Accumulation and excretion of morphine by Calliphora stygia, an Australian blow fly species of forensic importance

Journal of Insect Physiology 57 (2011) 62–73

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Accumulation and excretion of morphine by Calliphora stygia, an Australian blow fly species of forensic importance Samuel Parry a,*, Stuart M. Linton a, Paul S. Francis a, Michael J. O’Donnell b, Tes Toop a a b

School of Life and Environmental Sciences, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3217, Australia Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 October 2009 Received in revised form 31 August 2010 Accepted 23 September 2010

This study examined the ability of the forensically important blow fly, Calliphora stygia to actively excrete morphine, thereby maintaining a low morphine level within its body when fed on a diet containing morphine at low (7 pmol g1) and high (17.5 pmol g1) concentrations. Morphine was accumulated within the bodies of maggots (70% within the tissues) at concentrations which were lower than that of the meat (3–24%). The morphine content of the initial developing stages (second and third instar maggots) maintained on the high morphine diet was higher than those on the low morphine diet. Morphine was cleared from the body with negatively exponential kinetics (High morphine group: Morphine (pmol g1 wet weight) = 8425e0.014t. Low morphine group: Morphine (pmol g1 wet weight) = 2180e0.010t). Clearance constants for morphine by animals in both groups were similar and thus both groups had a similar ability to excrete morphine. The Malpighian tubules of maggots were able to actively secrete morphine using a transport mechanism that transports small type II organic cations, such as morphine and quinine. The rate of morphine secretion by the Malpighian tubules could explain the clearance of the drug by the maggots. As the morphine was transported across the Malpighian tubules cells, a significant proportion was metabolised into a compound that is yet to be fully characterised. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: Calliphora stygia Morphine Forensic entomology Malpighian tubule Morphine metabolism

1. Introduction Forensically important insects such as blow flies are used to estimate the post mortem interval in legal investigations where the time of death is greater than 72 h and the temperature of the cadaver can no longer be used for such estimates. The post mortem interval is determined by collecting immature larvae from the body which allows the insect to be identified, along with its size and stage. An Australian species of forensic importance, the golden haired blow fly Calliphora stygia, lays its eggs around natural and un-natural orifices of the body, such as the mouth or lesions from gunshot wounds. Thereafter the insect goes through a life cycle consisting of three larval stages, a puparial and an adult stage. The larval stages occur whilst feeding upon the cadaver, and at the completion of the third larval instar, the larvae leave the food source to pupate, which is followed by the adult emergence from the puparium. The duration of each of these stages can be altered by a range of biotic and abiotic factors.

* Corresponding author. Tel.: +61 3 5227 3449; fax: +61 3 5227 1040. E-mail address: [email protected] (S. Parry). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.09.005

The post mortem interval is calculated using larval growth and succession rates of insects, the determination of which must be as accurate as possible to be used in legal proceedings. The rate of development in forensically important species can be affected when the food source contains drugs, but this effect appears to be dependent upon the species and the drug involved. When larvae of the flesh fly, Boettcherisca peregrine, were fed rabbit liver containing either heroin or cocaine, growth rates, larval size and time required for metamorphosis increased, and when fed Amitriptyline the time required to complete development increased, due to a prolonged non-feeding stage (Goff et al., 1989, 1991, 1993). In contrast, the rate of development decreased in the sacrophagid fly, Parasarcophaga ruficornis, maintained on liver containing 3,4-methylenedioxymethamphetamine (MDMA) (Goff et al., 1997). This alteration to the growth and development of the insect may affect the estimation of the post mortem interval by between 18 and 48 h (Goff et al., 1992). Studies on the Australian blow fly, C. stygia, maintained on minced pet meat containing morphine suggest their development is not affected by the presence of the drug (George et al., 2009). This may be indicative of their ability to actively excrete morphine, thereby keeping the concentration of the drug within their bodies at concentrations low enough to prevent disruption of normal development.

S. Parry et al. / Journal of Insect Physiology 57 (2011) 62–73

The potential of a drug to affect development and its reliability as a toxicological marker depend on its concentration and metabolism within the insect. The internal concentration depends on the uptake and excretion rates of the drug and sequestration of the drug within various body compartments. Uptake, sequestration and excretion of illicit drugs such as morphine and heroin within the body of the developing larvae, pupae and adults have received little attention, especially in native Australian blow fly species, such as C. stygia. The ability of these insects to handle and metabolise morphine is of particular importance as morphine/ heroin overdoses are a forensically important form of death, with heroin and opioid-related deaths accounting for approximately 43% of drug related deaths in Australia (ABS Deaths collection 2007). Insects may be capable of efficient drug excretion and thus may be able to maintain the concentration of the drug at lower levels than that of their food. This may explain why the development of C. stygia is unaffected by morphine. Larvae of the blow fly, L. sericata, fed on a substrate containing morphine, codeine and norcodeine contained low concentrations of these drugs in later development, which suggests that these drugs are efficiently excreted by this species (Kharbouche et al., 2008). Similarly the concentration of drugs, amitriptyline and temazepam within the pupae of another blow fly species, C. vicinia, were less than that of the feeding larval stages maintained on muscle containing these drugs (Sadler et al., 1995). The physiological mechanism of morphine secretion by blowflies is unknown. However, the Malpighian tubules are a likely candidate for active secretion since these insect organs are capable of secreting a wide range of organic compounds (reviewed by O’Donnell, 2009). The transporters involved in morphine transport by the Malpighian tubules may include p-glycoproteins or multi-drug resistance associated proteins (MRP) which are involved in type II organic cation transport (Gaertner et al., 1998; Karnaky et al., 2003; Leader and O’Donnell, 2005; O’Donnell, 2008). At physiological pH, morphine is classified as a type II organic cation given it is an amphipathic compound with a positively charged tertiary nitrogen situated close to a large aromatic group (Wright and Dantzler, 2004). Both the p-glycoprotein and the MRP2 transporters are ATP-dependent transport pumps which belong to the ATP-binding cassette superfamily of transporters and are involved in transporting a wide variety of organic compounds (Tarnay et al., 2004). The p-glycoprotein of insect Malpighian tubules transports compounds such as nicotine, vinblastine and danorubicin while the MRP2 transports compounds such as Texas Red (Gaertner et al., 1998; Karnaky et al., 2003; Leader and O’Donnell, 2005). The rate at which Malpighian tubules from a forensically important insect secrete a drug may increase if the insect is feeding on a substrate containing that substance. Malpighian tubules from the larvae of the fruit fly Drosophila melanogaster, for example, secrete either the organic cation, tetraethyl ammonium, or the organic anion, salicylate, at increased rates when the larvae are fed diets containing these compounds (Bourel et al., 2001; Wallman, 2001). The current study examined the accumulation, metabolism and excretion of morphine by the Australian blow fly, C. stygia when it was grown on meat containing morphine at concentrations of 0, 7 and 17.5 nmol of morphine per gram meat. In particular, the concentration of morphine within the insect was measured throughout development to determine if it was capable of actively keeping the concentration within the body low. The potential of morphine to be sequestered within the body of second instar larvae and the extent to which the adult fly excretes significant amounts of morphine in the meconium and puparial casings were determined. The ability of the Malpighian tubules of larvae to secrete morphine and the possible contribution of p-glycoproteins,

