The in vitro maintenance technique described in this article has been used successfully to rear Cimex lectularius (L.) by feeding for >2 yr all nymphal stages and adults through parafilm "M" sealing film on different types of blood. Using this feeding technique, the subsequent egg production of female bedbugs was remarkably high. The blood was maintained at 37°C to enhance the attachment of the bugs. The effect of anticoagulation methods for the blood meal was investigated, and heparinized blood was found the most suitable for feeding bugs. All stages of the bugs fed weekly on blood in the artificial feeding system remained attached for up to 0.5–1.0 h, until completion of their blood meals, and all reached engorged weights. More than 90% of the bugs fed artificially on whole blood, and they molted or laid eggs successfully.
Laboratory culture of parasites and studies on pathogens transmitted by ticks and insects have depended on the use of experimental animals (Bailey 1960, Branagan 1969, Norval et al. 1992). The bed bug, Cimex lectularius (L.), is a common bloodsucking parasite in temperate and subtropical climates that attacks humans, poultry, and other mammals (Kettle 1995). Although they have not been linked to transmission of any disease, they have been shown to harbor the causative organisms of plague, relapsing fever, tularemia, Q fever, and hepatitis B (El-Masry and Kotkat 1990). The transmission of hepatitis is theoretically possible by contamination from crushing the bug, contamination from infected feces, or from regurgitation during the bite (Jupp et al. 1983). Transmission of trypanosomes has been demonstrated for bats (Gardner and Molyneux 1988). For decades, this parasite has been used as an experimental model, representing arthropods, to detect ectoparasite activities of different drugs tested. The need for an inexpensive production of large numbers of this species at synchronized ages for infection and life-cycle studies, as well as for conducting screenings of insecticides for control purposes, led us to develop an effective and standardized artificial membrane blood-feeding method that could feed all five instars. The use of in vitro feeding techniques for the mass rearing and maintenance of ectoparasites has major advantages in terms of convenience, productivity and financial expenditure over alternative in vivo procedures (Sonenshine 1991). This work describes the development of a membrane feeding technique that permits the large-scale production of C. lectularius, feeding all instars on fresh heparinized chick blood through Parafilm sealing film (Laboratory Film, American National Can, Greenwich, CT).
Materials and Methods
Bed bug Colony.
The bed bugs, C. lectularius (Figs. 1A and B), were obtained from BASR (Basic Animal Science Research), Merck Research Laboratories (Rahway, NJ), where these parasites have been fed on Guinea pigs more than ten yr.
The colony was maintained, raised, and fed in wide mouth jars (20 cm high by 8 cm diameter) containing filter paper strips. The jars (two or three for each stage of the colony) were covered with fine mesh cloth for ventilation, which was held in place with a rubber band. Bugs were separated according to the instar and the jars maintained at a constant temperature of 28°C and 70% RH in a nonilluminated incubator as described by Schwan et al. (1991) to rear Ornithodoros moubata (Murray, 1877).
A source of CO2 was used to anesthetize the bugs for easy handling. After each feeding, ≈7–8 d later, the adult females laid eggs on the filter paper strips. The eggs hatched in 5–7 d. The different instars were separated after each molting and the molts removed. Each instar molted to the next instar in 5–7 d after the blood meal. They were then ready for the next blood meal. All instar groups were fed every week.
Blood Meal of Bedbugs.
The effect of anticoagulation methods for the blood meal was tested. Four different types of blood were used: heparinized blood recently extracted from chickens and cattle, commercial defibrinated lamb-blood (Oxoid, S.A.), and heparinized lamb-blood (Soria Melguizo). Under our conditions, the entire life cycle from the eclosion of eggs to adult stage ranged from 35 to 45 d.
Artificial Feeding System.
The feeding apparatus (made to measure by Afora, S.A.) consisted of a glass container with a parafilm "M" membrane placed at the bottom (Figs. 2 and 3). The membrane was stretched to hold in place and facilitate the feeding. To enhancethe attachment of the bugs, the blood, around 4–5 ml per feeder, was maintained at 37°C using a warm bath with a pump for circulating the water through the glass feeders. Bugs were fed on the respective blood types until they were replete and had detached themselves from the sealing film.
