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“Living Syringes”: Use of Hematophagous Bugs as Blood Samplers from Small and Wild

Article · May 2011 DOI: 10.1007/978-3-642-19382-8_11

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“Living syringes”: use of triatomines as blood samplers from small and wild animals

Günter A. Schaub1, Arne Lawrenz2 & André Stadler2 1Zoology/Parasitology Group, Ruhr-University Bochum 2Zoological Garden Wuppertal

Zusammenfassung: Die Blutabnahme ist bei kleinen Tieren und Wildtieren sehr schwierig. Bei der konventionellen Entnahme werden die Tiere sehr gestresst und evtl. verletzt. Auch eine vorherige Narkose ist problematisch. Triatominen (, ) sind die größten Blut saugenden Insekten und saugen an allen Endothermen, aber auch warmen Amphibien und Reptilien. Die fünf Nymphenstadien dieser Insekten nehmen – entsprechend ihrem Wachstum – zunehmend mehr Blut auf. Deshalb ist je nach erforderlicher Blutmenge beim Einsatz als „lebende Spritze“ ein entsprechendes Nymphenstadium einzusetzen. Das aufgenommene Blut wird im Magen gespeichert und durch den Entzug der wässrigen Blutbestandteile konzentriert, aber fast nicht verdaut. Das Blut kann direkt nach der Blutaufnahme der Raubwanze leicht mit einer Spritze aus dem Magen entnommen und zur Bestimmung der Blut- und physiologischen Parameter eingesetzt werden, außerdem zur Bestimmung von Hormon- und Antikörper-Konzentrationen sowie zum Nachweis von Parasiten. Besonders Larven von , Triatoma infestans und Dipetalogaster maxima werden bei dieser nicht-invasiven Methode zur Gewinnung von Blutproben verwendet. Die Nymphen werden auf die Tiere platziert, in speziellen Behältern angehalten oder saugen durch Perforationen im Boden von Schlafboxen oder in künstlichen Eiern. Neben Freilanduntersuchungen an Vögeln hat sich der Einsatz besonders bei Zootieren bewährt.

Key words: Artificial eggs, blood sampling, Dipetalogaster maxima, Rhodnius prolixus,

Zoology/Parasitology Group, Ruhr-Universität Bochum, Universitätsstraße 150, 44801 Bochum, Germany; E-Mail: [email protected]

Introduction The collection of blood samples from animals is often very difficult. In small animals injuries and subsequent bleeding have to be avoided, in large wild animals the anesthesia is risky. In humans, blood sucking bugs have been used for nearly 100 years as a tool in diagnosis, especially in Chagas disease (summarized by Me i s e r & Sc h a u b 2011). In such a xenodiagnosis, bugs from laboratory colonies suck blood of a patient suspected to be infected with . In case of an infection, the low number of parasites ingested with the human blood can multiply and be identified more easily microscopically. Presumably based on these procedures, ticks and then triatomines were used to investigate components of the blood of lizards (Wi l l 1975) and then for the sampling of blood from bats (v o n He l v e r s e n & Re y e r 1984). Meanwhile, the triatomines Rhodnius prolixus, Triatoma infestans and Dipetalogaster maxima are being increasingly used as “living syringes”, especially to avoid stress for the donor animals (summarized by Sc h a u b & al. 2011).

The “living syringes” – triatomines At present the subfamily Triatominae of the family Reduviidae contains 140 species, the majority living in Latin America (Sc h o f i e l d & Ga l v ã o 2009). All postembryonic developmental stages are obligatory blood suckers and usually require one full engorgement for the development to the next instar (Sc h a u b 2008). The period of time required from the engorgement to the moult is species- and temperature-dependent, and as in other hemimetabolic , this development correlates with an increase in size (Table 1).

