A Survey Of Calliphoridae Genera In Small Island Of Braila

A SURVEY OF CALLIPHORIDAE GENERA IN SMALL ISLAND OF BRAILA, HIGHLIGHTING THEIR PREFERENCES FOR DIFFERENT TYPES OF BAIT

AUTORI

Running title: Calliphoridae genera and bait type preferences

ABSTRACT

Due to the role that Calliphoridae (Diptera) plays in ecological processes as: food source for invertebrates and vertebrates,, pollinators and decomposers, their research is particularly useful in understanding the mechanisms by which anthropogenic pressures contributed to biodiversity loss and, thereby to erosion of ecological services. However, the composition and structure of Calliphoridae communities and their relation with provision of ecosystem services at different spatial and temporal scales is still poorly known. The experimental studies have been conducted only in some environmental conditions: biogeographic regions, habitats, and seasons. In addition, the lack of consensus regarding the methods for trapping blowflies, including animal tissues used as attractant further diminish the data comparability. This paper presents results obtained in a field research that deemed to assess the effects of different animal tissue used as baits – liver, fresh and decaying muscle, bones and feathers – on the assessed necrophilous Diptera communities, in terms of their density and relative relative abundance. Results demonstrate the presence of four genera of Calliphoridae: Calliphora, Lucilia, Protophormia and Pollenia, in the study area; Calliphora was the dominant one. Their density and relative abundance revealed Calliphoridae genera preference for liver baits. Moreover, the particularities of habitat and the specificity of climate and biogeographic conditions seems to be instrumental in controlling Diptera’ preferences for a certain bait type, making it difficult to extrapolate results to a larger spatial scale.

Key words: Calliphoridae genera, bait type, SPA site, Romania.

INTRODUCTION

The Insects of Diptera order (flies) represent an important component of any ecological system due to their great numerical abundance, species richness (over 150,000 species) and trophic spectrum diversity (Courtney et al., 2009). Calliphoridae, called blowflies, blue bottles or green bottles, are robust flies with body from medium to large size and lenght about 4 -16 mm distributed worldwide (Lehrer, 1972; Capinera, 2011). Except the negative impact that some species have as pests in animal and human health (Zumpt, 1965; Lehrer, 1972; Capinera, 2011; Greco et al., 2014), their role in ecosystems is particularly important. These insects are food source for invertebrates and vertebrates (Capinera, 2011), acting as food web links and plants pollinators (Heath, 1982; Clement et al., 2007; Huda et al., 2015), – thus contributing to the ecosystems resilience and supply of the support and production ecological servicesand production capacity. In addition, together with microorganisms, blowflies have a fundamental role in animal organic matter decomposition (De Carvalho, & Linhares, 2001; Byrd & Castner, 2010; Capinera, 2011; Merritt & De Jong, 2015) whose breakdown is a source of energy and nutrients (Moore et al., 2004), thus providing important ecosystem services. The presence on carcasses made them to be used mainly as forensic indicators in legal medicine (Zumpt 1965; Byrd & Castner, 2010 ).

The importance of Calliphoridae in regulating the size of populations, species richness and diversity of primary producers and consumers of different orders, makes their research essential for understanding the mechanisms by which anthropogenic pressures contributed to biodiversity loss and erosion of the ecological services. However, the composition and structure of Calliphoridae communities and their relation with provision of ecosystem services at different spatial and temporal scales is still poorly known. The experimental studies have been conducted only in some biogeographic regions, habitat type and seasons (Brundage et al., 2011; Tomberlin et al., 2012, Vasconcelos et al., 2015). Besides, the lack of consensus regarding methods for trapping flies, including type of animal tissue used as bait to attract the insects generate additional problems in the attempt to extrapolate the results at larger spatial scales (Boonchu et al., 2003; Moretti & Godoy, 2013; Farinha et al., 2014; Vasconcelos et al., 2015).

The paper presents the preliminary results obtained in a broader field research that deemed to assess the structure and role of necrophilous communities in a Natura 2000 Special Protected Area (SPA) along the Lower Danube River System. According to our knowledge, this is the first study of its kind in the specific environmental context of Danube islands and in Romania. The main objectives of the paper are: a) to assess the presence of the Calliphoridae genera in the Small Island of Braila and b) to assess their preferences for different animal tissue used as baits.