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multi-drug resistance associated proteins and organic cation transporters were also examined. To determine if prior exposure of the insect to morphine affected the ability of the Malpighian tubules to secrete it, the morphine secretion rates were measured in Malpighian tubules from larvae grown on meat containing different concentrations of morphine. 2. Materials and methods 2.1. Maintenance of adult fly stocks Adult flies, C. stygia (Diptera: Calliphoridae) (Fabricius, 1782) were caught from the wild, identified using the key of Wallman (2001), and maintained in the laboratory for culturing. Adult fly stocks were periodically replenished from the wild to prevent inbreeding. Adult flies were maintained in the laboratory at natural light and temperature (18–25 8C) in 31 L clear plastic storage containers, with mesh panels on the sides to allow adequate ventilation. Flies within the cage were provided ad libitum with tap water, sugar cubes and protein biscuits. The protein biscuits were made by mixing 2 chicken eggs, 1 cup powdered milk, 1 cup sugar, 16 g dry yeast, 50 mL water together on a paper plate. The mixture was allowed to dry at room temperature before being cut into wedges and placed into the cage. 2.2. Culturing of maggots To obtain eggs, egging dishes composed of 70 mL plastic containers (Genfac Plastics, Australia) were filled with 75 g of cat food, primarily kangaroo meat (V.I.P. Petfoods, Australia) with a ball of cotton wool placed upon the meat. These containers were placed into the cages of the adult flies, checked every 2 h, and any eggs were removed and counted. Eggs were then placed onto a cotton ball, and allocated to a meat-filled 60 mL specimen jar (Sarstedt, Germany) or a 250 mL polystyrene cup (Dart, Australia). A square piece of damp paper towel was also placed upon the meat to prevent desiccation. The day that unhatched eggs were placed upon the meat was classified as day 0. The meat filled jar/cup was then placed into a round clear plastic 850 mL container with a lid containing a 50 mm mesh covered hole for ventilation (Genfac Plastics, Australia). The container was also filled with Breeder’s ChoiceTM kitty litter (Fibre Cycle, Australia) to a depth of 20 mm, which acted as a pupation substrate for the maggots. All samples were incubated in a temperature and light controlled incubator (Thermoline Scientific, Australia) at 22 8C with a 12:12 h day:night cycle. 2.3. Accumulation of morphine within the maggots during development Accumulation of morphine by insects grown on a substrate containing three morphine concentrations (0, 7 and 17.5 nmol morphine g1 meat, henceforth referred to respectively as control, low and high) was measured in C. stygia at different developmental stages (second instar, third instar and wandering maggots, pupae and adults). These experimental concentrations were similar to those reported to occur in heroin overdose victims (Bourel et al., 2001; George et al., 2009). For each experimental treatment, twelve 60 mL specimen jars (Sarstedt, Germany) containing 32 g of meat and 30 eggs were placed into an incubator and allowed to develop. At 4, 6, 7 and 10 days, which respectively represent the developmental stages of second instar, third instar, wandering maggots and pupae, three jars were removed from the incubator. Each jar represented one replicate. The animals contained in these jars were then processed as detailed below and morphine content determined.

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2.4. Preparation of meat containing radiolabelled morphine

2.6. Clearance and excretion of morphine from second instar maggots

Meat containing the low morphine concentration (7 nmol g1 meat) was prepared by adding 280 mL of 1.25 mmol L1 1 tritiated morphine (Morphine [N-Methyl-3H], American Radiolabelled Chemicals, USA, Cat No ART 659), 280 mL of 0.01 mol L1 non-radioactive morphine and 842 mL of distilled water to 400 g of pet food (V.I.P. Petfoods, Australia) which was then blended in a food processor (Sunbeam, Australia) to ensure even distribution of the morphine throughout the meat. Similarly, to create meat containing the high morphine concentration (17.5 nmol g1 meat), 701 mL of 1.25 mmol L1 tritiated morphine and 701 mL of 0.01 mol L1 nonradioactive morphine was added to the 400 g of meat. The control treatment of 0 nmol morphine per gram meat was created by adding 1402 mL distilled water to 400 g of pet meat. In the meat containing both the low and high concentrations of morphine the ratio of radioactive to non-radioactive morphine was kept at 1:8000. This ratio ensured that the radioactivity of the radioactive morphine could be reliably detected using beta scintillation counting.

The proportion of radioactivity accumulated within the body of a maggot and that cleared from the gut and excreted by the Malpighian tubules over 4 h was determined. Maggots were fed meat containing three morphine concentrations (control, low and high), each in triplicate plots. After 4 days, 10 second instar maggots were removed from each replicate and rinsed with distilled water. Five of these maggots were frozen in liquid nitrogen, ground to a fine powder with a mortar and pestle, and the morphine extracted. These five maggots acted as a control, and were used to quantify the level of radioactivity in second instar maggots prior to excretion. The five remaining maggots were placed into an empty 6 mL Pico Prias scintillation vial (Perkin Elmer, USA) for 4 h, to enable gut evacuation. At the end of the 4 h, the maggots were rinsed with 5 mL of 50% (v/v) methanol containing 10 mmol L1 acetic acid mix to remove the expelled gut contents and excreta and to solubilise any morphine adhering to the cuticle. The maggots were then removed from the vial, frozen in liquid nitrogen, ground into a fine powder with a mortar and pestle, and the morphine extracted as described above. The vial containing the 50% methanol/0.01 mol L1 acetic acid mix used to rinse the maggots was evaporated to dryness at 60 8C. Once cool, 2 mL of Ultima Gold AB scintillation fluid (Perkin Elmer, USA) was added to the vial, and the radioactivity of the sample was determined by beta counting. The amount of radioactivity contained within the adult fly and the amount it left behind in the cuticle and excreted meconium were also determined for insects maintained on meat containing low and high concentrations of morphine. To achieve this, maggots were grown on meat containing low and high morphine concentrations for 10 days to pupation. Immediately after pupation, five puparia were removed, frozen in liquid nitrogen and ground to a fine powder and extracted as described above. After a further 10 days, when flies emerged from the puparia, five newly emerged flies were killed and the puparial casings and meconium were collected. The flies, puparial casings and meconium were washed separately in distilled water, frozen in liquid nitrogen, ground into a fine powder, and the content of radioactivity determined as described above.

2.5. Processing of maggots for morphine determination At 4, 6, 7 and 10 days post hatching, triplicate groups of 1, 2 and 5 maggots/pupae were removed from the meat or kitty litter and rinsed with distilled water in order to remove any meat or morphine adhering to the cuticle. The maggots/pupae were then frozen in liquid nitrogen and ground to a fine powder using a ceramic mortar and pestle. Ground maggots/pupae were added to pre-weighed tube (either 1.5 mL, 2 mL microtube or 10 mL centrifuge tube) and wet mass determined. A solution of 50% methanol dissolved in 10 mmol L1 acetic acid mix was added to the ground maggots at a dose of 1 mL per 0.3 g powder to efficiently solubilise the morphine. Tubes were then vortexed for 30 s, sonicated for 30 min (2510 Sonicator, Branson, USA), and placed in an oven (Qualtex, Australia) for 12 h at 60 8C. Following incubation the samples were again vortexed and sonicated as described above, and then centrifuged. The 1.5 mL and 2.0 mL micro tubes were spun in a Model 16K microcentrifuge (Bio Rad, USA) for 10 min at 10,000  g at room temperature. The 10 mL centrifuge tubes were spun in an AllegraTM 6R Centrifuge (Beckman CoulterTM, USA), for 15 min at 3500 rpm at 15 8C. The supernatant containing the solubilised morphine was retained for morphine quantification. Morphine within the supernatant was quantified by determining the amount of radioactivity present in the extract. A 100 mL aliquot of the supernatant containing the solubilised morphine was added to 2 mL of Ultima Gold AB scintillation fluid (Perkin Elmer, USA) in a 6 mL Pico Prias scintillation vial (Perkin Elmer, USA) and the amount of radioactivity in the sample was determined by counting for 10 min in a Tri-Carb 2900TR scintillation counter (Packard Instrument Company, USA). The amount of tritiated morphine in the sample was estimated by comparing background corrected counts against the specific activity of the radioactive morphine (50,000 cpm pmol1). The morphine concentration was then calculated from the ratio of radioactive morphine to unlabelled morphine, 1:8000. It was assumed that the radioactivity was present within the morphine given the manufacturers specifications, product was greater than 97% pure (ARC Radiolabeled Chemicals Inc). Recently purchased radioactive morphine was used for the experiments. It was also assumed that the non-radioactive morphine was handled the same way as the radioactive species, and was therefore 8000 times more non-radioactive morphine than the radioactive morphine. Thin layer chromatography of the whole body samples qualitatively revealed that morphine and no other metabolites were present in the samples. The methods used for thin layer chromatography were as previously described (Yeh et al., 1979).

