Muscidae (Diptera) of medico-legal importance associated with ephemeral organic substrates in seasonally dry tropical forests

Muscidae (Diptera) comprises one of the most important taxa in medical, veterinary and forensic entomology, especially due to their association with decomposing carcasses and cadavers. Yet, knowledge on their distribution and behaviour is still incipient in several biomes, which is the case of dry tropical forests. This study aimed to evaluate the attractiveness of different organic substrates to species of Muscidae in areas of seasonally dry forests (Caatinga) in Northern Brazil. Sampling was carried out in five Caatinga areas be‐ tween 2015 and 2017, using suspended traps baited with bovine spleen, fish or human faeces. When all samplings were combined, 3,176 adults of nine species were collected. The assemblages of muscids had higher richness and abundances on bovine spleen, which attracted 66% of all specimens, when compared to the other substrates. Musca domestica was the most abundant species, and it was dominant in assemblages associated with spleen and fish. Assemblages attracted to spleen and fish had higher similarity, differing from those associ‐ ated with faeces. Differences in food attractiveness are presented for species of medical interest as pathogen vectors (e.g., Synthesiomyia nudiseta (Wulp, 1883)) and of forensic relevance as colonizers of human corpses (e.g., Ophyra aenescens (Wiedemann, 1830)). Key-Words. Atherigona orientalis; Caatinga; Carrion; Forensic entomology; Synthesiomyia nudiseta.


INTRODUCTION
Muscidae (Diptera) comprises one of the most important taxa in medical, veterinary and forensic entomology (Skidmore, 1985;Grzywacz et al., 2017).Yet, knowledge on their distribution and behaviour is still incipient in several biomes, which is the case of dry tropical forests (DTF), a complex set of environments that cover over one million km² in Africa, Americas, Asia and Oceania (Miles et al., 2006).In broad terms, DTF's are characterized by limited and irregular rainfall, high temperature throughout the year, low air and soil humidity and a set of specializations of local fauna and flora to adapt to xeric conditions (Bullock et al., 1995).Sudden shifts in resource availability in dry forests, regulated mostly by pulses in water availability, act as a selective force that favours insects that can promptly locate and colonize patchy substrates, such as carrion.
Dry tropical forests are among the most threatened environments in the world due to climate change, deforestation, fire and conversion to agriculture, particularly in Brazil (Miles et al., 2006;Santos et al., 2011).The Brazilian seasonally dry tropical forest (SDTF), known as Caatinga, covers ca.800,000 km² and it is exposed to severe droughts that can last decades.It is the least studied biome in the Neotropics (Santos et al., 2011), which encourages research on insect diversity and behaviour in order to understand the dynamics of assemblages in patchy, ephemeral resources.
In this study, we performed a short-term survey of Muscidae species associated with decomposing substrates in fragments of SDTF.Specifically, we aimed to compare the attractiveness of different baits to muscids, with emphasis on species of medico-legal importance.
Adult flies were collected using traps described by Oliveira et al. ( 2016), a low-cost device that relies on olfactory cues and positive phototropism.The traps were suspended 1.0 m above the soil and contained decomposing matter as bait (150 g, after 48 h exposure to 25°C).Three types of baits were used: bovine spleen, fish (sardine) and human faeces.Sixteen traps of each treatment separated from one another by 25 m were exposed in the field for 48 h in each environment, after which all traps were taken to the laboratory and adults were identified (Carvalho et al., 2002).
The assemblage of Muscidae associated with each substrate was characterized by: richness, relative frequency, Shannon-Wiener's diversity index, Simpson's dominance and Pielou's equity index, while abundance was compared using a Chi-square test (minimum of 100 adults/treatment).To test for similarities in the assemblages on each bait, we built a similarity matrix, through the index of Bray Curtis, after data transformation log(x + 1).The statistical packages Primer 5.0 and Biostat 5.0 were used, with 5% significance level.

RESULTS
When all samplings were combined, 3,176 adults of nine species were collected (Table 1).Overall, the most abundant species were M. domestica, Atherigona orientalis Schiner, 1868 and S. nudiseta, corresponding to 64.0%, 18.3% and 12.6% of all specimens, respectively.Insect abundance differed between treatments (χ² = 52.39;d.f.= 2; P < 0.01), as spleen was associated with the highest number of flies (66.0% of total).Higher abundance on spleen was observed for M. domestica and A. orientalis, while faeces were the most attractive substrate to S. nudiseta (Table 1).The number of female adults was significantly higher than their male counterparts for all species in all substrates (P < 0.0001) (Fig. 2).
Diversity indices ranged from 0.86 (spleen) to 1.16 (fish) and dominance varied from 0.39 (fish) to 0.50 (spleen), which is explained by the numerical dominance (73.2%) of M. domestica on spleen.Equity indices varied from 0.41 (spleen) to 0.68 (fish), showing higher unifor- mity in the relative frequency of the species on the latter.Cluster analysis evidenced high similarity between muscid assemblages on spleen and fish (Fig. 3).