MATERIALS AND METHODS temperatura

The present study is based on an experiment conducted for 13 days, in May 2012, in 13 random sampling stations located over the Harapu Island (44 º59’52’’N; 27 º54'31' E), mostly in its Northern forested part that is more accessible as compared to the Southern part. This island is a part of the Small Island of Brăila Natural Park, an international socio-ecological long-term research area (ILTER), a wetland area remained under the natural flooding regime, declared as a RAMSAR site (Adamescu et al., 2004), and it is characterised by a rich diversity of wild bird species (ROSPA0005) and habitats (ROSCI0006).

Five types of chicken baits – liver, fresh and decaying (two days old) muscle, bones and feathers, of 5±0.25 grams fresh weight were used. In order to increase the statistical relevance and coverage of the habitat diversity, small bait carrions were considered appropriate (Farinha et al., 2014). The carcasses were collected from an intensive poultry farm, immediately after chickens’ natural dead, sliced and frozen until they were used. A total of 101 traps made of 2-liter plastic bottles, fitted with holes (1 cm diameter) for blowflies access were suspended at two meters above ground, in trees (Fig. 1). A group of three traps was randomly installed in one tree. For statistical relevance between 6 and 12 replicates per sampling station were used (2-4 trees with suspended trap).

A small quantity of water supplied with few detergent drops for insect immersion was added at the bottom of traps. Because the immersion liquid leached from the trap, 28 traps were excluded from the analysis. The insects were collected from the traps at six times: 1, 2, 3, 6, 9 and 13 days after installation (due to a heavy rain the traps from some stations were not emptied in the first day). The adult insects were collected and stored in 70% alcohol. Calliphoridae were identified at genera level using Lehrer’ (1972) identification key under an Olympus stereomicroscope with 400x magnitude.

Classical calculation methods (Rîșnoveanu & Popescu, 2011) were used to assess densities (nr. ind./ trap*day) and relative abundances (%) in baited traps at family and genera level. Mann-Whitney U test (Statistica 10) was used to test the significant differences between genera densities on different baits.

RESULTS

Nine orders of insects were identified in the experimental samples: Hymenoptera, Diptera, Coleoptera, Mecoptera, Neuroptera, Trichoptera, Lepidoptera, Hemiptera, Dermaptera. The adult Diptera represented over 28% of the total number of trapped insects. Out of the 3367 trapped Diptera, Calliphoridae family represented 35%. Their densities were significantly higher on liver (2.55 ind/trap*day) than on each fresh muscle (1.38 ind/ trap*day; p=0.0040), decaying muscle (1.22 ind/ trap*day; p=0.0289), bones (0.66 ind./ trap*day; p=0.0000) and feathers (0.03 ind./ trap*day; p=0.0000) (Fig. 2). Significant differences were also recorded between feathers and each fresh (p=0.0006) and decaying muscles (p=0.0010). No significant differences were recorded between the other bait types (p>0.05) (Fig.2). The highest relative abundance of Calliphoridae individuals (56%), was recorded in the liver baited traps, followed by fresh (23%) and decaying muscle (14%), bones (6%) and feathers (~1 %) (Fig. 3).

Calliphoridae family was represented in samples by four genera: Calliphora (Robineau-Desvoidy, 1830), Lucilia (Robineau-Desvoidy, 1830), Protophormia (Townsed, 1908) and Pollenia (Robineau-Desvoidy, 1830). Calliphora represented over 80% of the total number of Calliphoridae (Fig. 4).

The density of Calliphora individuals was significanly higher on liver (2.20 ind/ trap*day; p<0.05) than on the other baits (Fig. 5). Lucilia density (0.32 ind/ trap*day) was slightly higher on liver baits, but there were significant differences only as compared to bones and feathers (p<0.001). No significant differences (p>0.05) were found for Protophormia and Pollenia (Fig. 5).

The highest relative abundances of Calliphora (100%) were recorded on feathers where this was the only one Calliphoridae genus present (Fig. 6). The relative abundances of Lucilia were higher on muscle: decaying (19.91%) and fresh (19.68%) (Fig. 6).

Out of the five types of bait, only liver and decaying muscle trapped the entire diversity of Calliphoridae genera (Fig. 6).

