with organs only after a period of decay.

The present study revealed that the pupation phase represents 47–53% of the total lifecycle of L. sericata. Specifically, at 24 °C (47%), skeletal muscle had the longest pupation period, while heart tissue had the shortest period at 30 °C (53%). This finding aligns with the study by Karabey and Sert (2018), which showed that the average pupation period of L. sericata reared on beef liver at 20, 25, and 30 °C was 50%. Both these studies documented the developmental milestones of the pupation phase, which is crucial, when puparia are collected from remains of forensic interest and considered for minPMI estimation.

Before the Karabey and Sert (2018) study only 2 other studies had been conducted on the pupal development timeline of L. sericata. Firstly, Grisendi et al. (2015) used a similar approach to the current study by capturing photographs over time of the external organ development of L. sericata puparium. This technique is cost-effective and time-efficient, making it accessible even to those with limited knowledge on pathology and DNA-based techniques. In contrast, the second study by Zajac and Amendt (2012) recorded the development landmarks of the pupation phase of L. sericata using morphology and histology methods. Unfortunately, these previous studies failed to use a color chart to make color comparisons. The present study incorporated the Munsell color system to identify color and color changes which will assist any future comparisons in other studies or casework. It is important to note that the color standard accounts for approximately one third of the elements required to accurately match color. The other two-thirds include a light source, which is standard and consistent, such as daylight, and observers who have the ability to see colors accurately. Only one other major study on the blow fly Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) by Brown et al. (2015) used a color recognition index (Photoshop CS5). However, this index was only used for eye color changes whereas other color changes pertaining to metamorphosis were broadly grouped. Again, future color comparisons will be arduous without a color coding system.

Aligned with Zajac and Amendt (2012), Grisendi et al. (2015), and Karabey and Sert (2018) studies, the current study used a range of morphological stages to characterize pupation timing. Karabey and Sert (2018) recorded 18 stages, Grisendi et al. (2015) 6, and Zajac and Amendt (2012) 10, while the present study focused on the 4 primary pupation landmarks. Generally, the number of stages in all studies associated with these landmarks were based on the sampling time followed by the characters considered for these demarcations. In addition, all authors agree that pupation is a temperature-dependent process that greatly affects the emergence and progression of pupal characters over time.

The present study fills a significant gap in the literature by documenting the growth stages of L. sericata immatures reared on 3 tissue types of same host species under varying temperatures. Previous research on L. sericata development has often been conducted in isolation, focusing separately on larval and pupal stages (Bambaradeniya et al., 2023b). The methods used in this study, incorporating a color system with detailed morphological stage characterization, contribute to a perspicuous understanding of pupal development and its association with temperature. Overall, this study integrates the complete developmental timeline of immature life history stages of L. sericata with a special focus on metamorphosis and offers a foundation for further research while also enhancing the accuracy of time since death estimations in case work.

Acknowledgments

Authors would like to thank Anjelique Raison for her valuable contribution in measuring the length and width of larvae. We would also like to thank Martin Hall for his comments and editorial on an earlier version of this manuscript.

Author contributions

Tharindu Bambaradeniya (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Project administration [equal], Writing—original draft [equal], Writing—review & editing [equal]), Paola Magni (Conceptualization [equal], Data curation [equal], Investigation [equal], Methodology [equal], Project administration [equal], Supervision [equal], Writing—review & editing [equal]), and Ian Dadour (Conceptualization [equal], Investigation [equal], Methodology [equal], Supervision [equal], Writing—original draft [equal], Writing—review & editing [equal])

References