See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/251383302 “Living Syringes”: Use of Hematophagous Bugs as Blood Samplers from Small and Wild Animals Article · May 2011 DOI: 10.1007/978-3-642-19382-8_11 CITATIONS READS 10 314 3 authors: André Stadler Christian Karl Meiser Alpenzoo Ruhr-Universität Bochum 20 PUBLICATIONS 41 CITATIONS 14 PUBLICATIONS 164 CITATIONS SEE PROFILE SEE PROFILE Guenter Schaub Ruhr-Universität Bochum 187 PUBLICATIONS 5,617 CITATIONS SEE PROFILE All content following this page was uploaded by Guenter Schaub on 14 December 2015. The user has requested enhancement of the downloaded file. HALLE (SAALE ) 2012 MITT . DTSCH . GES . ALL G . AN G EW . ENT . 18 “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 (Reduviidae, Hemiptera) 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 Rhodnius prolixus, 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, Triatominae 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 MEISER & SCHAUB 2011). In such a xenodiagnosis, bugs from laboratory colonies suck blood of a patient suspected to be infected with Trypanosoma cruzi. 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 (WILL 1975) and then for the sampling of blood from bats (VON HELVERSEN & REYER 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 SCHAUB & al. 2011). The “living syringes” – triatomines At present the insect subfamily Triatominae of the family Reduviidae contains 140 species, the majority living in Latin America (SCHOFIEL D & GALVÃO 2009). All postembryonic developmental stages are obligatory blood suckers and usually require one full engorgement for the development to the next instar (SCHAUB 2008). The period of time required from the engorgement to the moult is species- and temperature-dependent, and as in other hemimetabolic insects, this development correlates with an increase in size (Table 1). 349 MITT . DTSCH . GES . ALL G . AN G EW . ENT . 18 HALLE (SAALE ) 2012 Tab. 1 Developmental times1 and sizes of R. prolixus, T. infestans and D. maxima; data from MEISER & SCHAUB 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 (SCHAUB 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 RELL 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 MEISER & 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 SCHAUB & 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 SCHAUB & 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 SCHAUB & 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 350 HALLE (SAALE ) 2012 MITT . DTSCH . GES . ALL G . AN G EW . ENT . 18 carnivores, can be sampled in cages free of litter during feeding. The triatomines can freely approach the animal, 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 (VOI 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 SCHAUB & al. 2011). A bird’s egg in the nest is exchanged by an artificial egg and replaced again after about 15 minutes (e.g. BECKER & al. 2005, BAUCH & 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 SCHAUB & al.