Bionomics of Peckia (Euboettcheria) Anguilla and Peckia (Euboettcheria) Collusor (Diptera: Sarcophagidae) in The Laboratory.

Flesh-ies are important mechanical vectors that cause myiasis in man and animals and they also play an important role in forensic entomology. Postmortem interval can be estimated using data available in the literature on the biology of the species. This study aims to elucidate the bionomics of these two species in order to provide preliminary data for medical, veterinary and forensic entomology analyses. We analyzed the larval stage durations (L1–L3), weight of the mature larvae (L3), L1-pupae stage duration, L3-pupae stage duration, pupal stage duration, L1–adult duration, adult emergence, atrophies and the viability of larvae and adults. The mean duration of the L1–adult of Peckia anguilla was 22.6 days and 21.8 days, in the rst and second experiments. Mean lifespan for females and males was 75 and 69.6 days, respectively. The mean duration of the L1–adult of Peckia collusor was 25.9 days and 23.8 days, in the rst and second experiments. Mean lifespan for females and males was 77.5 and 73.5 days, respectively. Although the two species presented similar results in relation to their post-embryonic development, P. collusor showed an adult lifespan longer, laying 1983 larvae throughout the experiment, while P. anguilla depositing 2298 larvae.


Introduction
Forensic entomology is the branch of science responsible for studying insects and other arthropods associated with criminal and civil events. Knowledge about the life cycle and feeding habits of these organisms provides useful information to estimate the postmortem interval (PMI), which can help to solve crimes (Oliveira- Knowledge of the biology of these species is extremely important because of their close relationship with humans and this knowledge can be used to control the spread of these pathogen carriers. In addition, this knowledge provides support for forensic entomology and can be used in criminal investigations, such as at a crime scene or in cases of neglect of the elderly, children and the disabled (Benecke et al 2004). The comparison between bionomic data of species of the same genus and subgenus is essential for a good interpretation of the results, since these insects, even coexisting in the habitat, may have speci c ecological, morphological and biological characteristics.
This work aims to analyze the bionomics of P. (E.) anguilla and P. (E.) collusor through post-embryonic development, biotic potential, sex ratio and mean lifespan of adults in the laboratory, in order to provide primordial data for ecological, sanitary and medical-legal studies.

Materials And Methods
Colonies of Peckia (Euboettcheria) anguilla and Peckia (Euboettcheria) collusor were established from the adult samples collected on the campus of the Instituto Oswaldo Cruz (IOC / FIOCRUZ) (S 22° 51' 06" After collection, the insects were transported to the Laboratório de Entomologia Médica e Forense (LEMEF/IOC), where they were identi ed by a group-speci c key (Carvalho & Mello-Patiu 2008) and kept in wooden cages (30 x 30 x 30 cm ) coated with nylon mesh and conditioned in a climatic chamber regulated at a temperature of 27 ± 1 °C, relative humidity of 60 ± 10% and a 12h photoperiod. These muscoid dipterans received a saccharose solution of 80% and a putrefactive ground beef diet that also served to stimulate posture.
After larviposition, 400 neolarvas (L1), belonging to the rst generation of P. (E.) anguilla and P. (E.) collusor were removed from the meat with the help of ne brushes (number zero) and transferred to plastic containers (5 x 7 cm) containing 2g / larvae of putrefaction ground meat. Two experiments were done with four replicates, containing 50 larvae each. The experiments were performed at intervals of two weeks between them in order to con rm the results obtained.
These containers were placed in larger containers (10 x 10 cm) containing vermiculite (substrate for pupariation) and were kept in a climatic chamber with the same temperature, relative humidity and photoperiod as the colonies. In order to collect information on the bionomics of these species, the mature larvae (L3) were then weighed on a precision scale in order to obtain a biomass mean. After this, the larvae were individualized and packed in test tubes containing 1/3 of vermiculite and capped with cloth and elastic. Thus, the duration and viability of its larval, pre-pupal, pupal and neolarva periods to adult could be recorded.
To study the biotic potential and lifespan of the species, three wooden cages (30 x 30 x 30 cm) containing 15 couples from the rst four replicates of the tests were assembled. Ground beef in decomposition stage was offered daily to cages in order to record the number of L1 larvae laid by the females. These were recounted when they reached the L3 stage in order to record the viability of the postures. Mortality of males and females was also checked daily to generate their survival curves.
The survival curves for males and females were represented by the Weibull distribution model. This model shows if arthropods reared in laboratory are comparable with the expected survival curve to the wild ones. One of the advantages of using Weibull distribution for survival analysis is that, by estimating only two parameters, informations on both lifespan and type of survival curve are obtained (Sgrillo1982). Chi-square test was carried out to analyze the survival distribution of the insects in order to con rm if they followed the Weibull distribution model (Sgrillo 1982).