2.7. Secretion of morphine by Malpighian tubules The ability of the Malpighian tubules to secrete morphine, and the rate of secretion were measured using the Ramsay assay (Ramsay, 1953). To do this, Malpighian tubules from larvae of C. stygia were dissected under insect saline (pH = 7) containing (in mmol L1) 117.5 NaCl, 20 KCl, 2 CaCl2, 8.5 MgCl2, 10.2 NaHCO3, 4.3 NaH2PO4, 8.6 HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid), 1 cAMP, 20 glucose and 10 glutamine (O’Donnell et al., 1996). Lengths of tubule were then transferred to a 20 mL droplet of the same saline which contained a morphine mix, consisting of radioactive and non-radioactive morphine at a final concentration of 10.0625 mmol L1 (10 mmol L1 of non-radioactive morphine and 0.0625 mmol L1 of radioactive morphine). The morphine was added to the saline on the day of the experiment. Tubules were placed into the saline droplet already containing the morphine. The saline droplet was submerged under paraffin oil in a petri dish with a Sylgard base (#SYLG184, Dow Corning). The ends of the tubule lengths were tied around insect pins (0.15 mm minuten pins, Fine Science Tools Cat No. 26002-15), which were positioned just outside the droplet. Part of the tubule suspended between the droplet and the pin was punctured by gently pinching with fine forceps (Student No. 5 forceps, Fine Science Tools). A secreted droplet formed at the puncture and was left to accumulate for 60 min. After this time, the secreted droplet was removed from the

S. Parry et al. / Journal of Insect Physiology 57 (2011) 62–73

tubule using a fine glass rod and allowed to settle on the bottom of the petri dish. The diameter of the droplet was measured using an eyepiece micrometer and the volume calculated ((pd3)/6). The secreted droplet was collected using a micro-pipette, ejected under 3 mL of scintillation fluid (Ultima Gold AB, Perkin Elmer, USA) and its content of radioactivity determined by counting in Tri-Carb 2900TR scintillation counter (Packard Instrument Company, USA). The amount of morphine secreted by the tubule was estimated from the specific activity (50,000 CPM pmol1) of the radioactive morphine and ratio of non-radioactive and radioactive morphine. The specific activity of the morphine was determined from scintillation counting of radioactive morphine standards ranging from 0.0625 pmol to 1.25 pmol. Again it was assumed that the radioactivity was contained within the morphine given the manufacturers specifications and that recently purchased radioactive morphine was used for all of the Malpighian tubule experiments. It was assumed that both the non-radioactive and radioactive morphine were transported in proportion to their ratio in the bathing saline (Table 1). Rates of fluid and morphine secretion were calculated by dividing the volume of the secreted droplet, and amount of morphine, by the total time of the experiment, 60 min, and the length of the tubule in the droplet (measured with an eyepiece micrometer). A 10 mL aliquot of paraffin oil covering the bathing droplet was also collected and counted. Counts from the oil samples were similar to the background counts. 2.8. Qualitative detection of morphine in insect urine The form of the morphine in the secreted droplet was qualitatively determined. Two groups, experimental and control, each consisting of 60 tubules were set up in the Ramsay assay and allowed to secrete for 60 min. The bathing saline of experimental tubules contained 10.0625 mmol L1 morphine while the bathing saline of the control tubules contained 0 mmol L1 morphine. At the end of the incubation time, the secreted droplets from the control or experimental groups were separately collected and pooled by ejecting them under 100 mL of distilled water. These samples were then analysed for the presence of morphine using HPLC (1200 Series; Agilent Technologies) with acidic potassium permanganate chemiluminescence detection. The permanganate chemiluminescence reagent provides highly sensitive detection of various phenols and related species (Adcock et al., 2007) and has been extensively used for the detection of morphinan alkaloids (Francis et al., 2008; Costin et al., 2007). Samples were separated using a Chromolith Performance RP-18e monolithic column (100 mm  4.6 mm, i.d.; Phenomenex, Australia) with monolithic guard column (5 mm  4.6 mm, i.d.) under isocratic conditions (95% aqueous trifluoroacetic acid (0.1%, v/v) and 5% methanol) at room temperature. The flow rate through the column was 1 mL min1 and the injection volume was 5 mL. The chemiluminescence detection involved merging the post-column eluate and the reagent (750 mmol L1 potassium permanganate and 1% (m/v)

65

sodium polyphosphates, adjusted to pH 2.0 with orthophosphoric acid) at a T-fitting immediately prior to a ‘GloCel’ chemiluminescence detector (Global FIA, USA) with a single-inlet serpentine reaction channel (Terry et al., 2008). Samples were run in triplicate. The retention time of the morphine peak was determined from analysis of six morphine standards with concentrations ranging between 1 mmol L1 and 20 mmol L1. Blanks of distilled water were also run on the HPLC system. 2.9. Exposure of larvae to morphine in their diet To determine whether the consumption of morphine and developmental stage affected the rate of morphine secretion by the Malpighian tubules, the rate of morphine secretion was measured in Malpighian tubules from second and third instar larvae raised on meat containing morphine at control, low and high concentrations. Malpighian tubules were dissected out and mounted in the Ramsay assay, as described above, in a droplet of insect saline containing 10.0625 mmol L1 of morphine (10 mmol L1 nonradioactive morphine and 0.0625 mmol L1 radioactive morphine). The total length of the Malpighian tubules from second and third instar larvae maintained on meat containing control, low and high morphine concentrations was also measured using an eyepiece micrometer, in order to determine if the length of the tubules was affected by the consumption of morphine. 2.10. Rate of tubule morphine secretion with increasing morphine concentrations in the bathing saline The rate of morphine secretion with increasing concentrations of morphine in the bathing saline was measured for Malpighian tubules from third instar larve of C. stygia. Between 4 and 10 Malpighian tubules from third instar larvae were set up in the Ramsay assay, as described above, in a bathing droplet containing a mixture of non-radioactive and radioactive morphine at concentrations between 0.125 mmol L1 and 1000.625 mmol L1 (Table 1). Tubules were left for 60 min, after which time the secreted droplet was collected and the volume and amount of radioactivity measured and the morphine content estimated as described above. 2.11. Inhibitor experiments To investigate the type of transporters involved in transporting the morphine across the epithelium of the Malpighian tubules, competition and inhibition assays were conducted on the Malpighian tubules from third instar larvae. Between 14 and 20 tubules were set up as described above in a bathing saline containing 10.0625 mmol L1 morphine (10 mmol L1 non-radioactive morphine, 0.0625 mmol L1 radioactive morphine). Tubules were then divided into two groups, an experimental group of 7–10 tubules and a control group of 7–10 tubules. Potential competitors and inhibitors were added to the saline bathing the experimental tubules. The ratio of the competitor or inhibitor to the morphine

