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Aedes albopictus (Skuse) males in laboratory and semi-field cages: Release ratios and mating competitiveness Odessa Madakacherry, Rosemary Susan Lees 1 , Jeremie Roger Lionel Gilles ∗ Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, IAEA Laboratories, Seibersdorf A-2444, Austria
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Article history: Received 8 August 2013 Received in revised form 18 November 2013 Accepted 23 November 2013 Available online xxx Keywords: Sterile insect technique (SIT) Aedes albopictus Competition assays Induced sterility Radiation sterilization
a b s t r a c t To control the container-breeding mosquito and major vector of dengue and chikungunya Aedes albopictus, the sterile insect technique (SIT) is proposed as a component of integrated vector management programs in endemic areas. For the technique to be successful, released males, sterilized with 35 Gy of ionizing radiation during the pupal stage, must be able to compete for mating opportunities with wild counterparts and successfully copulate with wild females to induce sterility in the population. Any reduction in competitiveness can be compensated for by increasing the ratio of released sterile to wild males, a ratio which must be optimized for effectiveness and efficiency. Fruit fly SIT programs use field enclosures to test the competitiveness of sterile males to monitor the quality of the colony and adjust release ratios. This is laborious and time consuming, and for mosquito programs it would be advantageous if similarly useful results could be obtained by smaller scale laboratory tests, conducted on a more regular basis. In the present study we compared the competitiveness, as measured by hatching rate of resulting egg batches, of irradiated males measured in small and large laboratory cages and semi-field enclosures in a greenhouse setting, when competing in a 1:1, 3:1, and 5:1 ratio with fertile males. The sterile males were found to be equally competitive when compared to unirradiated counterparts, and a 5:1 ratio was sufficient to reduce, but not eliminate, the fertility of the female populations, irrespective of cage size. Variability in hatch rate in eggs laid by individual females and so-called indeterminate matings, when we could not be certain whether a female had mated a fertile or a sterile male, could be investigated by closer investigation of mating status and the frequency of multiple matings in Ae. albopictus. The laboratory results are encouraging for the effectiveness of the SIT using irradiated males of this species, and we support further assessment in the field. © 2013 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.
1. Introduction The chikungunya and dengue vector Aedes albopictus (Skuse) is an invasive pest in many parts of the world (Benedict et al., 2007) and a proposed target of area-wide integrated pest management (AW-IPM) operations with sterile insect technique (SIT) components, if feasibility trials currently underway in Italy (Bellini et al., 2007) and La Reunion (Boyer et al., 2011) prove successful. As with any other release program, the success of SIT relies on a steady and likely large scale production of high quality males able to compete with their wild counterparts for females. The production of large quantities of insects requires mass rearing facilities in which insects are reared under conditions
∗ Corresponding author. Tel.: +43 676 506 4818. E-mail addresses: [email protected] (O. Madakacherry), [email protected] (R.S. Lees), [email protected] (J.R.L. Gilles). 1 † Polo d’Innovazione Genomica, Genetica e Biologia S.C.a.R.L., Edificio D, 4ˆ piano Polo Unico di Medicina Santa Maria della Misericordia , Loc. S. Andrea delle Fratte, 06132 Perugia, Italy.
different from those in the field and are subjected to many unnatural processes including artificial diets, constant environmental conditions, high density rearing, colonization, and radiosterilization. Many of these treatments are thought to affect the overall quality of the adult insects produced and could hinder their ability to integrate into natural populations and effectively compete for and mate with wild females (Clarke and McKenzie, 1992). Sterilization through ionizing radiation can be especially damaging for the released adults (Patterson et al., 1977; Helinski et al., 2006). Though the effects of radiosterilization on longevity and mating capacity can be minimized by adjusting the dose and timing of irradiation (Andreasen and Curtis, 2005; Parker and Mehta, 2007), Oliva et al. (2012a,b) showed that the sterilization process may inhibit sperm production post-eclosion leading to fewer matings that result in successful insemination from radiosterilized males as compared to fertile males, thus decreasing the overall mating effectiveness. One strategy for counteracting any decreased mating ability is to release quantities of sterilized males that far outnumber existing males in the field, so-called ‘overflooding ratios’. For example, SIT releases of the Mediterranean fruit fly Ceratitis capitata are typically on the scale of 50–100 sterile males per wild male fly (C. Caceres,
0001-706X/$ – see front matter © 2013 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.11.020
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personal communication). This surfeit of sterile males, while individually less competitive than wild males will, on the basis of sheer number alone, be able to impact the fertility of wild populations. While the prospect of releasing vast amounts of males seems simple enough, the costs and labor associated with such high production would be prohibitive for many operations and a balance must be reached which ensures a sufficient number of sterile males are produced and released at the lowest cost possible. An additional concern when mass rearing any species is how colonization pressures may affect the strain. For example, one study found that the processes involved in colonization and laboratory rearing had a greater impact on the transcriptome of An. gambiae than the difference between the M and S molecular forms of the species complex (Aguilar et al., 2010), which exhibit at least partial genetic isolation (Tripet et al., 2001; Diabaté et al., 2009). Inbreeding depression effects have also been observed in Ae. albopictus (O’Donnell and Armbruster, 2010). Pressures for synchronous larval and pupal development, large size and longevity are all beneficial to facility management but may have far reaching effects that could lead to changes in the colony over time rendering them unsuitable for release (Bartlett, 1984). Artificial diets and rearing conditions can also lead to phenoand genotypic changes in the colony population (Bartlett, 1984). Most facilities, to monitor any changes that may occur, will have a series of quality control tests, which can easily be applied to assess certain parameters of strain health/quality (van Lenteren et al., 2003). Tephritid fruit fly release programs, for example, use frequent measurements of pupal weight as a means of assessing the quality of immature stages during rearing (FAO/IAEA/USDA, 2003). Any steady decrease in pupal weight could point to deficiencies in larval rearing—nutrition, crowding, temperature, etc.—that can then be quickly addressed. Quality control tests for mating ability, the results of which are used to establish the necessary proportion of sterile fruit flies released to the wild male population, are often performed using field cage trials (FAO/IAEA/USDA, 2003). Because of the labor and space necessary to conduct such large-scale trials, these tests are performed infrequently and any changes in male mating ability will not be immediately detected. Field cage trials are just as useful for monitoring mosquito mating ability, though just as in fruit flies, the practicability of doing large scale trials is a significant obstacle. Particularly convenient for mass-rearing facilities would be a smaller, laboratory-based approach, which could not only suggest field performance but also might serve as indicators of colony quality. To test the feasibility of using laboratory based mating trials to replace larger experiments, differing release ratios of sterile:fertile males were tested in both laboratory and semi-field cages.
