Toxicological effects of caffeine on blowfly fecundity and survival

Trabalho vencedor do 1º Concurso de Trabalhos Originais GICFinho 2017
por: Karen Mira (Estudante Pós-Graduada, Instituto Superior de Ciências da Saúde Egas Moniz, Monte de Caparica)

Abstract:

For many years insects were ignored in the field of forensic investigation but, with the development of the study of ecology and entomology, this group has become important for the estimation of postmortem interval (PMI). The science of studying arthropods and insects in criminal investigations is called Forensic Entomology. Insects are usually associated with the body after death because it is their natural tendency to colonize it, whether the corpse is inside a dwelling or in an open space. They play a very important role in the decomposition of organic matter, which makes them a potential source of evidence in cases of homicide or suspicious death. From soon after death, insects are attracted by a decaying body due to gases released by bacterial activity. Necrophagous flies, particularly blowflies (Diptera: Calliphoridae) are the first arrivals. As primary colonizers, they are the most relevant group with which to estimate the post mortem interval. However, the insects’ behaviour can be affected by ingested drugs or toxins present in the body. Entomotoxicology is a relatively new area of Forensic Entomology, which covers the analysis and identification of xenobiotics, especially in necrophagous diptera larvae. This science observes the effects of these toxic substances on the development of insects, which can lead to a discrepancy in the calculation of the PMI. This study aimed to observe the changes that caffeine causes in blowflies, particularly in oviposition and survival, and the results obtained showed that this toxin has effects on the larval stages, accelerating the development of the animals. Once the flies emerged, this toxin also has an effect on the flies, decreasing their longevity.

Keywords: Forensic entomology, Decomposition process, Calliphoridae, Blowfly life-cycle, Oviposition, Caffeine 

1. Introduction
1.1. Forensic entomology 

Forensic entomology is the science of studying insects and other arthropods (LeBlanc and Logan, 2010), by collecting and analysing them in a forensic context. 

Much information can be extracted from these insects. Sharma et al., (2015) suggests the importance of having the knowledge about their biology, behaviour and distribution because it can answer several questions providing information on where, when and how the crime was committed. The main aim of forensic entomology is to establish the postmortem interval (PMI) being a valid method to support chemical analysis or helping police investigations (Turchetto and Vanin, 2004). Anderson and Van Laerhoven (1996) defined postmortem interval of a body as being the time since death occurred until it is discovered. 

Klotzbach et al., (2004) stated the importance of collecting living insects from the corpse in order to study their development under laboratory conditions. According to Kashyap and Pillai (1989) analysing the corpse insects is the best and most accurate method to determine the PMI, when investigated up to 3 days from the moment of death. 

1.2. Decomposition process 

As soon as death occurs, all the cells become deprived of oxygen. The chemical reactions inside the cells accumulate and they start dying by self-destruction in a process called autolysis. Decomposition initiates when the interior enzymes start to digest dead cell membranes and tissues (Costandi, 2015).

The decomposition process occurs in distinctive phases. Wolff et al., (2001) studied the changes on pigs exposed carrion during the decay process and separated it into five different stages: fresh stage (1-2 days), bloated stage (2-6 days), decay stage (7-12 days), post-decay stage (13-23 days) and dry stage (24 or more days). The fresh stage starts when death occurs until the bloating of the corpse is observed. No external alterations are noted. Even without any odour obvious to humans, the chemicals released during autolysis attract insects. The bloated stage is considered to be the principal component of decomposition. Due to the production of gases by anaerobic bacteria activity the corpse becomes bloated raising the internal temperature. Calliphoridae are intensely attracted to the corpse during this stage. The decay stage begins when the abdominal wall is penetrated allowing the gases to escape, what causes the corpse to deflate and lose the bloating characteristic. In this phase the temperature of the corpse increases due to the formation of maggot masses which also increases the odour. The post-decay stage initiates when most insects abandon the body leaving the bones, some tissue and hair. Finally the dry stage is only characterized by bones and all remaining tissues dried (Tullies and Goff, 1987). 