Results
The values of chi-square (0,3869 for males and 0,2093 for females of P. (E.) anguilla; 0,1722 for males and 0,1559 for females of P. (E.) collusor) showed a concordance between observed values and expected values, therefore, the survival distribution followed the Weibull model.

Peckia (Euboettcheria) anguilla
The larval viability found for larvae of P. (E.) anguilla at 27 °C in the rst experiment was 86%, whereas in the second experiment it was 90% ( Table 1). The mean duration of the larval period of this species in the rst experiment was 11.8 ± 7.4 days ( Table 2), ranging from 4 to 29 days. In the second experiment the duration of this period was 10.0 ± 6.4 days (Table 2), ranging from 3 to 24 days.  After mature larvae L3 left the diet they took an average of 1.49 ± 0.48 days to begin the pupation process in the rst experiment and 1.13 ± 0.87 days in the second experiment (Table 2). In the rst experiment, the shortest time a mature larva took to start the pupation process was one day, while the longest was ve days. In the second experiment, while some of the larvae also started the pupation process after only one day, others took up to 11 days.
Mature larvae that were unable to continue their development had a mean larval mass of 89.1 ± 22.5 mg (Table 3), with a minimum mass of 26.5 mg and a maximum mass of 167.7 mg in the rst experiment, whereas in the second experiment they obtained 81.3 ± 27.7 mg (Table 3), with a minimum of 21.4 mg and a maximum of 163.0 mg. The larvae that initiated the emergency process, but did not nish it, were only present in Experiment 1. They obtained an average larval mass of 113.1 ± 24.5 mg (Table 3) in the rst experiment, ranging from 85.1 to 139.2 mg.
The pupal viability was 43.6% in the rst experiment and 53.3% in experiment 2 ( Table 1). The mean duration of the pupal periods was 13.3 ± 2.9 days and 14.0 ± 2.2 days in the rst and second experiments, respectively ( Table 2). While in the rst experiment, the minimum duration of this stage was 3 days and the maximum period was 23 days, in the second experiment, these values ranged from 6 days to 22 days.
In the neolarva period, the mean number of days obtained for the rst experiment was 22.6 ± 5.4 days ( Table 2), ranging from 15 to 41 days and with a total viability of 37.5% (Table 1). In the second experiment, this period averaged 21.8 ± 4.4 days (Table 2), with a minimum of 18 and a maximum of 35 days, with a total viability of 48.0% (Table 1). The gender ratio was 0.5 in both tests.
Atrophied specimens were observed only in the rst experiment. A total of 18.9% of atrophy was recorded, male adults presented 14.7% of total atrophy and females 14.6% of that same atrophy (Table 4). In the case of males, two specimens presented atrophies on the wings and three specimens were totally atrophied ( Table 5). The females only presented six fully atrophied specimens (Table 5).  The maximum lifespan for male adults was 75.0 ± 17.7 days, and the minimum was 14.3 ± 10.7 days. The rst death occurred on the 2nd day and the last occurred on the 95th day ( Fig. 1). The maximum lifespan for females was 69.6 ± 8.6 days, and the minimum was 5.6 ± 3.5 days. The rst recorded death occurred on the 2nd day and the last occurred on the 79th day (Fig 2).
During the experiment, 2298 larvae L1 were deposited, from day 8 to day 79. The number of larvae deposited per female ranged from 0.22 on the 18th day to 10.0 on the 79th day (posture peak) with a mean of 2.92 larvae per female (Fig 3). Of these larvae, only 2085 developed until the L3 stage, so that the viability of L1-L3 was 90.7% (Table 1).