2. Materials and methods The strain of Ae. albopictus used in the present study originates from Rimini, Italy and was provided by the centro agricoltura ambiente (CAA) in Crevalcore, Italy. It has been reared in the IAEA insect pest control laboratories (IPCL) in Seibersdorf, Austria since 2010. Eggs from the same generation (F 16) were used for all tests. Larval rearing took place in a climate-controlled insectary (T: 26 ◦ C, RH: 60%, light: 11L:11D with two, one-hour twilight periods simulating dawn and dusk) at a density of 1 larva/mL in 1 L of deionized water. Larvae were fed daily with a 4% solution of IAEA 2 diet (Puggioli et al., 2013) until pupation. Pupae were first separated for size by submerging a cylinder containing pupae in the bottom below two sieves of decreasing aperture (1.4 and 1.25 mm, VWR, Vienna, Austria) in a basin of room temperature deionized water and allowing the smaller (mainly male) pupae to swim to the top while trapping the larger (mainly female) pupae at the
bottom. Pupae trapped between the two sieves were a mix of males and females. Following sieving for enrichment, all pupae were manually separated under a stereomicroscope. After separation, female pupae and a portion of the male pupae were allowed to emerge into separate adult cages without further treatment (fertile), and a portion of male pupae were first sterilized with 35 Gy of ionizing radiation from a Co-60 source (Gammacell 220, MDS Nordion, Ottawa, Canada) when aged between 40 and 52 h (sterile). All treatments—fertile males, sterile males and virgin females—were maintained in separate cages with continuous access to a 10% (w/v) sucrose solution for 3 days following emergence to allow for sexual maturation. Separation of the sexes during the period following emergence was crucial to avoid any effect that the precocious emergence/sexual maturation of sterilized males noted by Bellini et al. (2013) might have on measurements of competitiveness. 2.1. Release ratios Competitive ability of sterilized males and the effect of release ratios on hatch rates were examined by testing three experimental ratios of sterile to fertile males: 1:1, 3:1, and 5:1. Assuming near parity in numbers between the sexes in wild populations, quantities of virgin females (n = 100) equal to the number of fertile males (n = 100) were used in all treatments. Control treatments with either only sterile or only fertile males paired with virgin females were also included. Experimental and control cages were established with virgin adults on the third day following emergence. In experimental cages, both sterile and fertile males were first added to cages before females were introduced. Mating was allowed for a 3 day period. A 10% (w/v) sucrose solution was provided for the duration of the experiment. Following the 3 day mating period, females were removed and transferred to new small lab cages and given access to a blood meal of fresh bovine blood for a 2 h period. The females were removed from experimental cages to prevent any further harassment by males, which often occurs during the blood feeding period (personal observation). After blood feeding, all females were individually isolated in Drosophila tubes (VWR International GmbH, Vienna, Austria) with moist filter paper (Sartorius Stedim Biotech GmbH, Göttingen, Germany) lining the bottom for oviposition. The tubes were checked daily for oviposition. If eggs were found, oviposition papers were collected, the number of eggs was recorded, and the papers were stored under laboratory conditions in Drosophila tubes for a minimum of 5 days to allow embryonation to occur. If no eggs were observed by 4 days post-blood feeding, females were offered a second, 2 h blood meal and replaced into individual tubes. All oviposition papers were hatched in their tubes using a standard hatch solution (Balestrino et al., 2010), which had been diluted with 5 L deionized water to prevent film formation in the small tubes. The number of L1 larvae present in each tube was recorded 24 h after adding the hatch solution and used to calculate the individual hatch rates for each female. Since it was assumed that any loss of L1 between adding the hatch solution and counting would be negligible, the number of L1 after 24 h was equivalent to the number of eggs that hatched. To test the applicability of such experiments as indicators of actual field competitiveness, the above experiment, including all ratio treatments, was performed in both small and large sized aluminum-mesh laboratory cages (30 and 60 cm square, respectively; Bioquip, Rancho Dominguez, Ca.) and 1 semi-field cage (∼1.75 m square, Live Monarch, Boca Raton, Fl.); each cage experiment was replicated three times. The experiments in laboratory cages were conducted in the same climate-controlled insectary and under the same conditions as is used for rearing, described above; the experiments in field size cages were conducted in a
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climate-controlled greenhouse on site at the IPCL in Seibersdorf. Mean temperature in the greenhouse fluctuated between 24.65 ± 0.6 ◦ C in the morning and 26.67 ± 0.2 ◦ C in the evening with a relative humidity of approximately 50%. Light in the greenhouse is provided by ambient light with approximately 8 h 47 min of daylight for replicates 1, 2, and 9 h 3 min for replicate 3 (the difference being due to changing day length over the course of the experiment). Three replicates of each experimental ratio and each control were tested for each cage size. Due to practical constraints, a maximum of two complete replicates (i.e., two replicates of all treatments of a given cage size) were performed at one time. Timing of the replicates was randomized to avoid any seasonal effects. 2.2. Data analysis The competitive index, ‘C’, defined by Fried (1971) was calculated for each cage size using hatch rates from the fertile control, sterile control and the 1:1 experimental cages. Because C is calculated based on en masse egg collection, hatch rates for each replicate were defined as the total number of L1 divided by the total number of eggs produced from each cage. Hatch rates were then averaged for the treatment mean and variance calculated according to Hooper and Horton (1981) and Iwahashi et al. (1983). For the remaining calculations, data were angle (arcsine) transformed and replicates were pooled. Mean hatch rates for treatments are presented as the average of the hatch rates of individual egg batches. Proportions of fertile, sterile, and unknown matings were determined by comparing the hatch rate of an individual egg batch (i.e., that of a single female) with the mean hatch rate (±CI) of the fertile and sterile controls. It was assumed that any individual egg batch with a value within or exceeding the CI of the fertile control hatch rate would also be the result of a fertile mating and