According to Tullies and Goff (1987) the physical appearance, internal temperatures and the type of insects are different in each stage of decay process. The rate of postmortem decay can be influenced by several factors (Anderson and Cervenka, 2002). Campobasso et al., (2001) suggests that there are different factors, which can be intrinsic and extrinsic. Intrinsic factors concern the body itself including age, constitution, cause of death and integrity of the corpse. External factors are from the external environment and include the ambient temperature, ventilation and air humidity. The decomposition process is faster in children than in adults (Di Maio, 2001) but is slower in foetuses and newborns (Campobasso et al., 2001). Campobasso et al., (2001) also observed that in obese people the rate of decay is faster due to the larger quantity of liquid in the tissues that increases the extent of bacteria activity. The presence of traumatic lesions also accelerates significantly the decomposition process due to easier entrance of bacteria and flies (Pinheiro, 2006). 

Among the different extrinsic factors the ambient temperature is the most important. There can be a variation in the corpse temperature due to the activity of the maggots present, which can increase the corpse temperature around 1-3 °C above the normal ambient temperature (Charabidze et al., 2011), accelerating the decomposition process. In contrast, low temperatures inhibit the growth of bacteria slowing the decay process substantially (Gordon et al., 1988). The putrefaction process is mainly caused by the bacterial activity (Spitz and Fischer, 1980). The presence of bacteria in the gastrointestinal tract leads to the destruction of the soft tissues of the body producing liquids and gases (LeBlanc and Logan, 2010). These volatile substances, named apeneumones, are released and when escaping from the body, attract the insects. The volatile substances released during each stage of decomposition can alter insect behaviour (LeBlanc and Logan, 2010). Based on the studies done by Ashworth and Wall (1994) the attraction of flies to the decomposing carrion is caused by sulphurbased compounds and oviposition is induced by ammonium-rich compounds. 

1.3. Entomofauna 

Four different types of insects are attracted to carrion. According to Smith (1986) and Goff (1993) the cadaver fauna is divided into four groups: i) necrophagous insects, ii) parasites and predators, iii) omnivorous and iv) adventitious species. 

Necrophagous insects are the first species reaching the decomposed body feeding directly from the tissue or from the fluids released during the decomposition process (Goff, 1993). Joseph et al., (2011) observed that Calliphoridae are the predominant species in this group. Being the earliest insects to appear on the cadaver, they have an important role in understanding decomposition (Pohjoismäki et al., 2010). Parasites (e.g. wasps) and predators (e.g. carrion beetles and rove beetles) are the second group of insects arriving at the corpse. These insects do not feed directly from the carrion or fluids (Smith, 1986). According to Keh (1985), parasites use the cadaveric fauna to take resources for their own development and predators feed on other cadaveric insects. Omnivorous species (e.g. ants, wasps and some beetles) feed on the carrion but also on the associate fauna (Keh, 1985). Adventitious species (e.g. spiders, centipedes and mites) are found randomly in the body (Keh, 1985). They are not necessarily attracted to the corpse, using it only as part of their habitat or as a locale to hide (Campobasso et al., 2001). 

1.4. Oviposition and life cycle 

Studying the insect life cycle is the most accurate way to establish the time since death occurred (Sharma et al., 2015). According to Ireland and Turner (2006) the blowflies, like many other flies, have a specific life cycle (fig.1), which involves an egg, larva, pupa and adult stages (Sharma et al., 2015). When the adult fly lands on a decomposed body hundreds of eggs are laid. The eggs are laid near to a food source usually in the natural body orifices (mouth, ears and nostrils) or in open wounds (Goff and Lord, 1994). In approximately 24 hours the eggs will hatch releasing a larva typically called a maggot (Ireland and Turner, 2006). 

