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Longevity, Fertility and Fecundity of Adult Blow Flies (Diptera: Calliphoridae) Held at Varying Densities: Implications for Use in Bioconversion of Waste

Longevity, Fertility and Fecundity of Adult Blow Flies (Diptera: Calliphoridae) Held at Varying... Abstract Large numbers of flies are needed to produce the quantity of larvae required for insect bioconversion of waste. However, this ‘mass-rearing’ may negatively affect adult survival and reproductive output. This study assessed the suitability for mass-rearing of four blow fly species, Chrysomya chloropyga, Chrysomya chloropyga (Wiedemann), Chrysomya megacephala (F.), Chrysomya putoria (Wiedemann) and Lucilia sericata (Meigen). Flies were kept at densities of 20, 50, 100, 250, 500, and 1,000 flies per 30 × 30 × 30 cm cage with an even sex ratio. Time to 50% mortality (LT50) was recorded, and the effects of density, species, and sex on LT50, fecundity, and fertility were determined. Females survived longer than males across all species. There was evidence for a trade-off between survival and high fecundity in L. sericata and C. chloropyga at density 250. C. megacephala had low fecundity across all densities. At high densities, C. putoria had the lowest mortality and highest fecundity, making it the most suitable for mass-rearing. mass-rearing, bioconversion, Calliphoridae, density, mortality Bioconversion is a nutrient cycling process that can reduce agricultural, industrial, and household organic waste and produce value-added products. The bioconversion process can be applied to a variety of different waste types, and different organisms ranging from bacteria and yeast to insects have been used to facilitate this process (Kumar et al. 2008, Madhavan et al. 2012, van Huis 2013, Čičková et al. 2015). Flies (Insecta: Diptera) are often used in bioconversion due to their known association with decaying material, high fecundity, rapid generation times, and ease of rearing (van Huis 2013). Proteins and lipids recovered by bioconversion of organic waste can be fed to livestock (van Huis 2013) or converted into biodiesel (Li et al. 2011). Large-scale conversion of waste products by insects requires that they be mass-reared. Mass-rearing of insects has been used to produce large numbers for release in sterile insect technique (SIT; Parker 2005) and biological control programs (Sørensen et al. 2012). This has included various fly species including tephritid fruit flies such as Bactrocera tryoni (Froggatt) (Weldon et al. 2013) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (Pereira et al. 2013), as well as tsetse flies (Glossina species; Vreysen et al. 2011, Abd-Alla et al. 2013) and mosquitoes (Gilles et al. 2014). Insects that are kept in mass-rearing facilities should ideally not experience high mortality or suffer decreased egg production (fecundity) or egg hatch (fertility) when there is an increase in adult density within cages. However, different species of flies respond to the high density conditions of mass-rearing in different ways (Robinson 2005), which has implications for their suitability in large-scale bioconversion facilities. The effects of the stress associated with crowding in cages where adults are held at high density has been documented for a number of different fly species. This increased stress can lead to reduced longevity and physical injury, as well as changes in interactions between the different sexes, which then affects fecundity and fertility (Rull et al. 2012). Fecundity refers to the total number of eggs that are produced by a female, whereas fertility is the number of eggs that hatch. Male mating success and therefore male reproductive success can be reduced by increased density due to changes in mating behavior, decreased territoriality, and interrupted mating with females (Gaskin et al. 2002, Díaz-Fleischer et al. 2009). Drosophila melanogaster (Meigen) show decreased levels of territoriality at high densities, leading to fewer interactions between males (Hoffmann and Cacoyianni 1990). In C. capitata, male longevity is more negatively impacted by an increase in density than that of females, possibly due to increased aggression and increased behavioral costs to mate successfully (Gaskin et al. 2002). Some of the changes in behavioral interactions that can occur under high-density conditions have been observed in Anastrepha ludens (Loew) (Diptera: Tephritidae), where females mate sooner in high-density conditions, whereas males at lower densities mate more often (Díaz-Fleischer et al. 2009). Males of A. ludens also require a minimal area for males to establish their territories and to reduce male–male aggressive interactions that would negatively affect their reproductive success (Díaz-Fleischer et al. 2009). Egg production is one of the bottle necks in the process of mass-rearing. However, few studies have sought to maximize egg production by determining an optimal adult density for fly species that can be mass-reared in bioconversion facilities (Pastor et al. 2015). This situation demands that further research be pursued to ensure high levels of fecundity and fertility while minimizing the space, cost, and handling of mass-reared adults (Pastor et al. 2015). Limited research has been conducted on the use of blowflies (Calliphoridae) or flesh flies (Sarcophagidae) for mass-rearing (Čičková et al. 2015, Pastor et al. 2015). One exception is the screwworm fly, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), which is a major pest of cattle (Vargas-Terán et al. 2005), and has been reared successfully at large scales for release in SIT programs. In C. hominivorax, adult density does not affect survival, fecundity, or fertility (Berkebile et al. 2006). However, there was an observable difference in behavior between wild flies and those reared in the laboratory due to the selection pressure experienced during