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that an egg batch with a value within or less than the CI of the sterile control hatch rate would be the result of a sterile mating. Any individual egg batch with an intermediate value was deemed to be of unknown provenance. 3. Results 3.1. Competitive index The C values for sterilized males competing in 60 cm and 1.75 m cages were both over 1 (C60 = 1.61, variance60 = 0.19 and C1.75 = 1.07, variance1.75 = 0.05, respectively). Since the measurement of interest is the reduction in competitiveness of sterilized relative to unirradiated males, these values were truncated to 1 and equal competitiveness between sterile and fertile males was assumed. In 30 cm cages, the C value indicated that 1.17 sterile males would be needed to equally compete with a single fertile male (C30 = 0.85, variance30 = 0.03). According to Hooper and Horton (1981) due to the low value of calculated variability, the differences in C among cage sizes is considered insignificant, and so the sterilized males can be considered equally competitive as their unirradiated counterparts under these experimental conditions. 3.2. Mean hatch rates The average hatch rate of individual egg batches was significantly different for each experimental ratio and both fertile and sterile control treatments within each cage size (Fig. 1). As expected, fertile control cages had the largest mean hatch rate followed, in descending order, by 1:1 (average induced sterility = 57 ± 6%), 3:1 (average induced sterility = 71 ± 5%) and 5:1 (average induced sterility = 81 ± 4%); mean hatch rates for sterile
Fig. 1. Mean of individual hatch rates (angle detransformed data ±CI) from cages containing either a 1:1, 3:1 or 5:1 ratio of sterile to fertile males and from cages containing only sterile or fertile males (controls). Treatments were repeated for 30 cm, 60 cm and 1.75 m (semi-field) cages. Within each cage size, all hatch rates were significantly different than each other, as represented by different lower case letters (p < 0.01). Mean hatch rates for all treatments in large lab cages were significantly lower than in small lab cages or field cages (p < 0.01).
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Fig. 2. Distribution of hatch rates for individual females recollected from cages containing either a 1:1, 3:1 or 5:1 ratio of sterile to fertile males or from cages containing only sterile or fertile males (controls). Each series of treatments was repeated in small and large lab cages (30 and 60 cm) and in a semi-field cage (1.75 m). Upper dotted lines represent the value, above which, females are assumed to have mated with a fertile male and were derived from the lower CI for the mean hatch rate observed in the fertile control treatment. Lower dotted lines represent the value, below which females are assumed to have mated with a sterile male and were derived from the upper CI for the mean hatch rate observed in the sterile control treatment.
control cages were the lowest. With respect to cage size, mean hatch rates from small laboratory cages and field cages were not significantly different from each other, but were both higher than mean hatch rates observed in the large laboratory cages (Fig. 1). 3.3. Individual hatch rates Using the mean hatch rates from fertile and sterile control cages, plus or minus their respective CI, individual egg batches were classified as resulting from matings with either a fertile or sterile male. Any individual egg batch with a hatch rate above the lower confidence interval of the fertile control mean (small lab cage = 76.1%, large lab cage = 65.7%, semi-field cage = 82.1%) was deemed to have originated from a fertile mating; hatch rates which fell below the upper confidence interval of the sterile control mean (small lab cage = 4.8%, large lab cage = 1.6%, semi-field cage = 5.1%) were deemed to be sterile. Any value between these two cut-off points was considered to be of indeterminate parentage. As the ratio of sterile to fertile males increased, the proportion of egg batches resulting from sterile matings increased while the proportion from fertile matings decreased (Fig. 2). For all experimental ratios in all cage sizes the proportion of indeterminate matings was relatively similar (ranging between 40 and 50%). 4. Discussion Many consider the negative effects of radiation a hindrance to the overall mating performance of radiosterilized males used in mosquito SIT programs (Alphey, 2002; Phuc et al., 2007). In fact, release ratios in many other species must be highly skewed in favor of sterilized males due to their low competitiveness, whether due
to irradiation or other factors. Based on field tests performed with Glossina palpalis gambiensis in Burkina Faso, Sow et al. (2012) calculated that sterile males would need to outnumber wild males at least 14:1 to induce complete sterility in a wild population, but acknowledge that actual release ratios might potentially need to be much higher. Routine releases of Ceratitis capitata (medfly) in Guatemala did not see significant induced sterility until the ratio of sterile to wild males exceeded 100:1 (Rendon et al., 2004) and overflooding ratios of Cydia pomonella (L.codling moth) released at the beginning of the growing season are 40:1 (Bloem and Bloem, 2000). In these pest insects previously or currently controlled by sterile releases the necessary release ratios were discovered based on field tests and suppression trials. In mosquitoes, however, the path to open release has been more gradual with a greater amount of laboratory data being generated in the absence of significant field data, though recent trials with genetically modified Ae. aegypti (Harris et al., 2012) and Wolbachia-infected Ae. polynesiensis (O’Connor et al., 2012) provide some initial data in other Aedes species. Thus, in order to estimate the expansion factor required to determine necessary release ratios based on values calculated from contained trials it would be valuable to compare these data to field release data as they become available. In accordance with a previous study on Ae. albopictus mating competitiveness (Bellini et al., 2013), males in the present study were shown to be equally competitive with fertile males in all cage sizes. This high competitiveness in males sterilized as pupae with a dose of 35 Gy, especially in field cages, is encouraging for SIT programs because it indicates that, if dose and time of sterilization are optimized, the potential damaging effects of irradiation can be minimized (Andreasen and Curtis, 2005) and the number of sterile males required for releases need not potentially be as