Goff and Lord (1994) observed as maggots feed on the cadaver and grow, three different stages or instars of development are reached. The authors also observed once they reach the full size they stop feeding from the decomposed body and then leave it in order to find a safe location to pupate. At this phase the larvae develops a hard capsule, known as the puparium, in order to protect the fly through metamorphosis until it becomes an adult and emerge as a fly (Goff and Lord, 1994). 

1.5. Entomotoxicology 

Entomotoxicology studies the application of necrophagous insects in toxicological analysis to identify xenobiotics (foreign chemicals to a biological organism or system) present in a tissue. The science also investigates the effect of these substances on the development of insects (Introna et al., 2001). 

The number of deaths related to accidental consumption or suicide with drugs and toxins have grown in several countries (Introna et al., 2001). In many cases, such deaths are slow to be discovered or reported, remaining hidden for several days (Goff et al., 1994) which makes it difficult to analyse for toxic substances due to the decomposition of the body. 

In the 1950s it was observed that the flies were attracted in different ways by carcasses of rats, according to the poison that caused the death of the animal (Utsumi, 1958). Since then it has been found that, depending on the substance, the entomological succession could be changed (Gunatilake and Goff, 1989). According to Goff and Lord (2001) the necrophagous insects remain in the body even after the stage at which tissue samples, blood and urine become unviable for analysis, so toxicological data obtained from the analysis of these insects, can provide information to determine the cause of death. As the larva feeds on the carrion it can accumulate toxins, so larvae can be analysed by several techniques similar to those used for human tissue analysis, including gas chromatography, liquid chromatography, high-performance liquid chromatography or gas chromatography-mass spectrometry (Introna et al., 2001). Chromatography techniques are usually chosen due to their precision in the separation, identification and quantification of chemical substances (Collins et al., 1997). The absence of a drug in the larvae does not necessarily indicate absence of that drug in the body (Tracqui et al., 2004) so, only positive results can be considered, as verified by Sandler et al., (1997). 

In addition to the possibility of using insects in toxicological analysis, other entomotoxicology research is studying how the xenobiotic present in body tissues modify the behaviour of necrophagous insects, which is the aim of this project. Although limited, studies already done on the effects of xenobiotics on larval development have shown that the presence of drugs in the body tissues alters the life cycle of necrophagous larvae (Introna et al., 2001). The presence of chemicals can influence the life cycle of insects sufficiently enough to change the estimate of the postmortem interval. Studies by Chen et al., (2004) showed that toxins ingested by the larvae can influence their behaviour. For example, cocaine (Goff et al., 1989) and heroin (Goff et al., 1991) can accelerate the development of larvae, but on the other hand morphine slows it down (Bourel et al., 1999). 

However, some research studies showed the resistance of several insects and animals to mutagenic and toxic agents. Even though it has been shown in some studies that caffeine produces various effects on insects, in 1969 Petersen noted a resistance to caffeine in mice. 

1.6. Caffeine 

Caffeine, known as 1,3,7-trimethylpurine-2,6-dione, is a methyl xanthine alkaloid that affects the central nervous system by stimulating it (Luo and Lane, 2015).

According to D’Amicis and Viani (1993) caffeine occupies a prominent position among the drugs most consumed in the world being usually used by humans who consume coffee and/or soft drinks. It may be the most neuroactive compound consumed (Ashihara et al., 2008). The assumption that caffeine in general increases the performance of any activity has caused great scientific interest and was the basis for the development of numerous studies about its effect particularly on insects and animals (Nehling and Debry, 1994). Ishay and Paniry (1979) observed in their studies with bees an increase collection of resources. The authors suggest that it was influenced by the action of the caffeine, which acts on the central nervous system stimulating it. They also believe that the inclusion of caffeine in the diets of bees can be an alternative resource to increase the population of their colonies and, consequently, the production of honey. 

