Abstract
Mass production is an important component of any pest or vector control program that requires the release of large number of insects. As part of efforts to develop an area-wide program involving the sterile insect technique (SIT) for the control of mosquitoes, the Insect Pest Control Laboratory of the Food and Agriculture Organization–International Atomic Energy Agency (FAO–IAEA Joint Division) has developed a mass production cage (Aedes MPC) for brood stock colonies in a mass production system for Aedes albopictus (Skuse, 1895). A preliminary experiment using Plexiglas cages was carried out to estimate the impact of cage volume on egg productivity. Transparent Plexiglas cages of different dimensions but loaded with the same adult density were tested. Egg productivity (number of eggs laid per adult female) and adult survival were recorded and analyzed. According to the results, the optimal volume of 100 liters has been chosen to develop the Aedes MPC. The numbers of adults introduced into the Aedes MPC did not affect the egg production and adult survival in comparison with the Plexiglas cage experiment data, confirming the possible use of Aedes MPC for mass-rearing procedures. Finally, the modification of Aedes MPC and creation of a new prototype model of MPC (Anopheles MPC) to effectively contain Anopheles arabiensis (Patton, 1905) adults is discussed with major changes pioneered to oviposition devices and systems for automatic collection of the eggs.
Aedes albopictus (Skuse, 1895) is a mosquito native to the tropical and subtropical areas of Southeast Asia. In the last decades, this species has spread rapidly through Europe, North and South America, the Caribbean, Africa, and the Middle East and is currently considered to be the most invasive mosquito species in the world (Reiter and Sprenger 1987, Benedict et al. 2007, Scholte and Schaffner 2007). Ae. albopictus is an important vector for chikungunya virus (family Togaviridae, genus Alphavirus), dengue virus (family Flaviviridae, genus Flavivirus), and for several other arboviruses important to human health (Gratz 2004, Paupy et al. 2009). Even in the absence of disease transmission, the anthropophilic behavior of Ae. albopictus is a serious biting nuisance, particularly in urban areas. Owing to its urban and suburban distribution and its capacity to develop in artificial containers, the current control strategies based on source reduction and pesticide application are often ineffective and can become an economic burden on public administration (Carrieri et al. 2011). Therefore, new vector control strategies or integrative control measures need to be investigated to achieve an efficient control for this species.
The sterile insect technique (SIT) is a species-specific and environmentally friendly method for insect pest control based on the induction of sterility in the natural population through the release of a large number of mass-reared and irradiation-sterilized male insects. The approach of SIT for mosquito control, suppression, and elimination has been previously tested against several species with mixed outcomes (Benedict and Robinson 2003). Regardless of the species, many of the causes that contributed to failure of these mosquito release programs have been addressed, and new technologies have been proposed to improve the quality of released sterile insects and to significantly enhance the cost-effectiveness of the SIT (Benedict and Robinson 2003, Klassen 2009).The classic, transgenic, or paratransgenic approach to the SIT and more broadly to the genetic control of vectors has recently received a renewed interest with important laboratory and field demonstrations of their effectiveness (Alphey et al. 2010, Beech et al. 2012). Ae. albopictus has been deemed particularly suitable for classic SIT application (Bellini et al. 2007), and the feasibility of such a program has been successfully tested in preliminary mosquito control suppression trials (Bellini et al. 2013). In this approach, the released males were not genetically modified; they were sexed with mechanical methods and became sterile when subjected to the minimum effective dose of ionizing radiation (Balestrino et al. 2010). To move these and other promising results toward a more intense and sustainable field population reduction, there is the need to address further technical difficulties, including improvement of sex separation, refinement of release methods, and the development of a more vigorous and mechanized mass-rearing process able to ensure a large production of quality sterile males over a long period of time (Dame et al. 2009).