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high as previously thought. Of course, additional trials testing lab strain performance against wild or near-wild strains in outdoor field cages is critical because even strains which prove robust in lab testing can be unsuccessful when tested in such conditions. For example, a genetically engineered strain of Aedes aegypti successfully eliminated target populations in large field cages under laboratory conditions (de Valdez et al., 2011) but failed when tested in similarly sized cages on an outdoor field site (Facchinelli et al., 2013). The effect of such even competitiveness between sterile and fertile males in the present study led to significant decreases in mean hatch rate with increasing ratios of sterile to fertile males. Mean hatch rates were highest in fertile control cages and significantly decreased with each increasing ratio though further experiments would be required to meaningfully estimate the ratio expected to drive the fertility of the cage population to zero. While the highest ratio tested (5:1) did yield the lowest mean hatch rate of the experimental cages, they still differed significantly from results seen in the sterile control cages, indicating that larger ratios must be tested/released if population elimination were to be achieved. Of special note was the difference in mean hatch rates based on cage size. All cages followed a similar pattern in terms of hatch rate reduction at increasing ratios of sterile males, but mean hatch rates for large lab cages were significantly lower in all treatments and replicates. Because replicates were performed at different times and lowered hatch rates in the controls were only observed for large lab cages, many possible explanations for lowered hatch rates—human error during egg maturation/counting or problems with the blood used for feeding—are easily ruled out. While the reason for lower hatch rates in large lab cages is not immediately clear, the effect of cage size coupled with other external factors could be responsible. Although hatch rates did decrease with each increasing ratio of sterile:fertile males, it is interesting to note the broad distribution of hatch rates of individual egg batches and the many intermediate values seen. Unlike some Anopheline species (Helinski et al., 2008; Tripet et al., 2003) which can be inseminated by multiple males, Ae. aegypti have been shown to be largely monogamous with only very small numbers of multiple matings occurring (Gwadz and Craig, 1970). Results of a mating trial by Oliva et al. (Oliva, personal communication) indicate that while Ae. albopictus females are generally monogamous, insemination by multiple males is possible if the copulations occur within a short period of time (45 min). In the present study, the hatch rates of individual egg batches fell along the entire spectrum of fertility. While many of these points can be attributed to naturally occurring differences in fertility, the large proportion of intermediate matings in experimental treatments as compared to the controls suggests that a portion of these values could be attributable to multiple inseminations. In fact, although it was thought that larger cages would produce a smaller proportion of indeterminate matings as compared to smaller cages due to a lower overall density of adults, the actual proportion of indeterminate matings was relatively consistent for all experimental ratios in all cage sizes (roughly 50%). The high density of adults in the smaller cages was a condition similar to that of (Gwadz and Craig, 1970) who observed males interrupting the copulation of coupled pairs before complete semen transfer, possibly leaving females receptive to further mating. Because adults were sexually mature when released into experimental cages and mating began immediately, multiple inseminations could also be ascribed to several copulations within a short amount of time. What is important to note is that regardless of the high number of intermediate matings observed, higher ratios of sterile males do lead to many more egg batches that are definitely sterile, and thus influence the overall mean fertility of the cage population.
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Previous studies and the results presented herein indicate that irradiated male Ae. albopictus can effectively compete against fertile males indicating that both the dose rate and the life stage at which the males are irradiated are near optimal (Oliva et al., 2012a,b; Balestrino et al., 2010). With this key bit of the SIT puzzle in place, research can now be focused on developing specific, robust quality control measures for individual abilities (flight, sugar/mate seeking behaviors, mating initiation, sperm transfer) like those available to fruit fly and tsetse fly release programs (FAO/IAEA/USDA, 2003; FAO/IAEA, 2006). In the absence of these standardized quality control tests, mating trials such as those described herein are invaluable in their ability to indicate the general quality of the release population and give a broad idea of what results might be following open release. In fact, because results were not different based on cage size, experiments in small laboratory cages, which are less laborious and time consuming than field or semi-field trials, could be performed more often to establish an ideal overflooding ratio based on the both the current conditions in the field as well as the current status of the release colony. And although individual egg batches do show the range of potential hatch rates, for even simpler mating trials en masse egging would still provide much useful information. More frequent monitoring of the colony using these mating trials could even signal the need for out crossing or for establishing a new release colony altogether. As mentioned before, though mating trials are indeed valuable tools for assessing quality control, they are but one tool which should be combined with more in depth testing as it becomes available.
