Accepted Manuscript Title: Larval development rates of Chrysomya rufifacies Macquart, 1842 (Diptera: Calliphoridae) within its native range in South-East Asia Author: Surasuk Yanmanee Martin Husemann Mark Eric Benbow Guntima Suwannapong PII: DOI: Reference:
S0379-0738(16)30188-8 http://dx.doi.org/doi:10.1016/j.forsciint.2016.04.033 FSI 8449
To appear in:
FSI
Received date: Revised date: Accepted date:
25-3-2016 21-4-2016 27-4-2016
Please cite this article as: S. Yanmanee, M. Husemann, M.E. Benbow, G. Suwannapong, Technical Note, Forensic Science International (2016), http://dx.doi.org/10.1016/j.forsciint.2016.04.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
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Chrysomya rufifacies represents an indicator species in forensic entomology
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we investigated the development of the species from the east of Thailand
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we present isomorphen and isomegalen diagrams for nine temperatures
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the lower threshold temperature for total development was 9.5°C
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Technical Note:
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Larval development rates of Chrysomya rufifacies Macquart,
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1842 (Diptera: Calliphoridae) within its native range in South-
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East Asia
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Surasuk Yanmanee1, Martin Husemann2, Mark Eric Benbow3, Guntima Suwannapong4*
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Biological Science Program, Faculty of Science, Burapha University, Chon Buri 20131, Thailand General Zoology, Department of Biology, Martin-Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany
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Departments of Entomology and of Osteopathic Medical Specialty, Michigan State University, East Lansing, MI 48824, USA
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Department of Biology, Faculty of Science, Burapha University, Chon Buri 20131, Thailand
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*Corresponding author:
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Guntima Suwannapong
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Department of Biology
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Faculty of Science
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Burapha University
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Chon Buri 20131
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Thailand
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E-mail:
[email protected]
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Tel. 038-102222 Ext.3088
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Technical Note:
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Larval development rates of Chrysomya rufifacies Macquart,
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1842 (Diptera: Calliphoridae) within its native range in South-
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East Asia
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Abstract
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Chrysomya rufifacies represents an important indicator species in forensic entomology
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that is often used to estimate the minimum postmortem interval (PMImin) in crime scene
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investigation. However, developmental rates differ locally, so that estimates should be
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based on regionally generated development data. Therefore, we determined the
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developmental rates of C. rufifacies within its native range in Thailand under nine constant
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temperature regimes: 15, 18, 21, 24, 27, 30, 33, 36 and 39°C. Developmental times from
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egg to adult varied among the temperatures and were longest at 15°C (618 h) and shortest
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at 33°C (168 h). No pupae emerged at 39°C. We used linear regression models to estimate
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the minimum development threshold temperatures for each life stage: egg stage = 9.5°C,
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first to second instar = 10.8°C, second to third instar = 11.5°C, third instar to pupariation =
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11.4°C, pupariation to adults = 5.0°C; the minimum threshold to complete all larvae stages
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was 11.1°C and to complete all life stages from eggs to adult was 9.5°C. We further
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generated isomorphen and isomegalen diagrams that can be used to quickly estimate the
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PMImin for forensic applications.
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Keywords: Development time, isomegalen diagram, isomorphen diagram, minimum
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threshold temperature 3
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Introduction
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Forensic entomology is based on the use of developmental and distribution information of
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necrophagous insects, such as blow flies (Diptera: Calliphoridae), to support crime scene
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investigations. As insects are poikilotherms, their development rates are strongly
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temperature dependent and are usually predictable [1]. Further, insects require a certain
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amount of heat unit-energy units (degree-days) to develop from one life stage to the other
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[2] and do not develop below or above certain threshold temperatures [3,4]. The lower
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developmental threshold temperature is most important for PMImin estimation in forensic
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entomology. Forensic entomologists often use 0°C as lower threshold for estimates based
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on blow fly species; yet, some reports suggest that temperatures between 6 and 10°C are
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more appropriate for specific species [5].
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Generally, the temperature thresholds are species, or even population specific, and should
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be estimated for each taxon or regional population separately as insects adapt to the local
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conditions [6]. For instance, it has been reported that Phormia regina (Diptera:
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Calliphoridae) does not successfully complete development at temperatures below 10°C
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[7], whereas for Calliphora vicina (Diptera: Calliphoridae) a lower developmental
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threshold of 1°C was reported [8]. Marchenko (2001) provided lower developmental
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threshold temperatures for several blow flies and some other fly species further
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demonstrating interspecific variability: 2.0°C for C. vicina, 3.0°C for Calliphora
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vomitoria, 7.8°C for Protophormia terraenovae, 7.8°C for Lucilia sericata, 10.2°C for
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Chrysomya albiceps, 11.4°C for P. regina, 7.2°C for Muscina stabulans, 7.9°C for
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Muscina assimilis, 7.8°C for Boettcherisca septenrionalis and 6.4°C for Piophila
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foveolata [9]. Only few studies addressed variability of thermal thresholds within blow
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flies species; yet, such local estimates may be important, especially for geographically
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distant populations [7,10] of widespread taxa. This is specifically critical if widespread
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species are employed in forensic applications. Hence, studies are needed to better
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understand regional variability in developmental rates for individual species and their local
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populations.