The filter paper strips (Albet Ref. 101/240) have to be placed in such a way to ensure direct contact with the jar covering, to allow the bugs to reach the blood meal through the mesh cloth and the parafilm membrane. It was essential that all parts of the membrane were in contact with the blood pool, so that the bugs inserting their mouthparts could find blood rather than air pockets. All the stages of C. lectularius were fed with the different type of blood once a week. The system allows using four to six glass feeders disposed in parallel every time.
A new colony of C. lectularius was established and maintained more than 2 yr, starting from fourth stage nymphs. The artificial feeding through parafilm "M" sealing film was the unique method used to feed the different stages of development of this parasite. Every week, all the stages were fed and new eggs were obtained.
All stages of the bugs fed on blood in the artificial feeding system remained attached for up to 0.5–1.0 h, until completion of their blood meals and most of them reached engorged weight. They were readily recognized because of their distended bodies. Few bugs took only a partial blood meal (Fig. 1B). No bedbugs attached to the membrane when blood was at temperatures below 35°C, therefore blood was maintained at a constant temperature of 37°C for facilitating the attraction of the bugs.
Nymphs and adults achieved better engorgement and larger numbers of eggs when fed on whole blood with heparin as an anticoagulant than on defibrinated blood. Blood from three different animals were used: cattle, sheep, and chicken. Identical results were obtained when the blood was gotten from commercial sources or recently extracted and the anticoagulant used was heparin. On each feeding experiment, the average feeding rate for different instars was 90–100% when fresh heparinized blood was used as a diet. When commercial defibrinated lamb blood was used, the egg production decreased so dramatically that it was impossible to obtain a new generation.
We started our colony of C. lectularius from fourth-stage nymphs donated by our colleagues from Rahway, NJ. Le Sueur et al. (1993) obtained some of their colonies (e.g., C. lectularius) from other entomologists, and they consider adaptations to new food sources like anesthetized guinea pigs very important. These in vivo techniques and the experiments derived from them are expensive, imprecise, and normally require restraint facilities and cause discomfort to the experimental animal. By developing in vitro feeding techniques, it is possible to largely remove the necessity for the use of live experimental animals.
This study reports the maintenance of the entire life cycle of C. lectularius during more than 2 yr using artificial feeding. Schwan et al. (1991) reported the artificial feeding of O. moubata using a parafilm membrane; however, the design of their apparatus differed from ours. A very similar artificial feeder was described by Garcia et al. (1975) for the maintenance of Rhodnius prolixus.
In our study, nymphs and adults achieved better engorgement weights when fed on whole blood with heparin as an anticoagulant than on defibrinated blood. Similar observations were reported with Amblyomma variegatum (F.) by Voigt et al. (1993).
Moloo (1971) reported that the blood temperature was one of the most critical factors in membrane feeding of Glossina. Wallade et al. (1991) maintained a water bath at 42°C to heat the blood meal and membranes at 37°C, because they assumed that an important factor was the temperature gradient to attach the ticks to the membrane. In our study, we reproduced these conditions and efficient feeding was obtained.
Trials of artificial feeding of other insects, Glossinidae, through a synthetic silicone membrane were carried out by Pagot et al. (1973). These chemically inert membranes have physical qualities that allow them to be sterilized at high temperatures and conserved indefinitely. Although parafilm membranes cannot be sterilized, the cost is so low that a new membrane can be used each time. Slama and Williams (1966) described the action of the "paper factor" as an inhibitor of the embryonic development of an European bug, in contrast, we did not observe any effect derived from the filter paper strips.
This artificial feeding system could be applied to maintain different life cycles of other hematophagous arthropods. For instance, in our laboratory, all the stages of the life cycle of Ornithodoros erraticus (Lucas, 1849) are fed with this method. This parasite is being used to detect new drugs with activity against soft ticks in a new mouse model.