As the SIT relies on area-wide mass releases of competitive sterile males, efficient and effective mass rearing of the target insect is fundamental. Large-scale production of mosquitoes has been achieved on numerous occasions in the past decades with implementation of new rearing methods and technologies (McCray 1963, Morlan et al. 1963, Gerberg et al. 1969, Singh et al. 1975, Bailey et al. 1980) but without reaching daily operational level able to sustain indefinitely large operation activities (Dame 1985). Colonization is an essential unnatural process that can induce the artificial selection of genetically homogeneous individuals very different from wild populations and whose competitiveness with wild individuals is assumed to decline (Benedict et al. 2009a). Both forced confinement and the negative selective pressures on parental generations may induce the loss of characteristics that affect the fitness of released insects (Dame 1985). To minimize selective processes on colonized populations, the use of heterosis such as the induction of hybrid vigor by crossing strains of different origin (Craig 1964, Seawright et al. 1975) or regular periodic replacement of existing colonies with wild stocks obtained directly from the field, has been proposed (Whitten and Foster 1975, Saul and McCombs 1995). Together with the application of these methods, it is important to organize mass-rearing environment where natural conditions are simulated as much as possible and the biological need of the species are considered along with economical management and high insect productivity. The quality of colonies adult nourishment sources, the appropriate oviposition sites, and adequate space requirement for precopulative flight are some attributes that can be addressed in the development of a mass production cage. Although several attempts have been made to create mechanized and automated larval-rearing units (Morlan et al. 1963, Singh et al. 1975, Bailey et al. 1980, Balestrino et al. 2012), little attention was given to mosquito mass-rearing cages (Benedict et al. 2009a). Until now, cages used in SIT release programs were obtained simply enlarging typical laboratory cages while keeping rearing methods identical to those used in research laboratories.
At the Insect Pest Control Laboratory (FAO–IAEA Joint Division) in Seibersdorf, Austria, researchers are developing mosquito mass-rearing technologies to support Member States in the successful application of the SIT as an effective integrative tool for area-wide integrated pest management (AW-IPM) campaigns against mosquitoes. One important factor to be considered in mosquito cage construction is the size in relation to the species and number of mosquitoes to be caged. The number of adult mosquitoes per square centimeter of vertical resting surface in a cage (so-called Density-Resting Surface, or DRS) is considered an important parameter connected to mating, feeding, and longevity; a DRS value of 1.8 is generally reported to promote suitable adult mosquito-rearing conditions (Gerberg 1970). Several plastic, glass, or screened cages have been proposed for suitable mosquito rearing with effective DRS values ranging from 6.0 to 1.2, with smaller values usually preferred for species of mosquitoes that easily breed in a relatively small space under laboratory conditions (Gerberg 1970). The adaptation to a small or the requirement for a large space to successfully perform the precopulative flight has been named respectively stenogamy and eurygamy. The condition of eurygamy is considered a common selective barrier that can decrease competitiveness of mass-reared insect owing to adaptation to indoor confined environment during colonization (Weidhaas et al. 1962, Dame et al. 1964).
Mating is not only a matter of space availability, and several other cues can play an important role in the creation, location, and timing of mating swarms of eurygamous mosquitoes. Merely increasing the volume of standard laboratory and outdoor cages did not improve insemination rates of newly colonized Anopheles arabiensis (Patton, 1905) and Anopheles gambiae (Giles, 1902). In fact, it was the introduction of a twilight phase and an artificial horizon contrasting with a lighted background that stimulated swarming behavior and led to high insemination rate (Marchand 1985). The use of artificial twilight was also used to successfully stimulate mating in eurygamous Culex pipiens (L.) kept under laboratory condition in cages of different volumes (Vinogradova 2000). The condition of stenogamy, a trait observed in species such as Aedes aegypti (L.) and Ae. albopictus, is one of the common ecological prerequisites pertaining to invasive mosquitoes species, as it confers more capacity to colonize small habitats, which allows founding populations to effectively establish in new territories (Vinogradova 2012). This biological trait is also an important characteristic to ease the establishment of vigorous mass-rearing colonies. Both above mentioned species show remarkable vitality and are particularly resistant to the unfavorable environmental conditions (Hien 1976a), therefore facilitating the success of artificial confinement in relative high adult densities. The initial establishment of a colony may incorporate only a portion of the field population genetic variability and therefore adequate genetic composition may be proportional to the number of individuals used to start the colony. To maintain a fair amount of heritable variance in an Ae. aegypti strain, a breeding colony was reported to be maintained at a population of at least 700 individuals (Stahler and Terzian 1966). Only slight reduction on genetic variability was also observed in an Ae. albopictus laboratory strain originated from a mixture of different populations collected in northern Italy in 1993 and reared for ≈120 generations, with regular introduction of wild-collected material, at density of ≈1000 adults per cage (40 by 40 by 40 cm, DRS = 6.4), when compared with wild populations collected from the same area (Bellini 2005).