Table 1 Morphine concentrations in the bathing saline for the determination of the morphine secretion rate by Malpighian tubules bathed in different concentrations of morphine. Total morphine in the saline was a mixture of non-radioactive morphine and radioactive (tritiated) morphine (morphine [N-methyl-3H] (American Radiolabelled Chemicals Inc Cat No. ART 659)). Total morphine (mmol L1)

Non-radioactive morphine (mmol L1)

Radioactive morphine (mmol L1)

Ratio non-radioactive morphine: radioactive morphine

0.125 10.0625 50.125 100.0625 500.3125 1000.625

0 10 50 100 500 1000

0.125 0.0625 0.125 0.0625 0.3125 0.625

– 160 400 1600 1600 1600

S. Parry et al. / Journal of Insect Physiology 57 (2011) 62–73

M ¼ M 0 ekt where M represents the morphine content (pmol g1) of the animal, M0, the morphine content of the animal at time zero, k, the elimination rate constant (h1) and t, the time (h) of the developmental stage. This data was also converted into that

3.1. Morphine accumulation Morphine was detected within the bodies of C. stygia maggots when reared upon meat containing the drug (Fig. 1). During the feeding stages (second and third instars), maggots grown on meat containing the higher morphine dose (17.5 nmol g1), contained more morphine than those grown on meat at the lower dosage (7 nmol g1) (Fig. 1). The non-feeding stages of development (wandering maggots and pupae) had similar morphine concentrations within their bodies when reared on diet with either low or high morphine concentration (Fig. 1). Maggots fed the higher morphine dose, at each developmental stage contained less morphine within their bodies than that of the preceding stage (Fig. 1). When fed on the lower morphine diet, second instar maggots had a higher morphine concentration in their bodies than

[(Fig._1)TD$IG]

β

2400

Low morphine concentration High morphine concentration 2000

χ

1600

1200

α δ

800

γ γ

400

γ

ε

Pu pa e

W

an

In

de

st

rin

ar

g

0

3r d

where J represents the morphine secretion rate (fmol min1 mm1 tubule), Jmax is the maximum theoretical excretion rate, S represents the morphine concentration (mmol L1) in the bathing saline and Kt is concentration of morphine in the bathing saline at half the maximal transport rate. Data for the amount of morphine within the body of maggots and pupae against time were fitted to the following equation for exponential decay using non-linear regression and SigmaPlot 11:

3. Results

ar

J max S Kt þ S

dM ¼ kM 0 ekt dt

st

J¼

Data for both treatments was then refitted to a linear regression and the slopes compared using a t-test. To describe the rate of morphine excretion (dM/dt) by the maggots with time, the first derivative of the negative exponential clearance equation was derived, thus the equation became:

In

Data from the accumulation experiment were analysed statistically using a two-way ANOVA (P < 0.05). Factors tested were developmental stage (2nd, 3rd and wandering maggots, pupae and adult flies) and morphine concentration of meat (control (0 nmol g1), low (7 nmol g1) and high (17.5 nmol g1)). Where the interaction term was found to be statistically different, individual means were compared using post hoc custom hypothesis tests (SPSS for Windows 12.0). Excretion and tubule experiments were analysed statistically using one-way ANOVA (P < 0.05). If the means tested by the ANOVA were significantly different, then individual means were compared using Tukey’s post hoc tests. Statistical probabilities were calculated using SPSS V.15.0 for WindowsTM (SPSS Inc., USA). Data from the competition and inhibitor experiments were pooled to gain the best estimate for the rate of morphine secretion by the control tubules. This allowed for a more powerful statistical post hoc test, Dunnett’s post hoc test, to be used after a statistically significant one-way ANOVA. Data describing the rate of morphine transport by the Malpighian tubules with increasing concentrations of morphine in the bathing saline were fitted to the following equation for Michaelis–Menten transport kinetics using non-linear regression and SigmaPlot 11:

ln M ¼ ln M 0  kt

d

2.12. Statistical analysis

which fitted a linear form by calculating the natural log of the morphine content. Thus the above equation became:

2n

was between 100:1 and 10:1. Types and concentrations of competitors and inhibitors assayed were the p-glycoprotein inhibitor, verapamil (0.1 mmol L1), the p-glycoprotein substrates danorubicin (0.1 mmol L1), nicotine (1 mmol L1) and cimetidine, a small type I organic cation, tetra ethyl ammonium (TEA) (1 mmol L1) and Texas red (0.1 mmol L1), a substrate of the multidrug resistance protein 2 (Leader and O’Donnell, 2005; Gaertner et al., 1998; Karnaky et al., 2003). An equivalent volume of the vehicle solution (either 0.1% or 0.01% (v/v) ethanol) was added to the solution bathing the tubules. Tubules were then left for 60 min; the secreted droplet was then collected, its volume and radioactivity measured and the amount of morphine secreted estimated as described above. It was assumed that if morphine and a competitor were carried by a common transporter, then the rate of morphine secretion would be reduced in the Malpighian tubules from the experimental group compared to that from the controls. Similarly, if in the presence of an inhibitor, the rate of morphine secretion by the Malpighian tubules was reduced, then the morphine was assumed to be transported by the transporter that was inhibited.

Morphine concentration (pmol g-1 wet weight)

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Fig. 1. Morphine concentration (pmol g1 wet weight) within second instar, third instar, wandering maggots and pupae of C. stygia grown on meat containing morphine at either low (7 nmol g1) (open bars) or high (17.5 nmol g1) (black bars) concentrations. Data expressed as mean  SEM (n = 9). Within either the low or high morphine concentrations, different Greek letters (a, b, x, d, e, g) indicate that the morphine concentrations within the maggots differed between different developmental stages (P < 0.05, two-way ANOVA). Within a developmental stage, either wandering maggots or pupae, broken lines above the means indicate that maggots in the low or high morphine concentration treatments had similar morphine concentrations within their bodies (P > 0.05, two-way ANOVA).

[(Fig._2)TD$IG]

S. Parry et al. / Journal of Insect Physiology 57 (2011) 62–73

67

Morphine concentration (pmol g-1 wet weight)

3000 2500 -1

Morphine (pmol g )= 8425e-0.014t 2000 1500 -1

Morphine (pmol g )= 2180 e

-0.010t

1000 500 0 80

100

120

140

160

180

200

220

240

260

Time (Hours) Fig. 2. Morphine concentration (pmol g1 wet weight) within the bodies of maggots and pupae of C. stygia against time (hours). Animals were grown on meat containing morphine at low (*) (7 nmol g1) or high (^) (17.5 nmol g1) morphine concentrations. The equation: Morphine (pmol g1) = 8425e0.014t (non linear regression for exponential decay P(F1,11 = 93.35) < 0.01, r2 = 0.895) fitted data for animals fed meat containing the high morphine concentration while the equation: Morphine (pmol g1) = 2180e0.01t (non linear regression for exponential decay P(F1,11 = 72.26) < 0.01, r2 = 0.856) fitted the data for animals maintained on meat containing the low morphine concentration.