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Aedes albopictus (Skuse) males in laboratory and semi-field cages: Release ratios and mating competitiveness Odessa Madakacherry, Rosemary Susan Lees 1 , Jeremie Roger Lionel Gilles ∗ Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, IAEA Laboratories, Seibersdorf A-2444, Austria
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Article history: Received 8 August 2013 Received in revised form 18 November 2013 Accepted 23 November 2013 Available online xxx Keywords: Sterile insect technique (SIT) Aedes albopictus Competition assays Induced sterility Radiation sterilization
a b s t r a c t To control the container-breeding mosquito and major vector of dengue and chikungunya Aedes albopictus, the sterile insect technique (SIT) is proposed as a component of integrated vector management programs in endemic areas. For the technique to be successful, released males, sterilized with 35 Gy of ionizing radiation during the pupal stage, must be able to compete for mating opportunities with wild counterparts and successfully copulate with wild females to induce sterility in the population. Any reduction in competitiveness can be compensated for by increasing the ratio of released sterile to wild males, a ratio which must be optimized for effectiveness and efficiency. Fruit fly SIT programs use field enclosures to test the competitiveness of sterile males to monitor the quality of the colony and adjust release ratios. This is laborious and time consuming, and for mosquito programs it would be advantageous if similarly useful results could be obtained by smaller scale laboratory tests, conducted on a more regular basis. In the present study we compared the competitiveness, as measured by hatching rate of resulting egg batches, of irradiated males measured in small and large laboratory cages and semi-field enclosures in a greenhouse setting, when competing in a 1:1, 3:1, and 5:1 ratio with fertile males. The sterile males were found to be equally competitive when compared to unirradiated counterparts, and a 5:1 ratio was sufficient to reduce, but not eliminate, the fertility of the female populations, irrespective of cage size. Variability in hatch rate in eggs laid by individual females and so-called indeterminate matings, when we could not be certain whether a female had mated a fertile or a sterile male, could be investigated by closer investigation of mating status and the frequency of multiple matings in Ae. albopictus. The laboratory results are encouraging for the effectiveness of the SIT using irradiated males of this species, and we support further assessment in the field. © 2013 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.
1. Introduction The chikungunya and dengue vector Aedes albopictus (Skuse) is an invasive pest in many parts of the world (Benedict et al., 2007) and a proposed target of area-wide integrated pest management (AW-IPM) operations with sterile insect technique (SIT) components, if feasibility trials currently underway in Italy (Bellini et al., 2007) and La Reunion (Boyer et al., 2011) prove successful. As with any other release program, the success of SIT relies on a steady and likely large scale production of high quality males able to compete with their wild counterparts for females. The production of large quantities of insects requires mass rearing facilities in which insects are reared under conditions
∗ Corresponding author. Tel.: +43 676 506 4818. E-mail addresses: [email protected] (O. Madakacherry), [email protected] (R.S. Lees), [email protected] (J.R.L. Gilles). 1 † Polo d’Innovazione Genomica, Genetica e Biologia S.C.a.R.L., Edificio D, 4ˆ piano Polo Unico di Medicina Santa Maria della Misericordia , Loc. S. Andrea delle Fratte, 06132 Perugia, Italy.
different from those in the field and are subjected to many unnatural processes including artificial diets, constant environmental conditions, high density rearing, colonization, and radiosterilization. Many of these treatments are thought to affect the overall quality of the adult insects produced and could hinder their ability to integrate into natural populations and effectively compete for and mate with wild females (Clarke and McKenzie, 1992). Sterilization through ionizing radiation can be especially damaging for the released adults (Patterson et al., 1977; Helinski et al., 2006). Though the effects of radiosterilization on longevity and mating capacity can be minimized by adjusting the dose and timing of irradiation (Andreasen and Curtis, 2005; Parker and Mehta, 2007), Oliva et al. (2012a,b) showed that the sterilization process may inhibit sperm production post-eclosion leading to fewer matings that result in successful insemination from radiosterilized males as compared to fertile males, thus decreasing the overall mating effectiveness. One strategy for counteracting any decreased mating ability is to release quantities of sterilized males that far outnumber existing males in the field, so-called ‘overflooding ratios’. For example, SIT releases of the Mediterranean fruit fly Ceratitis capitata are typically on the scale of 50–100 sterile males per wild male fly (C. Caceres,
0001-706X/$ – see front matter © 2013 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.11.020
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personal communication). This surfeit of sterile males, while individually less competitive than wild males will, on the basis of sheer number alone, be able to impact the fertility of wild populations. While the prospect of releasing vast amounts of males seems simple enough, the costs and labor associated with such high production would be prohibitive for many operations and a balance must be reached which ensures a sufficient number of sterile males are produced and released at the lowest cost possible. An additional concern when mass rearing any species is how colonization pressures may affect the strain. For example, one study found that the processes involved in colonization and laboratory rearing had a greater impact on the transcriptome of An. gambiae than the difference between the M and S molecular forms of the species complex (Aguilar et al., 2010), which exhibit at least partial genetic isolation (Tripet et al., 2001; Diabaté et al., 2009). Inbreeding depression effects have also been observed in Ae. albopictus (O’Donnell and Armbruster, 2010). Pressures for synchronous larval and pupal development, large size and longevity are all beneficial to facility management but may have far reaching effects that could lead to changes in the colony over time rendering them unsuitable for release (Bartlett, 1984). Artificial diets and rearing conditions can also lead to phenoand genotypic changes in the colony population (Bartlett, 1984). Most facilities, to monitor any changes that may occur, will have a series of quality control tests, which can easily be applied to assess certain parameters of strain health/quality (van Lenteren et al., 2003). Tephritid fruit fly release programs, for example, use frequent measurements of pupal weight as a means of assessing the quality of immature stages during rearing (FAO/IAEA/USDA, 2003). Any steady decrease in pupal weight could point to deficiencies in larval rearing—nutrition, crowding, temperature, etc.—that can then be quickly addressed. Quality control tests for mating ability, the results of which are used to establish the necessary proportion of sterile fruit flies released to the wild male population, are often performed using field cage trials (FAO/IAEA/USDA, 2003). Because of the labor and space necessary to conduct such large-scale trials, these tests are performed infrequently and any changes in male mating ability will not be immediately detected. Field cage trials are just as useful for monitoring mosquito mating ability, though just as in fruit flies, the practicability of doing large scale trials is a significant obstacle. Particularly convenient for mass-rearing facilities would be a smaller, laboratory-based approach, which could not only suggest field performance but also might serve as indicators of colony quality. To test the feasibility of using laboratory based mating trials to replace larger experiments, differing release ratios of sterile:fertile males were tested in both laboratory and semi-field cages.