In this article, we present a new mass production cage (Aedes MPC) for brood stock Ae. albopictus colonies. Based on the results obtained in preliminary experiments, a 100-liter-volume cage has been produced and tested. The cage can be handled entirely from the outside, minimizing the risk of escape and facilitating the management of adult rearing colonies. Devices for the introduction of pupae, blood feeding, sugar feeding, egg collection, and for the cleaning of the cage are described. A stocking capacity of 13,000 adults with a DRS equal to 1.7 was successfully tested. Finally, the creation of a new prototype model of MPC to effectively contain An. arabiensis adults is discussed with major changes to oviposition devices and systems for automatic collection of the eggs.
Materials and Methods
Mosquito Stocks and Rearing Methods.
The mosquito strain used in this trial was obtained from field material collected in Rimini, northern Italy, and maintained for 20 generations under laboratory conditions (28 ± 1°C, 80% RH, and a photoperiod of 14:10 [L:D] h). Larvae obtained after standardized hatching procedures (Balestrino et al. 2010) were reared at fixed density (2 larvae/ml) in white plastic trays (41 by 31 by 11 cm) containing 2 liters of deionized water and were fed with IAEA liquid diet (6% wt:vol; Damiens et al. 2012, Puggioli et al. 2013). During the first 4 d of development, the trays received 40 ml of diet a day, respectively, equal to 0.6 mg/larva/d. Owing to the protandry observed in this species, the pupae produced were collected at 48 h from the beginning of pupation to retain balanced sex ratio within the population introduced into the tested cages. All cage trials were performed inside climatic controlled rooms at laboratory conditions.
Plexiglas Cage Description and Operation.
Plexiglas cages have been created by bending and gluing Plexiglas sheets into different cubic shapes. Each cage has circular openings that are closed with a plastic net. At least one of these openings in each cage is connected with tissue sleeve (polyester screen fabric, mesh aperture 300 μm) to access and operate inside the cage. Cages with dimensions (l by w by h) 46 by 46 by 46 cm (C1), 100 by 20 by 100 cm (C2), and 100 by 65 by 100 cm (C3) were constructed. These cages are simple containers where many devices for sugar and blood feeding, pupal introduction, cleaning, and egg collection have to be introduced and removed periodically for the management of adult colony with concomitant risk of insect escape (Fig. 1). Cage devices are listed and described as follows.
Plexiglas cage (C1; left). Blood feeder (BF), sugar feeder (SF), plastic cup for pupal introduction (PC), and two beakers for egg collection (EC) are shown. The three cages used in the current study C1, C2, and C3 were constructed in the same way but with different dimensions (right). (Online figure in color.)
Sugar Feeder. A transparent plastic jar containing 80 ml of 10% sugar solution and plugged with a polyester filter fabric (mesh aperture 21 μm, Tekpro Srl, Appiano Gentile, Italy) is hung upside down inside the cage (Benedict 2007). This device can be checked from the outside and is the only component not to require the continuous action of the operator (Fig. 1).