1

Morphine ðpmol g

Þ ¼ 8425e0:014t

(1)

for the high morphine group and 1

Morphine ðpmol g

Þ ¼ 2180e0:010t

(2)

for the low morphine group (Fig. 2). From these equations the times calculated for maggots raised on meat containing high and low concentrations of morphine to excrete half of their morphine were respectively 50 and 69 h. By calculating the natural log of the morphine concentration within the body this data was transformed into that which was fitted to linear regressions. The above equations transformed into a linear form were: 1

Lnðmorphine ðpmol g

ÞÞ ¼ 9:039  0:014  t

(3)

for the high morphine treatment group and 1

Lnðmorphine ðpmol g

ÞÞ ¼ 7:687  0:01  t

(4)

for the low morphine treatment group. The slopes of both of these equations were similar (t-test on the slopes, P(t0.05(2)22 = 2.074) > 0.9) and thus animals in both treatments had similar clearance constants (respectively 0.014 h1 and 0.010 h1 for the high and low treatment groups), indicating a similar morphine excretion potential. The first derivative of the equations for the clearance of morphine from the body of C. stygia for animals in the low and high morphine groups were respectively:

3.2. Morphine excretion efficiency Second instar maggots that were starved for 4 h to facilitate the clearance of their gut retained approximately 70% of the morphine and removed 29–31% of the drug from their systems (Fig. 3). These proportions were similar for maggots maintained on meat containing either the low or the high concentration of morphine. However in terms of absolute amounts, maggots grown on meat containing the high concentration of morphine retained and removed more morphine than those reared on the lower morphine dose (Fig. 3).

[(Fig._3)TD$IG] 3000

Morphine concentration (pmol g-1 wet weight )

that of the last three developmental stages (third instar and wandering larvae, and pupae), which had similar concentrations (Fig. 1). Overall, when compared to the concentration in the meat, the concentration of morphine within the bodies of the maggots was low, ranging from 3.30% to 23.76%. Morphine was cleared exponentially from the bodies of the maggots in both treatment groups (Fig. 2). The equations describing this exponential clearance were:

2500

Maggots with full guts Maggots with cleared guts Excreta

δ

ε

2000

1500

α 1000

φ

β χ

500

0

dm ¼ 21:8e0:010t dt

Low

(5)

High

Morphine concentration

and 1

dm ¼ 117:95e0:014t : dt

(6)

These equations describe the rate of morphine excretion with time by animals in the two treatment groups.

Fig. 3. Morphine (pmol g animal wet weight) content of second instar maggots with full guts (open bars), maggots with cleared guts (grey bars) and morphine excreted (black bars) over 4 h. Maggots were grown on meat containing either low (7 nmol g1) or high (17.5 nmol g1) concentrations of morphine. Data are expressed as Mean  SEM (n = 3). Different Greek letters (a, b, x, d, e, w) indicate that the means were significantly different (P < 0.05, two-way ANOVA).

[(Fig._4)TD$IG]

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Morphine Concentration (pmol g-1 wet weight)

68

δ

400

Table 2 Calculated free energy change (kJ mol1) (mean  SEM (n)) for the transport of morphine by Malpighian tubules bathed in saline containing different concentrations of morphine.

Puparium Exuvium + Meconium Adult Fly

300

ε α

200

γ

γ

γ 100

Morphine concentration in the bathing saline (mmol L1)

Calculated free energy change (kJ mol1)

0.125 10.0625 50.125 100.0625 500.3125 1000.625

5.6  1.3 9.4  0.5 7.2  0.5 7.9  0.5 6.4  0.4 7.1  0.5

(4)a (6)a (8)b (9)c (13)d (11)e

Malpighian tubules were from third instar larvae of Calliphora stygia. Free energy changes were calculated using the equation: 

DG ¼ ðRTÞ ln

0 Low

High

Morphine concentration Fig. 4. Morphine (pmol g1 wet weight pupae) excreted with the combined exuvium and meconium (grey bars) and within the fly (black bars) upon emergence of the adult from the pupae. Open bars represent the morphine content of the pupae before eclosion of the adult. Larvae of C. stygia were raised on meat containing either low (7 nmol g1) or high (17.5 nmol g1) concentrations of morphine. Data are expressed as Mean  SEM (n = 3). Different Greek letters (a, d, e, g) indicate that the means differed significantly (P < 0.05, two-way ANOVA).

3.3. Sequestration and excretion of Morphine in the Pupae When larvae were maintained on meat containing low and high morphine concentrations, and allowed to pupate and emerge, the resulting adult flies respectively contained 40.4% (low morphine diet) and 30.5% (high morphine diet) of the total amount of morphine present in pupae before emergence (Fig. 4). This respectively left 59.6% and 69.5% of the total morphine in the combined puparial casing and meconium (Fig. 4). The morphine content of the puparium and exuvium plus meconium, but not that of the adult flies themselves, was higher for insects grown on the high morphine diet (Fig. 4). 3.4. Morphine secretion by the Malpighian tubules 3.4.1. Kinetics of morphine secretion Fluid secretion rates of Malpighian tubules from third instar larvae of C. stygia ranged between 0.41 and 1.22 nL min1 mm1 tubule (Fig. 5). Fluid secretion rates of tubules bathed in saline containing high concentrations of morphine (500.3125 mmol L1 or 1000.625 mmol L1) were either higher than, or similar to those of tubules bathed in saline containing low concentrations of morphine (0.125 mmol L1 to 100.0625 mmol L1) (Fig. 5). Thus the tubules were functioning normally and were not affected by any potentially toxic effects of morphine. The rate of morphine secretion by the Malpighian tubule increased with an increase in the concentration of morphine in the bathing saline (Fig. 5b). At higher concentrations of morphine, the morphine secretion rate appeared to saturate, as tubules bathed in saline containing either 500.3125 mmol L1 or 1000.625 mmol L1 morphine, secreted the drug at similar rates (Fig. 5b). A Km of 484 mmol L1 and a Vmax of 6105 fmol min1 mm1 tubules were calculated on fitting the data to Michaelis–Menten transport kinetics (Fig. 5b). The concentration of morphine within the secreted droplet was calculated by dividing the rate of morphine secretion by the fluid section rate and ranged between 1 and 11,000 mmol L1 (Fig. 5c). The concentration of morphine in the secreted droplet was 8–24 times that of the bath, showing that morphine was transported against its concentration gradient.



morphine in secreted droplet morphine in bathing saline

þ zF  TEP

where DG is the free energy change, R, T, F, z and TEP are respectively the universal gas constant (8.315 J K1 mol1), the temperature (293 K), Faraday’s constant (96485 J V1 mol1), valency and transepithelial potential (Horton et al., 2006). For this calculation, the transepithelial potential was assumed to be 20 mV lumen positive. Superscript letters indicate that the calculated free energy change was significantly different from zero (equilibrium) (P < 0.05, one sample t-test).