However, conflicting results have been obtained. In the research paper of Itoyama and Fig 2. Caffeine structure. (Luo and Lane, 2015) 7 | P á g i n a Bicudo (1992) four concentrations of caffeine (50 μg/ml, 100 μg/ml, 1000 μg/ml and 1500 μg/ml) were administered in the medium culture of Drosophila prosaltans. The aim of Itoyama and Bicudo’s (1992) study was to detect possible effects on fertility, oviposition, adult longevity and speed of development from egg to adult. As the concentration of caffeine increased the authors observed a decrease of fecundity, oviposition, longevity and development time. In 1980, Ashburner and Wright studied as well the effect of caffeine on blowfly oviposition. While mated flies were laying their eggs quickly, virgin flies were retaining their eggs and only laying them on the 5th to 7th days after mating. The authors observed that the rate of egg production of the virgin flies was delayed and concluded that the caffeine ingested blocked the production of eggs causing a decrease of oviposition. 

Numerous studies in rats also have been done in order to observe the effect on development of maternal ingestion of caffeine (Itoyama and Bicudo, 1992). Tanaka et al., (1987) administered caffeine through the animal’s drinking water and observed a decrease of placental, foetal body and brain weights. Delays in foetal development and high mortality at birth were also observed in the studies of Pollard et al., (1987). 

Aims 

The present study intends to observe the egg-laying capacity of blow flies in a control culture medium and in media containing different concentrations of caffeine. The aim of the research is to observe the effects of caffeine on blowfly fecundity and survival. 

Material and Methods 

The maggots used in each method presented below were provided by the laboratory technicians. They were the larvae of blowflies (Calliphora species), commonly found in the early stages of carrion decomposition (Joseph et al., 2011). 

The first method used started by rearing some maggots until they emerged as flies. The maggots were placed into plastic cups containing sawdust, since it is a good absorbent, keeps the culture slightly damp and doesn’t contain anything that will affect the animals and influence their survival. The sawdust was previously moistened with tap water so that the environment was suitable for their development since larvae prefer, and develop rapidly, in humid environments. 100 cups were prepared, containing 10 maggots each, and then covered with cling film previously holed to permit oxygen to circulate. The plastic cups were stored in the greenhouse to be kept at optimal temperature. The flies took about three weeks to emerge, longer than the expected two weeks (Okuda and Stephenson, 2014) because it is believed that the development environment was sometimes too dry. After emerging the flies were being fed with soaked cotton with sugar-water solution until the preparation of the next method. 

The second method was intended to recreate an environment similar to a decomposing body, which has previously ingested caffeine in order to observe the fly behaviour towards this toxic, which is the main aim of this study. Pork liver was bought and homogenized in a blender, distributing 25 g per cup. 70 cups were prepared, divided into 7 different concentrations of caffeine (C, 10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L and 800 mg/L). The caffeine was previously added and dissolved in agar solution. After obtaining a homogeneous solution, 20 mL was added to each cup at the respective concentration, mixed, settled down until a consistent mixture and then stored in a low temperature room overnight to cool down and maintain the properties. The following day, the cups with the flies were placed on ice for these to calm down, making it easy to transfer them to the cups containing the liver. 6 flies were transferred to each cup using plastic forceps to ensure that they were not hurt. The cups were covered with cling film and then kept in the greenhouse. 

However, this method was not successful since in an interval of two weeks all flies died, without laying any eggs, including the control. Since there were no results from the previous method, a new method was used. For this method, the remaining flies from method 1 were used. These were not many since their life cycle was at the end being able to only use 15 flies. 3 concentrations of caffeine were defined (C, 100 mg/L and 800 mg/L) containing 5 flies each. For the preparation of this method, instead of using the pork liver it was used ground pork chop, keeping the quantity of 25 g. The caffeine was added directly in the meat and no agar was used in this method. The cups were stored in the greenhouse. 

In addition to the initial experiment of observing the effect that caffeine has on the behaviour of flies, the effect of this toxin on their final larval stages was also investigated. Once again the 7 different concentrations of caffeine (C, 10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L and 800 mg/L) were prepared. For each concentration, 5 replicates were prepared containing 13 g of maggot meal in each cup. The maggot meal used was ground up wheat bran, consisting of the outer husk of wheat seeds removed in milling. It is a sustainable protein source with a significant variation in nutrient composition (Makinde, 2015). After the addition of meal, 6 maggots were added per each cup and then stored in the greenhouse. 