2. Materials and methods The strain of Ae. albopictus used in the present study originates from Rimini, Italy and was provided by the centro agricoltura ambiente (CAA) in Crevalcore, Italy. It has been reared in the IAEA insect pest control laboratories (IPCL) in Seibersdorf, Austria since 2010. Eggs from the same generation (F 16) were used for all tests. Larval rearing took place in a climate-controlled insectary (T: 26 ◦ C, RH: 60%, light: 11L:11D with two, one-hour twilight periods simulating dawn and dusk) at a density of 1 larva/mL in 1 L of deionized water. Larvae were fed daily with a 4% solution of IAEA 2 diet (Puggioli et al., 2013) until pupation. Pupae were first separated for size by submerging a cylinder containing pupae in the bottom below two sieves of decreasing aperture (1.4 and 1.25 mm, VWR, Vienna, Austria) in a basin of room temperature deionized water and allowing the smaller (mainly male) pupae to swim to the top while trapping the larger (mainly female) pupae at the
bottom. Pupae trapped between the two sieves were a mix of males and females. Following sieving for enrichment, all pupae were manually separated under a stereomicroscope. After separation, female pupae and a portion of the male pupae were allowed to emerge into separate adult cages without further treatment (fertile), and a portion of male pupae were first sterilized with 35 Gy of ionizing radiation from a Co-60 source (Gammacell 220, MDS Nordion, Ottawa, Canada) when aged between 40 and 52 h (sterile). All treatments—fertile males, sterile males and virgin females—were maintained in separate cages with continuous access to a 10% (w/v) sucrose solution for 3 days following emergence to allow for sexual maturation. Separation of the sexes during the period following emergence was crucial to avoid any effect that the precocious emergence/sexual maturation of sterilized males noted by Bellini et al. (2013) might have on measurements of competitiveness. 2.1. Release ratios Competitive ability of sterilized males and the effect of release ratios on hatch rates were examined by testing three experimental ratios of sterile to fertile males: 1:1, 3:1, and 5:1. Assuming near parity in numbers between the sexes in wild populations, quantities of virgin females (n = 100) equal to the number of fertile males (n = 100) were used in all treatments. Control treatments with either only sterile or only fertile males paired with virgin females were also included. Experimental and control cages were established with virgin adults on the third day following emergence. In experimental cages, both sterile and fertile males were first added to cages before females were introduced. Mating was allowed for a 3 day period. A 10% (w/v) sucrose solution was provided for the duration of the experiment. Following the 3 day mating period, females were removed and transferred to new small lab cages and given access to a blood meal of fresh bovine blood for a 2 h period. The females were removed from experimental cages to prevent any further harassment by males, which often occurs during the blood feeding period (personal observation). After blood feeding, all females were individually isolated in Drosophila tubes (VWR International GmbH, Vienna, Austria) with moist filter paper (Sartorius Stedim Biotech GmbH, Göttingen, Germany) lining the bottom for oviposition. The tubes were checked daily for oviposition. If eggs were found, oviposition papers were collected, the number of eggs was recorded, and the papers were stored under laboratory conditions in Drosophila tubes for a minimum of 5 days to allow embryonation to occur. If no eggs were observed by 4 days post-blood feeding, females were offered a second, 2 h blood meal and replaced into individual tubes. All oviposition papers were hatched in their tubes using a standard hatch solution (Balestrino et al., 2010), which had been diluted with 5 L deionized water to prevent film formation in the small tubes. The number of L1 larvae present in each tube was recorded 24 h after adding the hatch solution and used to calculate the individual hatch rates for each female. Since it was assumed that any loss of L1 between adding the hatch solution and counting would be negligible, the number of L1 after 24 h was equivalent to the number of eggs that hatched. To test the applicability of such experiments as indicators of actual field competitiveness, the above experiment, including all ratio treatments, was performed in both small and large sized aluminum-mesh laboratory cages (30 and 60 cm square, respectively; Bioquip, Rancho Dominguez, Ca.) and 1 semi-field cage (∼1.75 m square, Live Monarch, Boca Raton, Fl.); each cage experiment was replicated three times. The experiments in laboratory cages were conducted in the same climate-controlled insectary and under the same conditions as is used for rearing, described above; the experiments in field size cages were conducted in a
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climate-controlled greenhouse on site at the IPCL in Seibersdorf. Mean temperature in the greenhouse fluctuated between 24.65 ± 0.6 ◦ C in the morning and 26.67 ± 0.2 ◦ C in the evening with a relative humidity of approximately 50%. Light in the greenhouse is provided by ambient light with approximately 8 h 47 min of daylight for replicates 1, 2, and 9 h 3 min for replicate 3 (the difference being due to changing day length over the course of the experiment). Three replicates of each experimental ratio and each control were tested for each cage size. Due to practical constraints, a maximum of two complete replicates (i.e., two replicates of all treatments of a given cage size) were performed at one time. Timing of the replicates was randomized to avoid any seasonal effects. 2.2. Data analysis The competitive index, ‘C’, defined by Fried (1971) was calculated for each cage size using hatch rates from the fertile control, sterile control and the 1:1 experimental cages. Because C is calculated based on en masse egg collection, hatch rates for each replicate were defined as the total number of L1 divided by the total number of eggs produced from each cage. Hatch rates were then averaged for the treatment mean and variance calculated according to Hooper and Horton (1981) and Iwahashi et al. (1983). For the remaining calculations, data were angle (arcsine) transformed and replicates were pooled. Mean hatch rates for treatments are presented as the average of the hatch rates of individual egg batches. Proportions of fertile, sterile, and unknown matings were determined by comparing the hatch rate of an individual egg batch (i.e., that of a single female) with the mean hatch rate (±CI) of the fertile and sterile controls. It was assumed that any individual egg batch with a value within or exceeding the CI of the fertile control hatch rate would also be the result of a fertile mating and