The calculated free energy change for the transport of morphine across the Malpighian tubule epithelium was significantly greater than zero for tubules bathed in saline containing morphine at concentrations of 0.125–1000.625 mmol L1 (Table 2). Thus morphine transport was an endergonic and hence active process. This calculation assumed that the transepithelial potential across the Malpighian tubules from C. stygia was 20 mV lumen positive. The TEP of 20 mV is a conservative estimate and is much lower than the TEP measured for Drosophila 55.20 mV (O’Donnell et al., 1996). Thus the contribution of the electrical gradient to the free energy change for transport is most likely an underestimate. 3.4.2. Detection of Morphine using HPLC with chemiluminescence detection In an examination of pooled secreted droplets using HPLC with acidic potassium permanganate chemiluminescence detection, three major peaks were observed for tubules exposed to 10.0625 mmol L1 morphine (Fig. 6). Peak 1 at 3.2 min (and several minor peaks at 1.7, 2.1, 2.4, 4.2 and 5.7 min) were also observed in the control sample (Fig. 6). The retention time of Peak 3 (5.3 min) was identical to that of a morphine standard (Fig. 6). Peak 2 was only observed in chromatogram (a) (corresponding to pooled droplets secreted from tubules exposed to morphine) and was therefore attributed to an unidentified morphine metabolite. Using a calibration prepared with six morphine standards between 1 mmol L1 and 20 mmol L1 (R2 = 0.9989), the concentration of morphine (peak 3) in the pooled secreted droplets was found to be 2.8 mmol L1. Moreover, based on the assumption that the metabolite (peak 2) detected with permanganate is structurally similar to morphine and elicits a similar chemiluminescence response with that reagent, we can estimate its concentration at 8.7 mmol L1, which would mean that approximately 24% of the morphine excreted by the tubules was unchanged and 76% was metabolised. It should, however, be borne in mind that subtle changes to the structure of morphine can have a significant effect on the chemiluminescence intensity with this reagent (Francis et al., 2008). 3.4.3. Effect of potential inhibitors and competitors on morphine secretion Out of all of the compounds tested, only quinine at a concentration of 0.1 mmol L1 (10 times that of morphine) reduced the rate of morphine secretion, compared to that of the control Malpighian tubules. None of the other compounds tested

[(Fig._5)TD$IG]

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Fluid secretion rate (nL min-1 mm-1 tubule)

1.6 1.4

69

χ

a δ χ χ

1.2 1.0 0.8

δ

0.6

δ

δ

0.4 0.2 0

200

400

600

800

1000

1200

-1

Morphine secretion rate (fmol min-1 mm-1 tubule)

Morphine concentration (μmol L ) in the bathing saline

6000

b

α

5000

α

4000 3000 2000

β

1000

-1

-1

Jmax= 6105 fmol min mm tubules

β β

-1

Kt= 484 µmol L

0 0

200

400

600

800

1000

1200

-1

Morphine concentration (μmol L-1) in the secreted droplet

Morphine concentration (μmol L ) in the bathing saline

16000 14000 12000 10000 8000 6000 4000 2000 0

c

0

200

400

600

800

1000

1200

-1

Morphine concentration (μmol L ) in the bathing saline 1

1

Fig. 5. Fluid secretion rates (nL min mm tubule) (a), morphine secretion rates (fmol min1 mm1 tubule) (b) and morphine concentration (mmol L1) (c) within the droplet secreted by the Malpighian tubules from third instar larvae of C. stygia. Tubules were bathed in insect saline containing different concentrations of morphine. The dotted line in (b) represents the Michealis–Menten plot when the data was fitted to Michaelis–Menten transport kinetics (non linear regression for a hyperbola, P(F1,45 = 57.29) < 0.0001, r2 = 0.55). Data are expressed as mean  SEM. Morphine concentration = 0.125 mmol L1, n = 4; Morphine concentration = 10.0625 mmol L1, n = 6; Morphine concentration = 50.125 mmol L1, n = 8; Morphine concentration = 100.0625 mmol L1, n = 9; Morphine concentration = 500.125 mmol L1, n = 13; Morphine concentration = 1000.625 mmol L1, n = 11. Similar Greek letters (a, b, d, x) indicate statistically similar means (one-way ANOVA, P > 0.05 followed by Tukey’s Post hoc hypotheses tests), for either the fluid secretion rates or rates of morphine secretion.

had any effect on the morphine secretion rate (Fig. 7). This included the p-glycoprotein inhibitor, verapamil at 10 times the morphine concentration, the p-glycoprotein substrates daunorubicin, cimetidine and nicotine, at 10, 10 and 100 times the morphine concentration respectively, and the MRP substrate Texas Red at 10 times the morphine concentration (Gaertner et al., 1998; Karnaky et al., 2003; Leader and O’Donnell, 2005). 3.4.4. Effect of developmental stage and exposure to morphine in the diet on morphine secretion rate The Malpighian tubules from third instar maggots (31.46  0.70 mm) were longer than those of second instar maggots (18.73  0.45 mm) (P < 0.05, two-tailed t-test). Tubule lengths from either second instar or third instar maggots were similar, irrespective of the level of morphine in the diet (control, low or high). Fluid secretion rates of Malpighian tubules ranged from 0.29 to 0.49 nL min1 mm1 tubule, and were unaffected by either developmental stage (second or third instar) or morphine concentration within the diet (Fig. 8a). Malpighian tubules from second and third

instar maggots secreted morphine at rates between 65.34 and 146.40 fmol min1 mm1 tubule (Fig. 8b). Like fluid secretion rates these morphine secretion rates were similar regardless of developmental stage or dietary morphine level (Fig. 8b). 4. Discussion The larvae of C. stygia assimilated morphine when maintained on meat containing the drug. The majority of morphine contained within the maggot was assimilated and only a minority remained within the gut contents. Of the morphine within the body of the insect only morphine was detected, with no substantial metabolites. The concentration of morphine within the body was proportional to the concentration in the diet, with maggots maintained on meat containing a high morphine concentration having a higher morphine concentration than maggots maintained on meat containing a low concentration of morphine (Fig. 1). The concentration of morphine within the bodies of C. stygia maggots was lower than that in the meat, demonstrating that these insects

[(Fig._6)TD$IG]

S. Parry et al. / Journal of Insect Physiology 57 (2011) 62–73

Chemiluminescence intensity (arbitrary units)

70

2

50

peritrophic membrane separates the gut contents from the cells (Boonsriwong et al., 2007). Morphine is likely to be assimilated across the midgut. It is known to be permeable to small organic cations such as tetra-ethyl ammonium and most likely morphine, which has a similar molecular mass and positive charge (Bijelic et al., 2005). If morphine is absorbed with similar characteristics, this route of uptake would be unregulated, and morphine would be expected to diffuse passively across the epithelium, the uptake rate being dependent upon the concentration gradient between the gut and the haemolymph (Bijelic et al., 2005). The other potential route for uptake of morphine could be by diffusion across the cuticle of the body surface or fore- and hind-gut. We suggest that this is unlikely as the cuticle is highly impermeable to water-soluble substances (Ebeling, 1974).

40 3 30

1 (a)

20

(b)

(c)

10

(d) 0

4.2. Distribution of the morphine within the body of the larvae 0

2

4

6

Second instar larvae removed from their food source for 4 h retained the majority of morphine within their bodies and lost only a minority of the drug. The morphine that was removed represents the amount cleared from the gut (via faeces) and that which is excreted by the Malpighian tubules over a 4 h period. This seems reasonable, since a related calliphorid species, C. vicinia has been reported to take 65 min to clear its gut (Greenberg and Kunich, 2002). Hence 4 h would be more than sufficient for C. stygia maggots to clear the gut of food material. This suggests that at the time larvae were killed, the majority of the morphine was contained in body tissues and only a minority was contained within the gut lumen. Once inside the body of the larvae, morphine may be distributed in various compartments such as the haemolymph, fat body and cuticle. As morphine is a hydrophilic compound, existing as an organic cation with a positive charge on its tertiary nitrogen, the majority is likely to be dissolved within the haemolymph and indeed this has been demonstrated in another calliphorid species, C. vomitoria (Bourel et al., 2001).