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Chrysomya rufifacies has a wide distribution and is a blow fly species of special interest
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for forensic science in many countries of the world, including Thailand [11-16]. The
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larvae of C. rufifacies are commonly used in forensics [17], but often based on
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developmental data generated in distant geographic regions. However, in order to more
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accurately estimate PMImin, local or regional developmental data are needed as
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populations in different latitudes and climates may vary in development due to plasticity
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or local adaptation [18]. Although developmental rates of Ch. rufifacies have been studied
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under ambient temperature in Chiang Mai, northern Thailand [13], the variability of
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developmental parameters under different temperatures has not been explored in Thailand.
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Therefore, the objective of this study was to determine the developmental variation of a
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local population of Ch. rufifacies from central Thailand at a range of temperatures to
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generate one of the first developmental data sets for this species within its native range in
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South East Asia.
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Material and Methods
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Study species, sampling and colony establishment and maintenance
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Adults and larvae of Ch. rufifacies were collected from chicken carcasses in Chon Buri,
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Thailand between 2012-2015. Adults were identified using the key by Marshall et al. [19]
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and third instars were identified according to Sukontasan et al. [13] and Smith [20]. One
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hundred Ch. rufifacies adults (50:50 male and female) were caged (6 x 18 x 13 cm3) under
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nine different temperature regimes (15, 18, 21, 24, 27, 30, 33, 36 and 39°C) with
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approximately 60-70% RH and a photoperiod of 12:12 (L.D). Each cage was supplied
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with water, 50% sucrose and granular sugar ad libitum. Flies were fed with blended swine
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liver to facilitate ovary development, and on the fourth day were provided ad libitum with
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beef muscle tissue.
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Egg deposition and larval development
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To study developmental time from oviposition to hatching at different temperatures (15,
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18, 21, 24, 27, 30, 33, 36 and 39°C), 100 adult individuals (sex ratio of 1:1) were reared in
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plastic cages (6 x 18 x 13 cm3). Egg batches were collected within 30 min of oviposition
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and transferred to fresh beef muscle placed in a plastic container (7.0 x 9.5 x 5 cm3).
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Specimens were kept at the same condition as described above. For the first 100 hatched
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larvae the time of emergence was recorded.
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Another 100 freshly hatched larvae were transferred to 20 g beef muscle placed on an
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aluminum boat (4 x 5 x 1 cm3) within the plastic container. To evaluate the developmental
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time from the first to the second instar, from the second to the third instar and from the
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early third instar to the post feeding stage, 10 larvae from each instar were parboiled
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(100°C) for 30 s before determining the larval stage under a stereomicroscope [21]. To
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minimize any potential effects of changing densities, larvae at the exact same stage were
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replaced from an extra cage keeping the total number of larvae constant at 100 individuals
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throughout experiment. 20 g of fresh beef muscle were replaced daily. For length
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measurements, the four largest larvae in each cage were removed from the plastic
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container every three hours following the methods of Grassberger and Reiter [10] and
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Byrd and Butler [22]. Larvae were parboiled for 30 s and measured [21] immediately after
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cooling under a binocular microscope in 0.1 mm units using a Vernier caliper [10]. When
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the third instars entered the post feeding stage, they were moved into 200 g of sterile soil
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sawdust (50% w/w) in a new plastic container (13 x 18 x 6 cm3) and the time of larval post
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feeding migration and emergence of each adult was recorded. Three replicates were
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performed for each rearing temperature.
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Statistical Analyses
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The relationship between the developmental rate and temperature was modeled according
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to a linear developmental rate model (ADH) [23]. The lower developmental threshold for
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each stage was estimated by the x-intercept method [24]. The reciprocal (1/b) of the slope
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of the regression line represented the sum of degree hours (ADH) defined as the K-value
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that insects required above the lower developmental threshold [25]. Developmental times
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and larval body sizes were compared across temperatures using an analysis of variance
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(ANOVA), and Duncan’s Multiple Rang test (α = 0.05). Statistical analyses were
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performed with PAST [26].
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Results
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Development curves from constant temperature regimes
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The mean maximum body lengths of all instars were plotted against time at each
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temperature (from hatching until the end of the third instar) (Fig. 1). The growth curve had
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a sigmoidal in form. As expected, larvae grew faster at higher temperatures. The periods
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of each developmental stage under all temperature regimes are presented in Table 1.
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Isomegalen- and Isomorphen-diagrams
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We plotted all developmental data from egg hatching to eclosion at nine constant
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temperature regimes as isomegalen and isomorphen diagrams (Fig. 2, 3). Isomegalen-
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diagrams plot the time from egg hatching to post feeding against temperature. Each line
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represents an identical larval body length (Fig. 2). Further, time of all stages from
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oviposition to eclosion was plotted against different constant temperatures, where each
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line represents morphological changes such has molting from one instar to the next. Areas
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between lines define developmental stages (Fig. 3).