A few days later, all the 15 flies in the cups prepared with the pork chop had died, giving no results to the experiment. However results were observed in the cups with the maggot meal. 

Results 

The first three methods used in this experiment did not produce any observations or results, since all flies eventually died after a few days. However, it was possible to obtain some conclusions through the last method used, which aimed to observe the effect of caffeine in larval stages. The results were collected based on the observations made and on the monitoring and counting the flies per concentration. The following results are presented as the sum of flies in the 5 cups per different concentrations on days 22nd, 25th and 27th April. 

Discussion 

From the results presented it is possible to observe the effects that caffeine has on the behaviour of the flies, especially the larval stages. Given the results obtained on April 22nd, the cups prepared with the maggot meal containing the highest concentration of caffeine, specifically 400 mg/L and 800 mg/L, obtained the highest number of emerged flies. The contact with this toxin has enabled a rapid larval development, accelerating the emergence and making these flies the first to emerge. This result was expected because caffeine is known as a stimulant, which normally increases the performance of any activity (Nehling and Debry, 1994). The first results obtained are considered the most relevant because it is possible to observe the greatest differences between the various concentrations prepared. Some days after the first observation, and over time, the values for each concentration begin to standardize since the flies begin to emerge in 0 5 10 15 20 C 50 200 800 Number of flies Caffeine concentration (mg/L) Alive Fig 3. Number of flies per concentration on 22nd April. 0 5 10 15 20 25 C 50 200 800 Number of flies Caffeine concentration (mg/L) Emerged Alive Dead 0 5 10 15 20 25 C 50 200 800 Number of flies Caffeine concentration (mg/L) Emerged Alive Dead Fig 4. Number of flies per concentration on 25th April. Fig 5. Number of flies per concentration on 27th April. 10 | P á g i n a accordance with their life cycle. Thus, the conclusions are no longer so evident. This was the case of counting flies on 25th and 27th April represented above. During this time the flies were being fed with a solution of sugar-water soaked in cotton wool. 

On April 25th, concentrations of over 800 mg/L still had a greater number of flies emerging as did the concentrations of 0 mg/L and 10 mg/L, however, with some associated deaths. The sum of the number of dead flies in the control (0 mg/L) is the smallest. However, as the caffeine concentration increases, the values for the dead flies tend to rise. With these results it is possible to observe a reduction in longevity of the flies as the caffeine concentration increases. This observation was also noted in the studies of Itoyama and Bicudo in 1992. The observations made on April 27th are similar to the observations made on 25th April. The number of emerged flies continues to be the greatest at concentration of 800 mg/L and the number of dead flies tends to increase with increasing concentration. The difference of both days resides in the fact that, in the April 25th at concentrations of 0 mg/L, 10 mg/L and 400 mg/L, the number of live flies was greater than the number of dead, however in April 27th the number of dead flies was greater than the living flies at all concentrations. 

In addition to the observations made during the experiment, it is also important to reflect on the reason why the other methods used did not produce any results. The first method used, cups containing sawdust, I would not recommend since the substrate is a potent absorbent, leaving the environment too dry for the development of the larvae. This leads to a delay of emergence of flies. 

However, this method is acceptable if moistened periodically. The method with the pork liver also did not go well because all flies died, including the control. The reason for this is still a mystery, but it is believed that the environment was too wet. Because of this, another method used was the substitution of the pork liver by pork chop. In this method all flies also died, including the control. When this method was used, the life cycle of the flies was at the end, so they could have died because of their age. To conclude, the results showed the effects that caffeine has during the larval stages, accelerating their development, as well as the effects it has on the flies, decreasing their longevity. To reach conclusions about the effect of this toxin in oviposition, future experiments will have to be developed. 

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