Blood Feeder. A thermostatically controlled electrically heated aluminum cup (4 cm in diameter and 2 cm in height) is used to heat the blood offered to the mosquitoes. A natural swine intestine is fixed externally on top of the cup that is then filled with defibrinated fresh swine blood through the use of a pipette. The heated cup filled with blood is offered to the mosquito by introducing the aluminum cup into the cage (Bellini et al. 2007; Fig. 1).
Pupal Introduction and Cleaning. The loading of the cage is performed by introducing square plastic cups (10 by 10 by 6 cm) containing ≈2,000 pupae in 200 ml of dechlorinated water. The removal of such containers is a delicate operation and must be performed before providing the adults with the bloodmeal. These containers provide an excellent substrate for oviposition, distracting the females from laying the eggs in the dedicated containers (Fig. 1). When necessary, the procedures for cage cleaning are performed manually by wiping the bottom of the cage and collecting the dead adults by means of aspirators. For this reason, the cleaning procedures are generally performed when the cage is not in operation, thus limiting the possibility of a continuous use.
Eggs Collection. The eggs are laid and collected on filter paper strips positioned inside a 250-ml beaker filled with 100 ml dechlorinated water (Fig. 1).
Mass Production Cage (Aedes MPC) Description and Operation.
Based on the results obtained in preliminary experiments with Plexiglas cages, we created a stainless steel mass production cage (Aedes MPC) with a volume of 100 liter (or 100,000 cm3) and overall dimensions of 100 by 10 by 100 cm. The two largest sides have openings of 95 by 90 cm covered with polyester screen fabric (mesh aperture 300 μm). The edges of the screen were sewn with strips of Velcro, which can be fixed on the cage frame on Velcro strips previously glued (Fig. 2). The cage is a self-contained adult-rearing unit and has devices for sugar and blood feeding, pupal introduction, cleaning, and egg collection. These devices have been designed to be operable from outside of the cage. The cage was designed to be suspended and to be easily stackable in a rack system from where it can be pulled out to be operated. Including a minimum distance of 5 cm between cages to ensure adequate ventilation and lighting conditions, it is possible to obtain a stacking density equal to six to seven cages per linear meter. Cage devices are listed and described as follows.
Technical drawing with top, front, and side views (left) and picture of the Aedes MPC (right). Devices for blood and sugar feeding, pupal introduction (inlet valve), and cleaning (outlet valve) are represented. Metal plates for egg collection equipped with oviposition filter papers are shown in the picture and includes the top, front, and side views of the oviposition plate. Filter for dead and exuviae collection is not shown. (Online figure in color.)
Sugar Feeder. This device is made of a stainless steel cylindrical tube (6 cm in diameter) sealed on each end. On one side of the tube, at 5 cm from the end, an inlet pipe with a watertight stopper has been connected for adding sugar solution from outside of the cage. A transparent Plexiglas window on the front side of the tube shows the level of the sugar solution inside the tube. Thirty holes (2.5 cm in diameter) were created along the tube on the same side as the inlet pipe and covered with 20-micrometer stainless steel mesh. When filled with sugar solution (1.2 liter maximum capacity) and rotated by 180 degrees, the adults are able to feed through the mesh (Fig. 2—sugar feeder). This device is held in position along the length of the lower half of the cage by holders attached to the cage sides. The sugar solution can be refilled when needed and can be maintained for the entire duration of the cage.
Blood Feeder. The top front of the cage has two flanged holes (3 cm in diameter) into which 20-cm-long tubes of gauze with a knot on one end are inserted and fixed. When these gauze tubes are hung in the cage, collagen sausage casings (Edicas 23NC, FIBRAN S.A., Girona, Spain) filled with defibrinated fresh swine blood heated at 38–39°C can be inserted for blood feeding of the caged females (Fig. 2—blood feeder). The immediate response of females to the blood source assures an effective engorgement rate after the first 20 min of exposure. During this time, the collagen sausage casing is able to maintain optimal temperature to trigger a blood feeding response.