DISCUSSIONS

The experimental study revealed that out of the total number of insects trapped on the carrion baits dipterans represented 28%. Calliphoridae accounts for as much as 35% of the total dipterans. The results clearly highlight their preferences for liver baits, with both the average numbers (density) and the relative abundance of individuals in liver baited traps significantly higher than on fresh and decaying muscle, bones and feathers baits. The same density pattern in individuals’ preferences was observed at genus level for Calliphora and Lucilia with the only exception that in the case of Lucilia, the differences between their densities and relative abundances on liver and muscle baits were not statistically different. Regarding the relative abundances, Calliphora were the dominat genus on all types of bait, with higher abundances on feather and lower on decaying muscle. Instead, relative abundance for Lucilia was higher on muscle (decaying and fresh) than on liver baits. These issues could support the existence of competition for resource between blowflies (Williams et al., 2014), knowing that the larger flies as Calliphora could removed/ exclude (ei folosesc cuvantul OUSTED) the smaller ones such as Lucilia (Von Aesch et al., 2003). The higher relative abundance of Calliphora species are also reported in other studies, for all seasons while Lucilia genus, with some thermophilous species, was abundant in summer/ higher temperature (Baz et al., 2007; Zabala et al., 2014).

In an attempt to compare these preferences with those existing in the literature, we found only few studies that tested the effectiveness of various tissues and organs in capturing blowflies (Boonchu et al., 2003; Moretti & Godoy, 2013; Vasconcelos et al., 2015) and even less of them used different baits from the same animal species (Boonchu et al., 2003; Farinha et al., 2014). The most frequently used baits are pig tissues (Boonchu et al., 2003; Farinha et al., 2014). Differences between bait origin, biogeographic regions (Vasconcelos et al., 2015), habitat type (Hwang & Turner, 2005; Brundage et al., 2011; Ibrahim et al., 2013), abiotic parameters (Anderson, 2001), were reported to lead to differences in Diptera taxa identity and activity (Parmenter & MacMahon, 2009; Barton et al., 2013) and so, preferences for a certain bait (Vasconcelos et al., 2015). All of these are limiting the comparability of the results. However, consistent results are reported supporting the preference of some species of Chrysomya (Boonchu et al., 2003) and Lucilia (Farinha et al., 2014) for liver and not for muscle baits. In contrast, Calliphora species were more abundant on fresh muscle than on liver.

The preference of Calliphoridae for liver baited trap could be explained by autolysis, a natural decomposition process that was reported to be faster in organs (i.e. liver) than for instance in muscle (Vass, 2001; Kasper, 2013; Hau et al., 2014). At the end of this process, the tissues are softening and the decomposition mediated by micro-organisms (heterolysis) could begin (Vass, 2001; Kasper, 2013). The gases released by heterolysis act as an olfactory stimulus (Gennard, 2007) which attracts a large number of blowflies. Moreover, better vascularisation of liver made the odor to be stronger than in the case of muscle. The less smelly and poor quality food as bones and feathers attracted fewer blowflies.

Given its advantages in catching a larger number of individuals and species of Diptera (Vasconcelos & Araujo, 2012), the liver was one of the most commonly used bait for different purposes (Von Aesch et. al., 2003; Young, 2008; Brundage et al., 2011; Muirhead-Thompson, 2012; Vasconcelos & Araujo, 2012; George et al., 2012; Weidner et al. 2014; Khoso et al. 2015). According to some authors (DeJong & Chadwick, 1999; DeJong & Hoback, 2006), the level of abundance of flies on the carrion baits represents a good indication for the associated decomposition rates. Nevertheless, Farwig (2014) considers that the decomposition rates are rather influenced by the species composition then by the abundances of flies.

The four genera identified in our samples: Calliphora (Robineau-Desvoidy, 1830), Lucilia (Robineau-Desvoidy, 1830), Protophormia (Townsed, 1908) and Pollenia (Robineau-Desvoidy, 1830), are widely distributed across biogeographic regions and were previously cited in Romania as well (Lehrer, 1972).