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Developmental thresholds and thermal constants
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The developmental rates of all stages increased with temperature (Table S1). The highest
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developmental rate was found when the eggs were reared at 39°C (0.163 1/hours), while
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the lowest developmental rate was found for pupariation at 15°C (0.003 1/hours). The
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developmental rate from oviposition to eclosion was lowest at 15°C and highest at 36°C.
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Minimum development thresholds were extrapolated from regression lines for each model
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(Supplemental Figs. 1, 2, 3). The linear regression of the development from oviposition to
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pupariation was described as y = 0.0007x - 0.0078 (R2 = 0.99) with a temperature
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threshold of 11.14 ± 0.58. The rate from oviposition to eclosion was described by the
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equation y = 0.0002x - 0.0019 (R2 = 0.96) with a minimum thermal threshold of 9.50 ±
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1.04. All minimum threshold temperatures and thermal constants are presented in
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Supplemental Table S2.
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Accumulated Degree Hours (ADH)
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The ADH of Ch. rufifacies from egg to eclosion was highest at 36°C. Different base
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temperatures showed differences in ADH. All values are provided in Supplemental Tables
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S3-5.
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Discussion
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In this study we investigated several developmental parameters at different temperatures
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for the forensically important blow fly species Ch. rufifacies from Thailand. We generated
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isomegalen and isomorphen diagrams for the use in forensics with the motivation to
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provide much needed developmental data for this forensically important species from a
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tropical region.
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Comparison to other studies of the same species
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The development times and sizes of peak feeding larvae of Ch. rufifacies we generated
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here were relatively similar to those reported by Sukontason et. al. [13] at ambient
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temperatures suggesting consistency of rates within a geographic region and supporting
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the quality of our data. Byrd and Butler [27] studied the development of eggs, larvae and
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pupae of Ch. rufifacies under 4 mean cyclic temperatures (15.6, 21.1, 26.7, and 35.0°C)
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and one constant temperature (25.0°C). The development from egg to adult under all
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regimes ranged from 190 to 598 h. The constant temperature of 25°C produced a range of
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pupariation times from 134 to 162 h, with adult emergence ranging from 237 to 289 h.
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These results are similar to what we report in the present study (see Table 2). No pupae
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survived at the highest temperature (39°C), indicating that the upper threshold temperature
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for Ch. rufifacies is at or below this temperature. The upper threshold temperature reported
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here is lower than the previous upper thermal limits documented for this species, which
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was 43°C [28]. The developmental time from egg to eclosion at 27°C was similar to the
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findings of Byrd and Butler [27], but was faster compared to findings of Swiger et al. [29].
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Whether these differences have a genetic basis or are plastic responses to different
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environmental conditions needs further evaluation.
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Comparison to other taxa
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Our developmental data for Ch. rufifacies slightly differ from that of other blow fly
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species; Protophormia terraenovae, for example, has a lower threshold temperature of
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10.3°C for egg hatching, of 10.7°C for the first instar larval stage, of 10.7-11.0°C for the
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second instar larval stage, of 11.0°C for the third instar larval stage, of 11.5-11.7°C for
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pupariation, and of 11.7°C for adult emergence [30]. Further, the lower threshold
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temperature from oviposition to eclosion in this species differed among geographic
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regions: for a population from Austria it was estimated at approximately 9°C [10,31],
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while in a Russian population it was estimated to be 7.8°C [9] and in a population from
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British Columbia it was 9.8°C [4]. Similar differences between local populations were also
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found in other species. Davies and Ratcliffe found a threshold of 3.5°C for Calliphora
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vicina in England [32], which differs from the estimate by Donovan et al. [33], who
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reported a threshold of 1°C from the same country, whereas Marchenko recorded a base
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temperature of 2°C for the same species from the Soviet Union [9]. These examples show
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that temperature threshold may differ even within a country, further supporting the need to
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explore regional variation and to develop local data for forensically important taxa.
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Conclusions
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The developmental rate of Ch. rufifacies was significantly and positively associated with
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temperature. The longest development times of egg stage, larval stage and pupariation
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were found at the lowest rearing temperature of 15°C, while the shortest developmental
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times were found at the higher temperatures. However, no pupal survival was found at the
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highest temperature (39°C), indicating that the upper threshold temperature for this species
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in its native range must be below this temperature. Furthermore, larval body size was
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significantly influenced by temperature where the minimum threshold temperature was
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estimated to be 11.14°C for the transition from oviposition to pupariation, while it was
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9.5°C from oviposition to eclosion. The comparison to literature data showed consistency,
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especially with data from the same region, but also revealed local differences in this and in
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other species suggesting the need for locally adapted data sets for forensic studies.
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Table 1. Developmental times (mean ± SD) of different life stages of Ch. rufifacies at nine constant temperatures: egg stage, F8 = 100.83,
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