Time (mins) Fig. 6. Chromatograms for (a) pooled droplets secreted from tubules bathed in saline containing morphine, (b) pooled droplets secreted from tubules bathed in ‘control’ saline without morphine, (c) 2.5 mmol L1 morphine standard, and (d) blank solution (DI water), using HPLC with acidic potassium permanganate chemiluminescence detection. Peak 3 = morphine.

were capable of actively maintaining internal morphine concentrations below the concentration in the surrounding medium, presumably through the active excretion of morphine. Animals in both treatment groups excreted morphine at similar rates given the clearance constants from both treatments were similar. 4.1. Assimilation of morphine into the body Blowfly maggots possess a foregut, midgut and hindgut; both the foregut and hindgut are cuticle lined. Although the midgut epithelium has no cuticular layer at its luminal surface, a

[(Fig._7)TD$IG]

100

Morphine secretion rate (fmol min-1 mm-1 tubule)

80

60

40

α

20

ne oti Nic

ed Te

xa

sR

e eti din Cim

ici n no

rub

ne Da

ini Qu

A TE

il am rap Ve

Co

ntr ol

0

Fig. 7. Effect of various compounds (verpamil (0.1 mmol L1), danorubicin (0.1 mmol L1), nicotine (1 mmol L1), cimetidine (0.1 mmol L1), tetra ethyl ammonium (1 mmol L1), quinine (0.1 mmol L1) and Texas red (0.1 mmol L1)) on the morphine secretion rates of Malpighian tubules from third instar larvae of C. stygia. Tubules were bathed in insect saline containing 10.0625 mmol L1 of morphine. Data (mean  SEM) from a number of experiments were pooled to gain the best estimate of the rate of morphine secretion by the control tubules. Control, n = 44; verapamil, n = 5; tetra ethyl ammonium (TEA) n = 10; quinine n = 5; danorubicin n = 6; cimetidine n = 5; Texas Red n = 11; nicotine n = 8. Letters above the means indicate that the means were significantly different from that of the control (P < 0.05, one-way ANOVA followed by Dunnett’s post hoc tests).

[(Fig._8)TD$IG]

S. Parry et al. / Journal of Insect Physiology 57 (2011) 62–73

Fluid secretion rate (nL min-1 mm-1 tubule)

0.7

a

0.6

Control Low High

0.5

0.4

0.3

0.2

0.1

0.0

Morphine secretion rate (fmol min-1 mm-1 tubule)

2nd Instar

200

3rd Instar

b

150

100

71

keeping the internal morphine concentrations at lower concentrations than in the meat. The rate of morphine secretion by the Malpighian tubules in vitro was saturable and transport occurred against an adverse concentration gradient (morphine concentration in the secreted fluid was 41 times that in the bath) and also against the calculated electrochemical gradient. Therefore transport was most likely active and carrier-mediated. The ability of the tubules to maintain high fluid secretion and morphine transport rates even when the concentration of morphine in the bath was 1 mmol L1, indicates that they were not sensitive to the potentially toxic effects of morphine and are capable of dealing with high concentrations of this potentially toxic drug. The Malpighian tubules of the kissing bug, Rhodnius prolixus, are also capable of actively transporting morphine, and in that species morphine competes with nicotine for the same transporter (Maddrell and Gardiner, 1976). The excretion rates of third instar (144 h) C. stygia maggots in the low and high morphine treatment groups were estimated to be 262 fmol g1 wet weight min1 for animals in the high morphine treatment group, and 86 fmol g1 wet weight min1 for animals in the low morphine treatment group. From this, the mean wet mass of a third instar larvae (126  7 mg) and the total length of four Malpighian tubules (126  3 mm), we calculated the rate of morphine secretion by the Malpighian tubules to be respectively 0.262 fmol min1 mm1 tubule and 0.086 fmol min1 mm1 tubule for the high and low treatment groups. These rates are physiologically possible given they are at the low end of the dose response curve (Fig. 2). Thus the Malpighian tubules are capable of secreting morphine in vitro at rates which may explain the measured in vivo excretion rates. Hence they may be almost solely responsible for keeping the morphine concentration within the maggots at the observed low levels. Organic cation transporters such as pglycoproteins along the gut may also contribute to the excretory ability of the animal (Tapadia and Lakhotia, 2005). The calculated excretion rates also suggest that the transport mechanisms are not saturated.

50

0 2nd Instar

3rd instar

Fig. 8. Fluid excretion rates (nL min1 mm1 tubule) (a) and morphine secretion rates per mm tubule (fmol min1 mm1 tubule) (b) for the Malpighian tubules from second and third instars of C. stygia. Larvae were grown on meat containing either no morphine (control, open bars), low (7 nmol g1, grey bars) or high morphine (17.5 nmol g1, black bars). Malpighian tubules were bathed in insect saline containing 10.0625 mmol L1 morphine. Data are expressed as mean  SEM (n = 7). Dashed lines above the means indicate that they are similar for either fluid secretion rates or morphine secretion rates (P < 0.05, two-way ANOVA).

Entry of morphine into the central nervous system of the C. stygia is unlikely, given it is surrounded by the perineurium, a simple epithelium which envelopes the central nervous system and protects it from potentially toxic organic compounds (reviewed by O’Donnell, 2009). The perineurium possesses a p-glycoprotein transporter which actively prevents toxic compounds such as nicotine and perhaps morphine from reaching the nervous system by actively pumping them out (reviewed by O’Donnell, 2009). 4.3. Excretion of morphine by the Malpighian tubules 4.3.1. Saturable and active secretion of morphine by the Malpighian tubules The Malpighian tubules of C. stygia were capable of actively secreting morphine at high rates and this may be responsible for

4.3.2. Proposed mechanism of morphine transport in light of work on p-glycoproteins and MDR transporters Morphine appeared to be transported on a common transporter with quinine given quinine at concentrations of 10–100 times that of morphine in bath reduced the rate of morphine secretion (Fig. 7). None of the other tested compounds affected the morphine secretion rate. These included the p-glycoprotein inhibitor, verapamil, p-glycoprotein substrates daunorubicin and nicotine, and the substrate for the multi-drug resistance associated protein (MRP), Texas red (Gaertner et al., 1998; Karnaky et al., 2003; Leader and O’Donnell, 2005). These results indicate that morphine is being transported on an organic cation transporter other than a p-glycoprotein and a multidrug resistance protein. Both morphine and quinine are similar type II organic molecules because they both possess two aromatic rings and a positive charge on a tertiary nitrogen. Nicotine is different to these molecules in that it only possesses one aromatic ring. Given this, the organic transporter that transports morphine and quinine may be specific for organic molecules that possess two aromatic rings and a tertiary nitrogen. Malpighian tubules express a number of organic solute transporters other than a p-glycoprotein and MRP and this transporter may be one of those which is species specific and has yet to be characterised (Dow and Davies, 2006). Morphine may also be transported on a number of transporters with different kinetic properties given only 55% of the variability of the data in the dose response curve of the morphine secretion rates could be explained by the hyperbolic regression for Michaelis–Menten transport kinetics (Fig. 5b). In contrast nicotine, morphine and atropine are transported on a common alkaloid transporter by the Malpighian