While Calliphora and Lucilia were reported in most studies that have targeted decomposition of carcasses in different biogeographic regions (Byrd & Castner, 2010; De Carvalho & Linhares, 2001; Anderson, 2011; e Castro et al., 2011; Farinha et al., 2014), Protophormia and Pollenia are less frequently mentioned in such studies (Byrd & Castner, 2010; Anderson, 2011; Baz et al., 2007). In our samples the last two genera occurred in very low number that will not allow us to draw an accurate conclusion about their preferences for a certain bait type. The role of the species belonging to Calliphora, Lucilia and Protophormia genera in the decomposition of carcasses are unanimously recognized in the literature. Nevertheless, the role of species assemblage belonging to Pollenia genus still remains unclear as they are well known as being parasites on earthworms (Baz et al., 2007). More studies are needed to clarify both the species richness, their distribution and preferences and some inter- and intraspecific mechanisms, that were rarely mentioned as competition (Williams et al., 2014) and facilitation (Rosati & VanLaerhovea, 2008).. This become even more relevant if we consider that these genera include species considered to be pests. They are vectors for viruses and bacteria which produce diseases to both wild animals and humans (Lehrer, 1972; Fisher et al., 2004; Gubler, 2009; Wanaratana et al., 2013). For instance, transmission of Mycobacterium avium subsp. paratuberculosis to wild birds by species of the Calliphora and Lucilia genera is well known (Fisher et al., 2004). Calliphora species were reported as passive vectors for H5N1 virus to birds and humans (Sawabe et al., 2009, Wanaratana et al., 2013) and, together with Lucilia were associated with wound myiasis (Rognes, 1991; Little, 2008; Parchami-Araghi et al., 2015) in birds. As our research area is managed as a Natura 2000 SPA site, characterized by a high birds species diversity, understanding the Caliphoridae species distribution and their ecology in relation to wild animal species represents key elements for informing the managerial decision. It is also worth to be mentioned that the only baits that trapped all four genera of Calliphoridae were liver and decaying muscle. Their presence on only these two types of bait highlighted the importance that the type of bait used has on accuracy of biological diversity estimation for various habitats / types of ecosystems / biogeographic regions.

CONCLUSIONS

Calliphoridae were present in the study area by four genera: Calliphora, Lucilia, Protophormia and Pollenia; Calliphora was the dominant one.

The study revealed clear preferences of blowflies for certain bait; chicken liver was the preferred bait in terms of genera densities and/ or relative abundance. In addition, all four genera were presented only in liver and decaying muscle baited traps. This suggests that bait type influence the studies aiming to characterize/ assess the biodiversity of different regions and its relationship with ecological services. Moreover, the animal species that provide the experimental bait, together with the particularities of habitat and the specificity of climate and biogeographic conditions seems to be instrumental in controlling Calliphoridae’ preferences for a certain bait type, making it difficult to extrapolate results to a larger spatial scale.

Knowing these aspects, together with Calliphoridae as animal and human pests, made so studies to be relevant for understanding the mechanisms of diseases transmition pentru intelegerea mecanismelor de transmitere a bolilor

In order to have accurate conclusions regarding the preferences and facilitation of Calliphoridae for a certain type of bait is absolutely necessary to extend research on different seasons and habitats as well as to foster the analyses to the identification at species level.

Acknowledgements: we thank to our technicians Marius Bujor, Aglaia Parvu, Costel Amarioarei, Aristide Dimache, Anisoara Bobeica, Sandica Visinescu, who helped in the field experiment and to Geta Niculae for sorting individuals from samples. This work was supported by the strategic grant POSDRU/159/1.5/S/133391, Project “Doctoral and Post-doctoral programs of excellence for highly qualified human resources training for research in the field of Life sciences, Environment and Earth Science” cofinanced by the European Social Found within the Sectorial Operational Program Human Resources Development 2007 – 2013.

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List of figures:

Figure 1. The chicken baited traps suspended in trees

Figure 2. Figure 2. Densities of Calliphoridae (ind./ trap*day) on different types of bait. Fm = fresh muscle, dm = decaying muscle, l = liver, b = bones, f = feathers.

Figure 3. Figure 3. Relative abundances (%) of Calliphoridae on different types of bait

Figure 4. Relative abundances (%) of Calliphoridae genera in traps.

Figure 5. Densities of Calliphoridae genera (ind./ trap*day) on different types of bait. fm = fresh muscle, dm = decaying muscle, l = liver, b = bones, f = feathers. Point = mean, box = ±SE, Whisker = Mean±1.96*SE

Figure 6. Relative abundances (%) of Calliphoridae genera on different types of bait. fm = fresh muscle, dm = decaying muscle, l = liver, b = bones, f = feathers.

Figure 1. The chicken baited traps suspended in trees

Figure 2. Densities of Calliphoridae (ind./ trap*day) on different types of bait. Fm = fresh muscle, dm = decaying muscle, l = liver, b = bones, f = feathers.

Figure 3. Relative abundances (%) of Calliphoridae on different types of bait

Figure 4. Relative abundances (%) of Calliphoridae genera in traps.

Figure 5. Densities of Calliphoridae genera (ind./ trap*day) on different types of bait. fm = fresh muscle, dm = decaying muscle, l = liver, b = bones, f = feathers. Point = mean, box = ±SE, Whisker = Mean±1.96*SE

Figure 6. Relative abundances (%) of Calliphoridae genera on different types of bait. fm = fresh muscle, dm = decaying muscle, l = liver, b = bones, f = feathers.