Pupal Introduction and Cleaning. The cage has inlet and outlet valves on the front side (Fig. 2—inlet valve and outlet valve). The inlet valve is connected to a pipe that runs through the cage to the opposite wall where it opens away from the outlet side. Through the inlet valve, it is possible to fill the bottom of the cage with water and to introduce the pupae when needed. The maximum size of the initial batch of pupae to be loaded into the cage can be calculated as ≈20,000–25,000 (corresponding to 20–25 pupae per square centimeter) according to the water surface available at the cage floor. This pupae batch size also corresponds to the maximum adult stocking density advisable for this species (DRS = 1) and taking into consideration the vertical resting surface available in the Aedes MPC. The outlet valve may be opened periodically for cleaning, during which, to aid washing away dead adults or exuviae, water may be flooded in through the inlet valve. A cylindrical filter with appropriate mesh size (e.g., 60–100 wires per square centimeter) can be placed at the outlet valve exit to collect or volumetrically estimate the dead individuals collected from the cage and to avoid escape of adults during cleaning. Furthermore, the easy removal of the two side screens allows access to the interior of the cage for the cleaning of all internal structures.
Egg Collection. At the bottom of the cage, four 40-cm-long and 3-mm-wide slots are created, two on each of the long sides. Removable metal plates with a lip along the lower edge to support oviposition filter paper are inserted into each of these slots (Fig. 2—oviposition plate). When pulled out to collect the oviposition filter paper, the lip prevents the complete extraction of the plates and thus prevents adult escaping. When inserted inside the cage the plates touch the bottom, with the lower part submerged in water. In this position, the metal plates are locked against the cage by external latches and the oviposition filter papers are held against the cage side sealing the slots.
Effect of Cage Volume on Survival and Egg Productivity.
The effect of the cage dimension on egg productivity and survival rate of Ae. albopictus was evaluated in three Plexiglas cages (C1, C2, and C3) at stocking capacity of 2,000, 4,000, and 13,000 pupae, respectively. The pupae loaded in each cage were volumetrically estimated, and in all cages, the adult density was equal to 20 adults per liter of cage volume. The DRS value created in this cage at the different stocking capacity was equal to 4.2, 6.0, and 2.5, respectively, for treatment C1, C2, and C3.
The three cages—C1, C2, and C3—were provided respectively with one, two, and seven sugar feeders at the beginning of the tests, and no further adjustments for the sucrose solution levels were performed during the tests. On days 7 and 8, after pupal introduction, a bloodmeal was offered to each cage using aluminum cups filled with 10 ml of swine blood previously heated to 38–39°C. Either one, two, or seven blood feeders were placed in cage C1, C2, and C3, respectively. Twenty minutes after the beginning of blood feeding, the cups were reheated and placed in the cages for additional 20 min. With the exception of the treatment C1, where the blood was placed on the cage floor, one cup from C2 and three cups from C3 were elevated to 50 cm by transparent plastic supports. This scheme has served to standardize as much as possible the exposure of blood to the females.
Eggs were collected by placing oviposition beakers (C1 = 2, C2 = 4, and C3 = 14) in cages 2 d after the blood feeding. The number of sugar feeders, blood feeders, and oviposition beakers used was based on the adult rearing experience with laboratory cages of the type C1 and adapted to larger cages in proportion to their volume. Five days after introduction, the oviposition filter papers were removed, left to dry in the climatic chamber for 24 h, scanned and eggs automatically counted using computerized processing of the egg oviposition filter paper images (ImageJ, U.S. National Institutes of Health, Bethesda, MD; Bellini et al. 2007). The mean number of eggs laid per female for each cage was determined by dividing the total number of eggs produced per cage by the number of surviving females present during blood feeding. Mortality of male and female adults was recorded on the day of blood feeding (day 7) to determine the number of females present at blood feeding and at the end of the trial (day 20) to evaluate final adult survival. Eight replicates for each cage stocking capacity have been performed. The adult sex ratios of the cage populations were calculated as the proportion of all male to female adults emerging from a sample of pupae (≈200) collected from the same stocks from where the initial load was obtained.