Peckia (Euboettcheria) collusor
The larval viability found for larvae of P. (E.) collusor at 27 °C in the rst experiment was 75%, whereas in the second experiment it was 89% ( Table 6). The mean duration of the larval period of this species in the rst experiment was 11.3 ± 0.9 days (Table 7), with a variation of 6 to 13 days. In the second experiment the duration of this period was 6.3 ± 2.2 days (Table 7), ranging from three to 11 days. The mean larval mass obtained in the rst experiment was 82.90 ± 6.70 mg (Table 8), with a minimum of 57.7 and a maximum of 94.9 mg. In the second experiment the average larval mass obtained was 84.90 ± 10.40 mg (Table 8), ranging from 25.3 to 150.1 mg. In the rst experiment, L1 to pupa lasted on average 13.71 days, while in the second experiment it lasted 8.54 days ( Table 7). The L3-pupae stage lasted 2.3 ± 2.0 days in the rst experiment and 3.1 ± 2.5 days in the second experiment (Table 7). After mature larvae L3 left the diet they took an average of 2.34 ± 2.06 days to begin the pupation process in the rst experiment (Table 7) and 3.19 ± 2.51 days in the second experiment (Table 7). In the rst experiment, the shortest time that a mature larva took to start the pupation process was one day, while the longest was 12 days. In the second experiment while some larvae started the pupation process after only one day, others took up to 15 days.
Mature larvae that originated male adults had a larger mass than the L3 that originated females in both experiments. The mean L3 larval mass that originated male adults in the rst experiment was 84.0 ± 21.0 mg (Table 8), with a variation of 63.3 to 94.1 mg and the L3 that gave rise to females was 81.6 ± 6.8 mg (Table 8), ranging from 57.7 to 94.9 mg. In the second experiment the mean L3 larval mass that originated male adults was 86.6 ± 9.7 mg (Table 8), with a variation of 60.4 to 119.0 mg and of the L3 that gave rise to females the mean mass was 84.7 ± 11.0 mg (Table 8), ranging from 37.6 to 107.6 mg.
The mature larvae that were unable to continue their development had a mean larval mass of 83.0 ± 4.0 mg (Table 8), with a minimum of 78.4 mg and a maximum of 88.2 mg, in the rst experiment, whereas in the second experiment the mean mass obtained was 89.6 ± 27.7 mg (Table 8), with a minimum of 25.3 mg and a maximum of 150.1 mg. The larvae that started the emergency process, but did not nish it, had a mean mass of 87.9 ± 0 mg in the rst experiment (Table 8), without minimum and maximum, since there was only one specimen. In the second experiment, the mean larval mass for this category was 88.3 ± 10.3 mg (Table 8), with a minimum of 81 and a maximum of 95.7 mg.
The pupal viability was 96.7% in the rst experiment and 74.7% in the second experiment ( Table 6). The mean duration of the pupal period was 12.5 ± 1.3 days and 14.8 ± 1.3 days in the rst and second experiments, respectively (Table 7). While in the rst experiment, the minimum duration of this stage was three days and the maximum period was 17 days, in the second experiment, these values ranged from 12 days to 19 days.
In the neolarva period, the mean number of days obtained for the rst experiment was 25.9 ± 3.6 days (Table 7), with a variation of 11 to 39 days and a total viability of 72.5% (Table 6). In the second experiment, this period averaged 23.8 ± 2.7 days (Table 7), with a minimum of 19 and a maximum of 34 days, presenting a total of 66.5% of viability ( Table 6). The gender ratio was 0.5 and 0.6, in the rst and second experiments, respectively.
The percentages of male and female adult atrophies that emerged were recorded, with 8.2% total atrophy in the rst experiment and 19.4% in the second experiment (Table 4). In the rst experiment, the male adults presented 5.3% of atrophy (Table 4), and four specimens were in a totally atrophied state ( Table 5). The females of this experiment presented 10% of total atrophy (Table 4), and seven were totally atrophied ( Table 5). In the rst experiment, there was one atrophic specimen with undetermined sex, representing 0.6% atrophy in this category ( Table 4).
The total percentage of male adults atrophied in the second experiment was 13.7% (Table 4), of which two had atrophied wings and six were totally atrophied ( Table 5). The total percentage of female adults atrophied in the second experiment was 22.6% (Table 4), of which two also had atrophied wings and 15 were also totally atrophied (Table 5). There was an atrophied specimen whose sex could not be identi ed, so that the percentage of atrophy here was 0.7% (Table 4).
The maximum longevity of male adults was 77.5 ± 0.8 days (Fig 4), while for females it was 73.5 ± 7.5 days (Fig 5). The minimum longevity of male adults was 11.5 ± 9.3 days (Fig 4), while for females it was 6.2 ± 4.5 days (Fig 5). The rst death of the adult males occurred on the 2nd day and the last occurred on the 79th day (Fig 4). The rst death of the female adults was recorded on the 1st day, while the last was on the 79th day (Fig 5).