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that an egg batch with a value within or less than the CI of the sterile control hatch rate would be the result of a sterile mating. Any individual egg batch with an intermediate value was deemed to be of unknown provenance. 3. Results 3.1. Competitive index The C values for sterilized males competing in 60 cm and 1.75 m cages were both over 1 (C60 = 1.61, variance60 = 0.19 and C1.75 = 1.07, variance1.75 = 0.05, respectively). Since the measurement of interest is the reduction in competitiveness of sterilized relative to unirradiated males, these values were truncated to 1 and equal competitiveness between sterile and fertile males was assumed. In 30 cm cages, the C value indicated that 1.17 sterile males would be needed to equally compete with a single fertile male (C30 = 0.85, variance30 = 0.03). According to Hooper and Horton (1981) due to the low value of calculated variability, the differences in C among cage sizes is considered insignificant, and so the sterilized males can be considered equally competitive as their unirradiated counterparts under these experimental conditions. 3.2. Mean hatch rates The average hatch rate of individual egg batches was significantly different for each experimental ratio and both fertile and sterile control treatments within each cage size (Fig. 1). As expected, fertile control cages had the largest mean hatch rate followed, in descending order, by 1:1 (average induced sterility = 57 ± 6%), 3:1 (average induced sterility = 71 ± 5%) and 5:1 (average induced sterility = 81 ± 4%); mean hatch rates for sterile
Fig. 1. Mean of individual hatch rates (angle detransformed data ±CI) from cages containing either a 1:1, 3:1 or 5:1 ratio of sterile to fertile males and from cages containing only sterile or fertile males (controls). Treatments were repeated for 30 cm, 60 cm and 1.75 m (semi-field) cages. Within each cage size, all hatch rates were significantly different than each other, as represented by different lower case letters (p < 0.01). Mean hatch rates for all treatments in large lab cages were significantly lower than in small lab cages or field cages (p < 0.01).
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Fig. 2. Distribution of hatch rates for individual females recollected from cages containing either a 1:1, 3:1 or 5:1 ratio of sterile to fertile males or from cages containing only sterile or fertile males (controls). Each series of treatments was repeated in small and large lab cages (30 and 60 cm) and in a semi-field cage (1.75 m). Upper dotted lines represent the value, above which, females are assumed to have mated with a fertile male and were derived from the lower CI for the mean hatch rate observed in the fertile control treatment. Lower dotted lines represent the value, below which females are assumed to have mated with a sterile male and were derived from the upper CI for the mean hatch rate observed in the sterile control treatment.
control cages were the lowest. With respect to cage size, mean hatch rates from small laboratory cages and field cages were not significantly different from each other, but were both higher than mean hatch rates observed in the large laboratory cages (Fig. 1). 3.3. Individual hatch rates Using the mean hatch rates from fertile and sterile control cages, plus or minus their respective CI, individual egg batches were classified as resulting from matings with either a fertile or sterile male. Any individual egg batch with a hatch rate above the lower confidence interval of the fertile control mean (small lab cage = 76.1%, large lab cage = 65.7%, semi-field cage = 82.1%) was deemed to have originated from a fertile mating; hatch rates which fell below the upper confidence interval of the sterile control mean (small lab cage = 4.8%, large lab cage = 1.6%, semi-field cage = 5.1%) were deemed to be sterile. Any value between these two cut-off points was considered to be of indeterminate parentage. As the ratio of sterile to fertile males increased, the proportion of egg batches resulting from sterile matings increased while the proportion from fertile matings decreased (Fig. 2). For all experimental ratios in all cage sizes the proportion of indeterminate matings was relatively similar (ranging between 40 and 50%). 4. Discussion Many consider the negative effects of radiation a hindrance to the overall mating performance of radiosterilized males used in mosquito SIT programs (Alphey, 2002; Phuc et al., 2007). In fact, release ratios in many other species must be highly skewed in favor of sterilized males due to their low competitiveness, whether due
to irradiation or other factors. Based on field tests performed with Glossina palpalis gambiensis in Burkina Faso, Sow et al. (2012) calculated that sterile males would need to outnumber wild males at least 14:1 to induce complete sterility in a wild population, but acknowledge that actual release ratios might potentially need to be much higher. Routine releases of Ceratitis capitata (medfly) in Guatemala did not see significant induced sterility until the ratio of sterile to wild males exceeded 100:1 (Rendon et al., 2004) and overflooding ratios of Cydia pomonella (L.codling moth) released at the beginning of the growing season are 40:1 (Bloem and Bloem, 2000). In these pest insects previously or currently controlled by sterile releases the necessary release ratios were discovered based on field tests and suppression trials. In mosquitoes, however, the path to open release has been more gradual with a greater amount of laboratory data being generated in the absence of significant field data, though recent trials with genetically modified Ae. aegypti (Harris et al., 2012) and Wolbachia-infected Ae. polynesiensis (O’Connor et al., 2012) provide some initial data in other Aedes species. Thus, in order to estimate the expansion factor required to determine necessary release ratios based on values calculated from contained trials it would be valuable to compare these data to field release data as they become available. In accordance with a previous study on Ae. albopictus mating competitiveness (Bellini et al., 2013), males in the present study were shown to be equally competitive with fertile males in all cage sizes. This high competitiveness in males sterilized as pupae with a dose of 35 Gy, especially in field cages, is encouraging for SIT programs because it indicates that, if dose and time of sterilization are optimized, the potential damaging effects of irradiation can be minimized (Andreasen and Curtis, 2005) and the number of sterile males required for releases need not potentially be as