Effect of Adult Density on Aedes MPC Survival and Egg Productivity.
Pupae used for this experiment were obtained from the same cohort used in the previous tests. The Aedes MPC was tested using a stock of 2,000, 4,000, and 13,000 adults corresponding to a DRS value equal to 11, 5.5, and 1.7 respectively. The volumetrically estimated pupae were loaded into the Aedes MPC cages using the inlet valve and 3.5 liters of dechlorinated water. The adult density at the different stocking treatments was equal to 20, 40, and 130 adult per liter. Sugar feeders in all cages were filled with 1.2 liters of 10% (wt:vol) sucrose solution when pupae were introduced. No further adjustments for the sucrose solution level were performed during the tests. On days 7 and 8, after pupal introduction, a bloodmeal was offered to the females through the top gauze tubes using a 20-cm-long collagen sausage casings filled with 100 ml of defibrinated fresh swine blood. The collagen sausage casings were heated and offered to the different cages as described in the Aedes MPC Description and Operation section. Two days after the first bloodmeal, oviposition filter papers were positioned inside the cage using the oviposition plates. Egg quantification, adult survival, sex ratio and egg productivity were performed and evaluated as described in the previous test. Three replicates for each cage stocking treatment have been performed.
Statistical Analysis.
All statistical analyses were performed using MiniTab (MiniTab Inc., State College, PA). Egg productivity was evaluated by a generalized linear model (GLM). Data on adult survival at 20 d from pupal introduction were analyzed with GLM after angular (arcsinsqrt) transformation of the data expressed in percent.
Results
Effect of Cage Volume on Egg Productivity.
Plexiglas cages with different dimensions but equal adult densities (20 adult per liter of cage volume) showed no statistical differences in the number of eggs laid per female. A statistical difference was observed in the adult survival as a function of the differences in cage size (F2,21 = 7.51; P < 0.01; Table 1). Cage C3 showed a higher adult mortality at 20 d from pupal introduction as compared with treatments C1 (F1,14 = 11.42; P < 0.01) and C2 (F1,14 = 5.08; P < 0.05). When survival was analyzed as a function of sex, it was possible to observe that males (F 2,21 = 8.25; P < 0.01) and not females (F2,21 = 1.55; P > 0.05) showed different mortality rate at 20 d from pupation. Males from treatment C3 lived shorter compared with males coming from both treatment C1 (F1,14 = 12.01; P < 0.01) and C2 (F1,14 = 8.36; P < 0.05).
Data from mass production cage trials
Data from mass production cage trials
Effect of Adult Density on Aedes MPC Egg Productivity and Survival Rate.
Egg production and adult survival did not show any statistical difference as a function of the stocking treatment (Table 1). With the exception of treatment C3, these parameters are not statistically different from data collected from Plexiglas cages in all the different settings tested (Table 1).
The sex ratio achieved with the single introduction of pupae collected at 48 h from pupation was equal to 0.976 ± 0.041 (Mean ± SD).
Discussion
The egg production and the survival rates obtained with the mass production cage (Aedes MPC) confirmed the results obtained from the Plexiglas cage treatments without being affected by the different cage densities tested. Compared with the Plexiglas cages, the Aedes MPC allowed an easier handling of adult colonies with a minimized risk of escape.
With the exception of the increased mortality for males in the largest Plexiglas cage (C3), no difference in survival rate at 20 d or in the number of eggs per female was observed. Males usually did not respond to resting females, but they often chased and attempted to copulate with flying females (Clements 1999). When at the same density, a refractory or gravid female in large cage can therefore better elude male harassment during copulation attempts. We presume that once complete female insemination is reached, the continuous sexual responsiveness to refractory females requires increased energy expenditure from males attempting to copulate, causing increased male mortality in larger cages.