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tubules of Rhodnius prolixus and nicotine transport in the Malpighian tubules of Manduca sexta involves a p-glycoprotein (Maddrell and Gardiner, 1976; Gaertner et al., 1998). Perhaps different insect species possess different organic cation transporters with different chemical specificities. HPLC analysis of droplets secreted by tubules exposed to morphine in the bathing saline revealed a large chemiluminescence peak that was not present in the droplets secreted by tubules not exposed to morphine. The peak was hypothesised to be a metabolite of morphine. This suggests that morphine may be metabolised as it is transported across the Malpighian tubule cell. Malpighian tubules express high levels of genes for detoxification enzymes, notably cytochrome P450 and glutathione transferase (Dow and Davies, 2006; Dow, 2009). These enzymes may act on morphine to form a highly hydrophilic or charged molecule that may be more efficiently excreted by transporters on the apical membrane of the Malpighian tubule cell. A similar metabolism of the sulphonylurea drug glibenclamide has also been observed as it transported across the Malpighian tubule cells of Drosophila melanogaster (Evans et al., 2005). 4.3.3. Transporters present at a high constitutive levels within the tubules The transporters for morphine were constitutively expressed given the rate of transport by the Malpighian tubules and the clearance constants were similar in larvae regardless of the level of morphine in their diet or their developmental stage (Figs. 2 and 7b). Also the Malpighian tubules did not display any phenotypic plasticity given their length was unaffected by the presence of morphine in the diet. Fly larvae feeding on rotting meat live in an environment that contains high concentrations of toxic organic molecules resulting from the catabolism of protein by bacteria and their secretions (Campobasso et al., 2001). Recently, the Malpighian tubules have been discovered to highly express genes for organic solute transporters and the high rates of morphine transport by the Malpighian tubules of C. stygia may reflect this high expression (Dow and Davies, 2006). Malpighian tubules from other insects such as the tobacco hornworm, Manduca sexta, the bufferfly Pieris brassicae and the kissing bug, Rhodnius prolixus transport another plant alkaloid, nicotine, at high rates, although the animals may not necessarily encounter the compound (Maddrell and Gardiner, 1976; Gaertner et al., 1998; Rheault et al., 2006). In contrast, there is an up regulation of organic cation (TEA) and organic anion (salicylate) secretion by the Malpighian tubules of Drosophila when the larvae are exposed to these compounds in the diet (Bijelic et al., 2005; Ruiz-Sanchez and O’Donnell, 2007). The mechanism for salicylate transport results in an increase in both fluid secretion rate and transport rate while the mechanism for increased TEA secretion is an increase in the TEA secretion rate (Bijelic and O’Donnell, 2005; Ruiz-Sanchez and O’Donnell, 2007). These increases appear to be an adaptive mechanism for ridding the animals of unwanted compounds at an increased rate (Bijelic and O’Donnell, 2005). 4.3.4. Metabolism of morphine with development The morphine concentration within the body of the fly larvae is dependent upon the rates of uptake across the midgut and excretion across the Malpighian tubules. The rate of uptake is dependent on the concentration gradient across the midgut. C. stygia larvae maintained on meat containing a high concentration of morphine will experience a larger concentration gradient across the midgut and will therefore acquire a higher concentration of morphine within their bodies. The ability of the Malpighian tubules of the larvae to secrete morphine must be substantial given they are able to keep the concentration within their bodies at a lower level than that present in the meat (3–24%).

In the wandering stage, there is no route for morphine uptake as the animal is no longer feeding and the gut is empty. Indeed given the empty gut, the concentration gradient is reversed and morphine may diffuse back from the haemolymph into the gut. This is the proposed mechanism by which TEA is cleared from the body of Drosophila melanogaster, when larvae are transferred from food containing TEA to TEA free food (Bijelic et al., 2005). However, in the wandering stage, the Malpighian tubules are still secreting, and this represents a major route for morphine excretion. Morphine remaining in the insect after this time may be due to the tail of the exponential clearance (Fig. 2) and may be sequestered within the compartments such as the, haemolymph and in-between the endo and exocuticle (Bourel et al., 2001). In other species of carrion flies, Lucilia sericata and C. vicinia feeding on meat containing illicit drugs, a similar drop in the concentration of drugs within the body was observed once the maggots left the food source and entered into the wandering stage (Sadler et al., 1995; Kharbouche et al., 2008). 4.3.5. Puparial and adult stages When the larvae enter the pupal stage, any remaining morphine is retained within the pupae, as body tissues, including the Malpighian tubules, cease to function and are degraded and then reformed into adult tissues during metamorphosis (Wrigglesworth, 1984). Once the adult Malpighian tubules are reformed they may resume secreting morphine which is excreted with the meconium upon emergence of the adult fly. Alternatively, morphine could be incorporated into the cuticle and excreted with the exuviae (Bourel et al., 2001). Upon emergence of adult C. stygia, the majority (70%) of the remaining morphine was excreted with the pupae/meconium and only a minority (30%) was retained within the adult fly (Fig. 4). The absolute concentration of morphine within the body of the adult fly was low in comparison to the feeding larval instars. Acknowledgements This work was supported by a Deakin University central research grant awarded to S.M.L., P.S.F., T.T. and M.J.O. and an Australian Pacific Science Foundation grant to S.M.L. and P.S.F. References Adcock, J.L., Francis, P.S., Barnett, N.W., 2007. Acidic potassium permanganate chemiluminescence: a review. Analytica Chimica Acta 601, 36–67. Bijelic, G., Kim, N.R., O’Donnell, M.J., 2005. Effects of dietary or injected organic cations on larval Drosophila melanogaster: mortality and elimination of tetraethylammonium from the hemolymph. Archives of Insect Biochemistry and Physiology 60, 93–103. Bijelic, G., O’Donnell, M.J., 2005. Diuretic factors and second messengers stimulate secretion of the organic cation TEA by the Malpighian tubules of Drosophila melanogaster. Journal of Insect Physiology 51, 267–275. Boonsriwong, W., Sukontason, K., Olson, J.K., Vogtsberger, R.C., Chaithong, U., Kuntalue, B., Ngern-klun, R., Upakut, S., Sukontason, K.L., 2007. Fine structure of the alimentary canal of the larval blow fly Chrysomya megacephala (Diptera: Calliphoridae). Parisitology Research 101, 1417–1423. Bourel, B., Fleurisse, L., Hedouin, V., Caillez, J.-C., Creusy, C., Goff, M.L., Gosset, D., 2001. Immunohistochemical contribution to the study of morphine metabolism in calliphoridae larvae and implications in forensic entomotoxicology. Journal of Forensic Sciences 46, 596–599. Campobasso, C.P., Di Vella, G., Introna, F., 2001. Factors affecting decomposition and Diptera colonization. Forensic Science International 120, 18–27. Costin, J.W., Lewis, S.W., Purcell, S.D., Waddell, L.R., Francis, P.S., Barnett, N.W., 2007. Rapid determination of Papaver somniferum alkaloids in process streams using monolithic column high-performance liquid chromatography with chemiluminescence detection. Analytica Chimica Acta 597, 19–23. Dow, J.A.T., Davies, S.A., 2006. The Malpighian tubule: rapid insights from postgenomic biology. Journal of Insect Physiology 52, 365–378. Dow, J.A.T., 2009. Insights into the Malpighian tubule from functional genomics. The Journal of Experimental Biology 212, 435–445. Ebeling, W., 1974. Permeability of insect cuticle. In: Rockstein, M. (Ed.), The Physiology of Insecta, 2nd ed., vol. VI. Academic Press, New York.

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