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Tab. 1 Developmental times1 and sizes of R. prolixus, T. infestans and D. maxima; data from Me i s e r & Sc h a u b 2011 1 At 26±1°C and 50-60% relative humidity

Nymphal Developmental time [days] Body length [mm] instar R. prolixus T. infestans D. maxima R. prolixus T. infestans D. maxima 1rst 9 10 14 3 4 8 2nd 9 10 14 4.5 6 11.5 3rd 10 12 16 8.5 9 15 4th 13 17 21 10 12 20 5th 14 28 51 15 19 27 Male 20 26 35 Female 22 29 42

The major advantage for the use of triatomines as “living syringes” is the special anatomy of the gut and the mode of digestion. The digestive tract of triatomines is a simple tube. The blood is ingested into the strongly distensible stomach where it remains essentially undigested. By and by small portions of stomach content are passed for digestion into the small intestine (Sc h a u b 2009). Only two important changes occur in the stomach. Blood cells are concentrated and thereby the hematocrit levels change since blood compounds without nutritional value are excreted rapidly, e.g. R. prolixus excretes about 45% of the weight of the ingested blood within the first four hours after blood ingestion (Ma dd r e l l 1969). Therefore, the period of time between the end of engorgement of the bug and the withdrawal of the stomach content for the analyses should be very short. In addition, compounds in the saliva and/or the stomach of triatomines inhibit the classical and the alternative activation pathway of the coagulation system (summarized by Me i s e r & al. 2010). Consequently, the coagulatory factors in the blood cannot be determined easily.

Suitability of different species of triatomines as “living syringes” So far three species have been used, R. prolixus, T. infestans and D. maxima. Rhodnius prolixus has the shortest developmental times and is easily bred in many laboratories (summarized by Sc h a u b & al. 2011). In addition, nymphs are very aggressive, i.e. they attack the host rapidly to start blood ingestion. The aggressiveness increases with time after the molt, but after the optimal phase, increasing numbers of bugs refuse to suck. A disadvantage of R. prolixus is the behaviour of adults. Females glue the eggs to surfaces, and adults easily climb up container walls since they possess adhesive modifications at the legs. The nymphs cannot be stored for a long period of time before use as “living syringes” since their starvation resistance is lower than in the other species. This species is also the smallest one, and only older nymphs ingest sufficient blood for analyses.Triatoma infestans mainly has a middle position regarding the relevant parameters making it suitable for a “living syringe” purpose, but possesses the lowest degree of aggressiveness of these three species. We recommend the use of D. maxima. Like T. infestans, the female deposits eggs on the ground, enabling an easy start of new colonies. This species is of similar aggressiveness as R. prolixus. The long generation time and the time required for each nymphal stage to develop to the next stage (see above) might seem to be disadvantages. However, these can also be considered as advantages, since even young nymphs of this biggest species of triatomines can be used. In a single meal, the first, second, third, fourth and fifth instar nymphs ingest about 0.1, 0.2, 0.6, 1.2 and 2.5 g blood, respectively. A final advantage is the easier possibility to obtain sterile nymphs: egg surfaces can be disinfected more easily than in R. prolixus and the sterile nymphs require fewer feedings each with the risk of contamination before a stage is obtained that ingests sufficient blood (summarized by Sc h a u b & al. 2011).

Modes of applications The mode of application has to be modified according to the biology of the host species and the location of blood sampling (summarized by Sc h a u b & al. 2011). Small mammals in the field or in the zoo can be fixed, and the free active bugs placed near them. Large aggressive animals in the zoo, e.g. big

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carnivores, can be sampled in cages free of litter during feeding. The triatomines can freely approach the , leave it after engorgement and then be captured after the zoo animal is induced to leave the cage. Then the bug is held between the fingers or with forceps and the blood withdrawn directly from the stomach using normal syringes and a thick needle (Vo i g t & al. 2004). If a bug is lost, an insecticide can be applied in the cage. In shy animals, which can be attracted for a short time only, the position of the feeding bug is arbitrary. The nymphs are placed on the skin in a beaker that will be removed after the beginning of blood ingestion. After finishing blood ingestion, however, the triatomine has to be regained. This seeking procedure can be omitted if keepers can handle the animals. In this case – identical with xenodiagnosis – a beaker containing the nymphs and closed by a metal or cotton gauze can be placed on the skin until the bug is engorged, which usually lasts 15-20 minutes. If animals in the zoo sleep or rest in boxes or caves, a second floor with areas covered with metal-gauze can be included. The triatomines are then placed in beakers below the gauze. Specific containers, namely artificial eggs with perforations, are used in ornithological studies (summarized by Sc h a u b & al. 2011). A bird’s egg in the nest is exchanged by an artificial egg and replaced again after about 15 minutes (e.g. Be c k e r & al. 2005, Ba u c h & al. 2010). Thus, in up to 86% of trials blood of the breeding bird was successfully obtained without stressing the bird. This methodology is successfully used for common terns (Sterna hirundo), Montagus’ harriers (Circus pygargus) and common swifts (Apus apus) (summarized by Sc h a u b & al. 2011).