The similarity of egg production in the different treatments implies that in the Plexiglas cage and in the Aedes MPC feeding systems are similarly effective. In all treatments, the average number of eggs per female (±SE) was relatively low (13.3 ± 1.2), although values ranging from 42 to 88 eggs per female are typically reported for the first gonotrophic cycle in Ae. albopictus (Del Rosario 1963, Hawley 1988). The average number of eggs oviposited after a single bloodmeal depends on several factors such as resources available at emergence, amount of blood ingested, blood quality, or mating status (Hien 1976a, Blackmore and Lord 2000). Fecundity and fertility can also be affected by forced retention of mature eggs owing to the lack of a suitable oviposition site or by repulsive responses to oviposition stimulus (Mogi 1982, Bentley and Day 1989, Xue et al. 2005).To determine the most efficient method of maintaining Ae. aegypti colonies for egg production, studies were carried out comparing cycling and noncycling colony-operating procedures (Ansari et al. 1977). The average number of eggs per female obtained when pupae were only stocked once was 19.8 against 59.8 obtained in the cycling colony cages maintained with continuous insertion of pupae. Similar to this test, our cage experiments were performed with noncycling adult maintenance and we observed, after two consecutive days of blood feeding, the same low egg production. In comparative studies between Ae. aegypti and Ae. albopictus, it was reported that Ae. aegypti females lived longer and laid more eggs during their lifetime than Ae. albopictus females (Hien 1976a,b; Soekimani et al. 1984). In laboratory conditions both species took their first bloodmeal from a host placed inside the cage on the second or third day after emergence (Hien 1976b). However, when a live host is replaced by an artificial membrane feeder, 96% of Ae. aegypti have engorged the blood, while only 20% of Ae. albopictus females successfully completed a blood feeding (Lyski et al. 2011).
Artificial blood-feeding techniques based on the use on natural and artificial membranes have been proposed for several mosquito species (Tarshis 1959, Gerberg 1970, Rutledge et al. 1964) both for laboratory research and for efficient mass production purpose (Bailey et al. 1978). While in Plexiglas cages, blood was supplied through heated aluminum cup covered with natural swine intestine, the blood-feeding method used in the Aedes MPC is similar to the one proposed by Bailey et al. (1978) and uses collagen sausage casings vertically inserted into the cage to improve feeding (Lyski et al. 2011, Deng et al. 2012). The blood-feeding procedure for the Aedes MPC required the use of a larger amount of blood, but the position and the characteristic of collagen sausage casings guaranteed an engorgement rate comparable with standard feeding methods. Moreover, the use of collagen membranes is preferable over the use of natural membranes for its easier preparation, heating, disposal, and its resistance to handling. Collagen membranes are also commonly available on the market, are modest in cost, and are of standard and certified hygienic safety. However, further studies are needed to increase the response of Ae. albopictus to the bloodmeal provided with artificial feeding techniques.
In addition to the type of membrane used, the quality of blood supplied is a determining factor that affects blood engorgement in Ae. albopictus. The blood usually used for feeding is collected from slaughterhouses, mechanically defibrinated, and stored in a refrigerator at 4–6°C for no more than 5 d before being offered to females. This species, as well as Ae. aegypti, refuses to fed on frozen blood probably because of the hemolyzation caused by the freezing process, and the addition of sugar, such as sucrose or honey, was necessary to provide a stimulus to initiate feeding for Ae. aegypti (Liles et al. 1960). The use of these and other phagostimulants (e.g., ATP, L-lactic acid) together with the use of visual, thermal, and olfactory host stimuli must be investigated on Ae. albopictus to maximize blood feeding and thus eggs production of mass-reared colonies.