Page 11/18
The results of the survival curves of P. (E.) collusor were concordant with the χ 2 test, as well as the expected results, allowing the survival curves to follow the Weilbull distribution model. In the experiments done with P. (E.) collusor at 27 °C the value of χ 2 for males was 0.1722 and for females it was 0.159. This difference was not considered signi cant.
Throughout the experiment 1961 L1 larvae were deposited from the 9th to the 71st day. The number of larvae deposited per female ranged from 0.2 on the 40th day to 7 on the 25th day (posture peak), with a mean of 2.36 larvae per female (Fig 6). Of these larvae, only 1668 were able to develop until the L3 stage, so that the viability of L1-L3 was 85.1% (Table 6).

Discussion
Bionomic studies have shown that knowledge about the post-embryonic development of muscoid dipterans is essential to determine the immature ages, especially larvae, as well as the biotic potential and longevity of adults. This is a very important step for forensic entomology, since the PMI can be estimated from this data ( The experiments were carried out in an ideal temperature range for insect development, which makes the data obtained in this work act as the comparative basis of the PMI calculation. Kamal (1958)  ) collusor had a mean weight higher than the larvae that originated female adults, similar to that observed by Salviano et al (1996). According to Slansky & Scriben (1985), adult insect size and body mass are the two main factors that in uence its performance. Size also intervenes directly on mating behavior and dispersion. Body mass reveals the amount of energy and nutrients stored. The two factors together can in uence the nal fecundity of adult ies, which makes it necessary to include this type of information in bionomic studies. In the work of da-Silva-Xavier et al (2015) the larvae with body mass below 22 mg of P. (S.) lambens completed the pupation but the adult insects did not emerge, which can be considered as a limiting factor for adult development of this species. In the present study, similar results were obtained only in the second experiment of P. (E.) collusor, where the lowest larval body mass found was 25.3 mg, whose larva completed its pupation, but did not emerge. In the second experiments with P. (E.) anguilla at 27 ºC a larva presented 17.8 mg of larval body mass, differing from the mean of larval body mass, but was able to continue its development, giving rise to a male adult insect. In relation to the species O. amorosa, da-Silva-Xavier et al. (2015) did not observe a limiting weight, since adults emerged from pupae with a minimum weight of 14 mg. This nding was also observed in the experiments performed with P. (E.) collusor, the lowest value found for the body mass of an L3 was 60.4 mg, but this was able to carry out the pupation process, develop and generate an adult without anomalies.  Ferraz (1995) do not corroborate the a rmation of Salviano et al (1996), who justi ed a lower longevity of females in relation to males, due to the ovarian development. On the other hand we believe that males expend a lot of energy at the time of copulation and consequently have a lesser longevity.
Throughout the experiment, females of P. (E.) anguilla and P. (E.) collusor deposited a total of 1326 and 1983 larvae in ground meat, respectively. Values similar to those found by da-Silva-Xavier et al (2015) for P. (S.) lambens (1433). These same authors also reported 4781 larvae were deposited for O. amorosa. The females of O. amorosa had a much higher longevity than P. (E.) anguilla, which would explain the greater number of deposited larvae. The longevity of O. amorosa females was similar to that found for females of P. (E.) collusor, but the latter deposited a smaller number of larvae. These results corroborate the high biotic potential of O. amorosa under these laboratory conditions. The posture peak of P. (E.) anguilla and P. (E.) collusor, was 3.7 and 6.4 days, respectively, suggesting a greater preference of P. (E.) collusor in depositing its larvae in ground beef. On the other hand da-Silva-Xavier et al (2015)