Fields of applications “Living syringes” have been successfully used in several studies in the field, but more often in laboratories and zoos, in the latter so far for 44 species including great apes (summarized by Sc h a u b & al. 2011). Triatomines were first used to investigate the protein profiles of the blood of geckoes (Wi l l 1975), then for the energy and water budget of nectar-feeding bats using doubly-labelled water (v o n He l v e r s e n & Re y e r 1984, Vo i g t & al. 2003). In the first investigation, parameters of blood additionally taken conventionally from the tail of the geckoes were similar, in the second, the isotope enrichment in blood of the bats obtained conventionally was significantly higher (Wi l l 1975, Vo i g t & al. 2003). However, the doubly-labelled water methodology depends on the rate of isotope decrease over time and not on a comparison of isotope concentrations, and the changes of the rates were identical. Also an identification of species is possible using blood for karyological analyses or for the isolation of DNA followed by PCR amplification of microsatellite DNA (summarized byS c h a u b & al. 2011). In endocrinological studies, concentrations of the sex hormones testosterone and progesterone in the blood of domestic rabbits were not affected by the methodology if the blood was withdrawn from the triatomines within four hours after blood ingestion (Vo i g t & al. 2004). In gestation hormone analyses, the conventional blood sampling of lynxes and elephants confirmed the methodology using “living syringes” in initial studies. Most recently, it was shown that the concentrations of adrenocorticotropic hormone metabolites in blood samples of elands and Indian rhinos were higher in blood obtained from bugs than in blood collected conventionally. However, the methodology of hormone analysis of rabbits and elands differed. Investigations of the stress hormone hydrocortisone demonstrate the major advantage of the “living syringes”. Using the conventional method, the period of time between capture and blood collection must be very short, in birds about three minutes, otherwise the concentration in the blood is increased by the stress of handling the animals. In rabbits, the standard deviations of concentrations determined in blood obtained by triatomines were lower than in samples obtained conventionally (Vo i g t & al. 2004). In zoos, blood metabolites are usually determined to get information about the general condition of an animal and its illnesses. In routine analyses, 22 clinically relevant blood parameters are determined (e.g. sodium, calcium, potassium, chloride, total carbon dioxide, bicarbonate, phosphate, creatinine, urea, glucose, hematocrit, hemoglobin, albumin, alkaline phosphatase, alanine aminotransferase, amylase, total bilirubin, globulin, total protein) (St a d l e r & al. 2009). So far, the use of bugs have been shown to affect only some parameters, but much less so if the blood is withdrawn from the fed bugs rapidly after blood ingestion. In birds, concentrations of cholesterol, triglyceride and uric acid did not differ with statistical

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significance after conventional or bug sampling (Ba u c h & al. 2010). An important field of investigations of blood is the determination of antibody titers. These were identical in both methods determining titers of antibodies against hemorrhagic disease virus in rabbits or against rabies after vaccination of mice (summarized by Sc h a u b & al. 2011).

Conclusion The use of triatomines as “living syringes” offers great advantages in comparison to conventional veterinary blood sampling routine. The puncture by the mouthparts of the triatomines is much smaller than the puncture caused by a needle. The major advantages are the possibility to obtain blood without anesthesia and stress and from animals of which veins are inaccessible.

Acknowledgements We thank Dr. R. Cassada for correcting the English style.

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