We did not observe any variation of egg production as a function of the cage volume used, probably owing to the fact that the blood-feeding devices were inside the cages rather than placed on top. Moreover, the distribution of the blood at different heights through the use of plastic supports (Plexiglas cages) or through the introduction of long blood casings from the upper part of the cage has probably increased and equalized the response of females to the blood in the different treatments.
The oviposition system created by the entire lower portion of the Aedes MPC, promotes the deposition exclusively in the areas covered by filter papers, confirming the ability of this species to lay eggs inside several water reservoirs using only the wet walls (Hien 1976a).
All operating procedures in the Aedes MPC were completed faster and allowed fewer escaped adults when compared with standard Plexiglas cages. Procedures for sugar feeding in all cages tested did not need further maintenance after the first positioning of the sugar-feeding devices. Adding methylparaben (0.2% wt:vol) to the sugar solution can preserve the quality of the sugar diet by avoiding possible fungal growth (Benedict et al. 2009b). Egg collection devices appeared effective in simulating vertical oviposition substrate. The cage showed excellent performance for brood stock maintenance in an Ae. albopictus mass production system, and can likely be easily adapted for the mass production of other Aedes mosquito species.
The first model of the Aedes MPC was made of stainless steel, because of its mechanic and corrosion resistance and for the possibility to perform high-temperature or steam-cleaning procedures routinely. Anyway this cage may be too heavy for its movement when not in use and has high production costs. For these reasons, further models in plastic or aluminum are under evaluation. The design of the Aedes MPC could also be further developed, and some changes be made to make it appropriate for species outside of the Aedes genus. Current work with the MPC is focused on creating a new prototype for An. arabiensis (Anopheles MPC) adult rearing. The dimensions of the Anopheles MPC have been increased to 200 by 20 by 100 cm (l by w by h, 400-liter volume) and, like the Aedes MPC, it has devices for sugar and blood feeding, and pupal production and both egg collection and cleaning occur from outside the cage. While the net sides and the devices for sugar and blood feeding are identical to those described above, major changes have been undertaken to create an appropriate system for oviposition and egg collection. The bottom of the cage is V shaped and may be partially filled with water to create an appropriate oviposition site. A perforated pipe welded all around the bottom introduces water into the cage and helps to rinse the cage to collect the eggs and dead adults through an outlet valve. The outlet valve has a free-rotating elbow that can be used to both introduce the pupae (when turned upwards) and to collect the eggs and dead (when turned downwards). Two strainers of different sizes are used to filter away dead adults from eggs during egg collection without damaging the eggs. Preliminary tests have shown the formation of swarms in the bottom part of the cage, confirming that the environment is conducive to mating behaviors of An. arabiensis. Further studies need to be performed to evaluate the effect of the volume and adult density on egg productivity and survival rate in this new cage prototype. In Fig. 3, the technical drawing and the overall view of the Anopheles MPC devised for An. arabiensis are shown.
Technical drawing with top, front, and side views (left) and picture of the Anopheles MPC (right). Devices for blood and sugar feeding are the same described in the Aedes MPC model. In this Anopheles MPC model, the inlet valve is only used to spray water and to rinse the bottom of the cage while the free-rotating elbow of the outlet valve can be used to introduce the pupae (upwards position) and to collect the eggs and the adult dead (downwards position). Tube gauzes for blood feeding and sieves for selective collection of eggs from adult dead are not shown. The two middle metal supports at the top of the cage can be equipped with black resting box where adults can hide during the day (not shown). (Online figure in color.)
Acknowledgments
This work has been conducted as part of the activities that Centro Agricoltura Ambiente “G. Nicoli” is planning as IAEA Collaborating Centre, and it was supported by grants from the Emilia-Romagna Regional Bureau-Public Health Department and from the FP7 Infrastructure project “INFRAVEC” Research capacity for the implementation of genetic control of mosquitoes, grant 228421.
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