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high as previously thought. Of course, additional trials testing lab strain performance against wild or near-wild strains in outdoor field cages is critical because even strains which prove robust in lab testing can be unsuccessful when tested in such conditions. For example, a genetically engineered strain of Aedes aegypti successfully eliminated target populations in large field cages under laboratory conditions (de Valdez et al., 2011) but failed when tested in similarly sized cages on an outdoor field site (Facchinelli et al., 2013). The effect of such even competitiveness between sterile and fertile males in the present study led to significant decreases in mean hatch rate with increasing ratios of sterile to fertile males. Mean hatch rates were highest in fertile control cages and significantly decreased with each increasing ratio though further experiments would be required to meaningfully estimate the ratio expected to drive the fertility of the cage population to zero. While the highest ratio tested (5:1) did yield the lowest mean hatch rate of the experimental cages, they still differed significantly from results seen in the sterile control cages, indicating that larger ratios must be tested/released if population elimination were to be achieved. Of special note was the difference in mean hatch rates based on cage size. All cages followed a similar pattern in terms of hatch rate reduction at increasing ratios of sterile males, but mean hatch rates for large lab cages were significantly lower in all treatments and replicates. Because replicates were performed at different times and lowered hatch rates in the controls were only observed for large lab cages, many possible explanations for lowered hatch rates—human error during egg maturation/counting or problems with the blood used for feeding—are easily ruled out. While the reason for lower hatch rates in large lab cages is not immediately clear, the effect of cage size coupled with other external factors could be responsible. Although hatch rates did decrease with each increasing ratio of sterile:fertile males, it is interesting to note the broad distribution of hatch rates of individual egg batches and the many intermediate values seen. Unlike some Anopheline species (Helinski et al., 2008; Tripet et al., 2003) which can be inseminated by multiple males, Ae. aegypti have been shown to be largely monogamous with only very small numbers of multiple matings occurring (Gwadz and Craig, 1970). Results of a mating trial by Oliva et al. (Oliva, personal communication) indicate that while Ae. albopictus females are generally monogamous, insemination by multiple males is possible if the copulations occur within a short period of time (45 min). In the present study, the hatch rates of individual egg batches fell along the entire spectrum of fertility. While many of these points can be attributed to naturally occurring differences in fertility, the large proportion of intermediate matings in experimental treatments as compared to the controls suggests that a portion of these values could be attributable to multiple inseminations. In fact, although it was thought that larger cages would produce a smaller proportion of indeterminate matings as compared to smaller cages due to a lower overall density of adults, the actual proportion of indeterminate matings was relatively consistent for all experimental ratios in all cage sizes (roughly 50%). The high density of adults in the smaller cages was a condition similar to that of (Gwadz and Craig, 1970) who observed males interrupting the copulation of coupled pairs before complete semen transfer, possibly leaving females receptive to further mating. Because adults were sexually mature when released into experimental cages and mating began immediately, multiple inseminations could also be ascribed to several copulations within a short amount of time. What is important to note is that regardless of the high number of intermediate matings observed, higher ratios of sterile males do lead to many more egg batches that are definitely sterile, and thus influence the overall mean fertility of the cage population.
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Previous studies and the results presented herein indicate that irradiated male Ae. albopictus can effectively compete against fertile males indicating that both the dose rate and the life stage at which the males are irradiated are near optimal (Oliva et al., 2012a,b; Balestrino et al., 2010). With this key bit of the SIT puzzle in place, research can now be focused on developing specific, robust quality control measures for individual abilities (flight, sugar/mate seeking behaviors, mating initiation, sperm transfer) like those available to fruit fly and tsetse fly release programs (FAO/IAEA/USDA, 2003; FAO/IAEA, 2006). In the absence of these standardized quality control tests, mating trials such as those described herein are invaluable in their ability to indicate the general quality of the release population and give a broad idea of what results might be following open release. In fact, because results were not different based on cage size, experiments in small laboratory cages, which are less laborious and time consuming than field or semi-field trials, could be performed more often to establish an ideal overflooding ratio based on the both the current conditions in the field as well as the current status of the release colony. And although individual egg batches do show the range of potential hatch rates, for even simpler mating trials en masse egging would still provide much useful information. More frequent monitoring of the colony using these mating trials could even signal the need for out crossing or for establishing a new release colony altogether. As mentioned before, though mating trials are indeed valuable tools for assessing quality control, they are but one tool which should be combined with more in depth testing as it becomes available.
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Please cite this article in press as: Madakacherry, O., et al., Aedes albopictus (Skuse) males in laboratory and semi-field cages: Release ratios and mating competitiveness. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.11.020