DISCUSSION
The number of species registered in this study did not differ much from other studies performed in rainforest fragments, urban areas and agroecosystems in South America (Vasconcelos & Araujo, 2012) and was similar to that recorded in longer studies using large vertebrate carcasses as baits in fragments of seasonally dry tropical forests (Vasconcelos et al., 2016).This suggests that small baits can be surrogates for short-term field inventories of diversity of copronecrophagous Muscidae, without the ethical, logistical and economical restraints imposed by the use of animal carcasses.
The consumption of several types of decomposing substrates by Muscidae reported in the literature (Savage, 2002) is corroborated in this study, especially for M. domestica, A. orientalis and S. nudiseta, which are widely distributed and show adaptations to the harsh condition of the Caatinga (Carvalho, 2002).The high similarity of muscid assemblages associated with fish and spleen have been reported to occur in other environments such as rainforest and agroecosystems (Vasconcelos et al., 2015).Volatile organic compounds (VOC's) released by decomposing fish are thought to attract a high diversity of necrophagous dipterans of Muscidae and other families, including also Calliphoridae and Sarcophagidae (Carmo et al., 2017).Faeces, on the other hand, are nutritionally poorer than carcasses and tend to be used as food substrate for the development of fewer species (D'Almeida & Almeida, 1998).According to Blackith & Blackith (1993), flies visit faecal matter to oviposit, to gain water from fresh dung in dry climates, and to gain nourishment from the dissolved or finely particulate substances.This is critical in the Caatinga, where resources are short-lived and erratic.Considering the scarcity of large native mammals, goats raised in small-scale livestock tend to provide the main input of faecal matter.
The low attractiveness of faeces to M. domestica reported here diverges from previous records that suggest strong colonization of faeces by this species (Greenberg, 1971).Although S. nudiseta have been frequently reported on fish carcasses (D'Almeida & Almeida, 1998), this study reinforces the use of faecal matter as an alternative and/or complementary substrate.The overall low abundance of Ophyra aenescens and Ophyra chalcogaster (Wiedemann 1824) is probably due to their preference for large carcasses (Barbosa et al., 2009).A. orientalis was strongly attracted to animal baits in this study but it has also been shown to feed on decaying plant material (D'Almeida & Almeida, 1998).Interestingly, the species is reported in association with carcasses in urban and forested areas (Barbosa et al., 2009;Vasconcelos et al., 2016).
Coprophagy is a common feeding habit of Gymnodia species ( Uribe-M. et al., 2010) and it is confirmed for Gymnodia debilis (Williston 1896) in this study.The low abundance of G. debilis in the five dry forest fragments in Brazil may reflect a stronger association of the species with urban/modified environment.The low abundance of Graphomya maculata (Scopoli, 1763) may be due to the fact that its larvae often prey upon other dipteran larvae and adults are mostly flower feeders (Skidmore, 1985;Marques & Couri, 2007).The numerical dominance of females found in the Caatinga has been a common pattern in surveys based on baits and carrion performed elsewhere (Mulieri et al., 2015;Carmo et al., 2017) and reflects the selection of the substrates for the development of the immature stages.Furthermore, the capture of few   male specimens compromises the register of a few species of Muscidae whose identification depends on the examination of the male genitalia.Data presented here contribute to expand the knowledge on the biogeography of Muscidae species, especially given the shortage of information on DTF's in the Americas and other continents.The unfavourable conditions of DTF's (e.g., severe drought) act as selective forces for the development of physiological traits to cope with water-stressed environments and ephemeral resources.In the case of coprophagies species, the scarcity of large vertebrates in the Caatinga further stimulates fierce competition for patchy resources.
The non-discriminatory feeding behaviour of S. nudiseta, M. domestica and A. orientalis and their synanthropy highlights their medical importance due to their role as mechanical vectors of pathogens and as causal agents of myiasis (Greenberg, 1971;Skidmore, 1985;Uribe-M. et al., 2010).Brazilian DTF's suffer a radical transformation caused by urbanization and intensive agriculture so that field surveys are crucial for the monitoring and control of species of medical and veterinary importance.Unsanitary conditions observed in many breeding systems are likely to maximize the potential of muscid in causing myiasis on goats in Caatinga fragments (Barbosa & Vasconcelos, 2015).
Finally, according to Grzywacz et al. (2017), Muscidae have received relatively little attention in forensic field studies although they can colonize bodies in urban and forested areas, under sunny and shade conditions, in exposed and concealed situations.Recently, O. aenescens was found on the soil below a hanged cadaver in Northeastern Brazil (Vasconcelos et al., 2017), which suggests that the actual role of Muscidae in forensic entomology is far from being properly appraised.In this scenario, databases on species-habitat association may help in the evaluation of the potential of species as indicators of site of death.

Figure 3 .
Figure 3. Cluster analysis for the similarity between muscid assemblages, according to the type of bait.

Figure 2 .
Figure 2. Sex ratio (female/male) of muscids collected in SDTF's fragments in Brazil, according to the type of bait (A) and species (B).

Table 1 .
Abundance and relative frequency (%) of Muscidae species registered on three types of bait in fragments of seasonally dry forests in Brazil.