mass-rearing. These behavioral changes included an increased amount of time spent walking rather than flying and decreased competitiveness between males (Bush et al. 1976). A more sedentary phenotype may have led to the laboratory reared C. hominivorax being less susceptible to the negative effects of increased cage density that are apparent in other mass-reared fly species. Blow fly species have the potential to be used for bioconversion of a number of different waste products and should be studied in more detail to assess their relative utility. There are a number of organic waste streams that are not efficiently converted to biomass by the fly species currently used by most bioconversion facilities, predominantly black soldier flies, Hermetia illucens L. (Diptera: Stratiomyidae), and house flies, Musca domestica L. (Diptera: Muscidae) (van Huis 2013, Čičková et al. 2015), and research into finding more suitable and versatile species is required. In addition, blow fly species exhibit wandering behavior prior to pupating (Richards et al. 2009a), which can be utilized in a ‘self-harvesting’ system. This may lead to more efficient production than those using H. illucens, which require mechanical separation of larvae from the remaining waste (reviewed by Pastor et al. 2015). Potential blow fly species include Chrysomya megacephala (Fabricius), Chrysomya chloropyga (Wiedemann), Chrysomya putoria (Wiedemann), and Lucilia sericata (Meigen) (Calliphoridae). These species have not been well studied in terms of mass-rearing with little information available on the effects of density on their life history traits. Chrysmomya megacephala and C. putoria are reported to be associated with pit latrine wastes (Laurence 1986, Lindsay et al. 2013) and may be able to successfully breakdown municipal or manure waste. Additionally, C. megacephala larvae have been successfully raised on restaurant waste that contained a mixture of vegetable and meat scraps (Li et al. 2011). L. sericata is a carrion-associated blow fly that is often used in laboratory experiments (Clark et al. 2006, Čičková et al. 2010, Blystone and Hansen 2014) and is highly prolific and successful under laboratory conditions (Blystone and Hansen 2014). C. chlorpyga is considered to be a large mammal carcass specialist and can produce high numbers of eggs at a time (Richards et al. 2009b). This species may be useful in the conversion of large quantities of waste. This study assessed the suitability for mass-rearing of four species of carrion flies, specifically assessing the effects on survival, fertility, and fecundity of holding adults at high densities. This is an important but often overlooked consideration for the successful establishment and continued high output of bioconversion facilities that use insects for organic waste valorization to usable biomass. The four species tested were: C. cholorpyga, C. megacephala, C. putoria, and L. sericata. These four calliphorid species are abundant in Gauteng province, South Africa, where this study was conducted (Parry et al. 2016). They are also present across most of South Africa (Richards et al. 2009c). C. chloropyga and C. putoria have a wide Afrotropical distribution (Picard et al. 2012), while L. sericata and C. megacephala are widely distributed across most of the world (Stevens and Wall 1996, Williams and Villet 2006). It was hypothesized that increasing adult density would negatively affect survival of all species and both sexes, but male survival would be affected more than that of females due to the energetic demands and physical interactions associated with competition for mates. At higher densities it was also predicted that fecundity and fertility per female would be reduced due to an increase in stress and fewer or interrupted interactions between females and males. Materials and Methods Colony Establishment Adult carrion flies were collected from various areas around Gauteng province, South Africa, using modified Red-Top hanging traps (Miller Methods Ltd., Pretoria). The traps were modified by opening the base of the trap and attaching a 500-ml plastic cup with several elastic bands. Adult flies were attracted to the trap by placing a bait mixture of food-grade chicken livers and fish in the plastic cup. A square piece of white voile curtain fabric was placed over the top of the cup to stop the flies from becoming fouled in the bait. Traps were hung at a height of approximately 2 m above the ground from available trees and were checked daily until the appropriate fly species and numbers were collected. Numbers of between 20 and 100 were necessary to start the initial colony, and additional flies were caught throughout the duration of the study to maintain colony fitness and genetic variability. The species that were specifically targeted for this study were C. chloropyga, C. megacephala, C. putoria, and L. sericata. Laboratory Rearing The flies that were collected live from the traps were identified and kept in single-species rearing cages. The cages were made from 13-liter transparent plastic containers (MaxiMultiBox, Hobbylife, Istanbul, Turkey) with dimensions of 0.36 × 0.25 × 0.23 m (0.013 m3). A 0.2 × 0.1 m2 section was cut from the lid of the plastic container and covered with white voile curtain fabric for ventilation. One side of the cage had a circular hole cut out with a diameter of 15 cm, fitted with a 45-cm long sleeve of white voile curtain fabric for daily feeding, removal of eggs and pupae, and cleaning of the cage. The flies were provided with unrestricted access to sugar, milk powder as a protein source, and water from soaked cotton wool. The rearing cages were maintained at approximately 25°C in a room with a window to provide a natural day:night photocycle. Food-grade chicken liver was provided in a 150-ml plastic container (WestPack Lifestyle) as a breeding medium to maintain the cultures. A larger 500-ml plastic container (WestPack Lifestyle) with sterile river sand was placed underneath the breeding medium to be used by the flies for pupation. Effects of Density on Survival, Fecundity, and Fertility Pupae were collected from the breeding cages and placed in a separate breeding cage. Pupae were kept in an incubator (LTIE, Labcon, Gauteng, South Africa) at 25°C until eclosion. Newly emerged flies were collected and sexed by measuring the occipital sclerite and placed into insect cages with dimensions of 0.3 × 0.3 × 0.3 m (0.028 m3, BugDorm 43030, MegaView Science, Taichung, Taiwan). Flies were kept at different densities with an even sex ratio (Table 1). Each density was repeated five times for each species. The density of 1,000 flies per cage was as high as or higher than the high densities that are found within bioconversion facilities based on the available cage surface area and volume per fly. Reported values from bioconversion facilities are between 2.5 and 10 cm2 internal cage surface area per fly or a volume of between 25 and 200 cm3 per fly (Charlton et al. 2015). Each cage was checked daily for fly mortality. Sex of fatalities was recorded. From 8 until 32 d after adult emergence, a Petri dish containing 30 g of liver was placed in each cage every second day. Within 36 h, the liver was removed and assessed for eggs laid in order to determine female fecundity. If eggs were present, they were carefully collected, weighed to determine the approximate number of eggs, and then placed on fresh liver where they were allowed to hatch. The approximate mass of an egg per species is 0.101 ± 0.024 mg for C. chloropyga, 0.128 ± 0.021 mg for C. megacephala, 0.124 ± 0.023 mg for C. putoria, and 0.119 ± 0.031 mg for L. sericata. The number of eggs that did not hatch into first instar larvae were counted the next day to determine egg viability. This was used to assess fertility of the females present at different densities. Recording of mortality and assessment of fecundity and fertility continued until 45 d after adult emergence or until up to 80% of females had died. Table 1. The cage surface area and cage volume that is available per fly for each initial fly density Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Open in new tab Table 1. The cage surface area and cage volume that is available per fly for each initial fly density Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Open in new tab Data Analysis The time elapsed, until 50% of females and males were dead (LT50), was determined for each replicate cage of each species and density. A general linear model was used to determine the effect of density, species, sex, and their interactions on LT50. Generalized linear models (GLZ) were used to analyze the effect of density, species and time, and their interactions on the number of eggs per female that were laid over time. The time (in days) until peak production and the number of eggs that were laid per female on the day of peak egg production were also analyzed in relation to density, species, and their interaction. These measures of fecundity are informative for the harvesting of eggs in bioconversion facilities. Stepwise deletion of the least significant terms was used to minimize the model. Post hoc multiple comparisons were performed using Fisher’s least significant difference tests. IBM SPSS Statistics was used to perform these analyses. A logistic regression with a quasibinomial distribution was applied to the total number of eggs produced combined with the total number of hatched eggs, both accounting for the initial density of females (included as a covariate in the model), in order to determine the effect of density and species on egg fertility. A quasibinomial distribution was applied due to over dispersion of the data when using a binomial distribution. In all cases, the minimal adequate model was obtained by applying a model simplification procedure based on Akaike’s information criterion (Crawley 2007). Differences between groups were identified by inspecting model coefficients. These analyses were performed using RStudio version 0.99.903 running R version 3.3.1 (R Development Core Team, 2016). Results Survival Survival for each species at each density was determined by looking at the time in days until 50% of females and males had died (LT50) for each cage. LT50 differed significantly between females and males (Fig. 1; F1, 217 = 24.669, P < 0.001), with females reaching 50% mortality later than males. Significant differences in LT50 were also found between species (F3, 217 = 17.871, P < 0.001), between densities (F5, 217 = 9.261, P < 0.001), and within species by density interactions (F15, 217 = 4.763, P < 0.001). Fig. 1. Open in new tabDownload slide Time until 50% mortality (LT50) of females and males of four calliphorid species held at different initial densities. Significant differences between different densities within a species are indicated by letters. Solid white bar = male, dotted bar = female. Significant differences between different densities within a species are indicated by letters with homogenous groups sharing the same letters. Fig. 1. Open in new tabDownload slide Time until 50% mortality (LT50) of females and males of four calliphorid species held at different initial densities. Significant differences between different densities within a species are indicated by letters. Solid white bar = male, dotted bar = female. Significant differences between different densities within a species are indicated by letters with homogenous groups sharing the same letters. Due to differences in the effect of density within different species, there was no clear trend for the overall effect of increasing density on LT50 (Fig. 1). At density 250, C. chloropyga and L. sericata were significantly different from all other species, with LT50 reached before all other species and densities. LT50 was reached last by C. putoria females at density 250 and 100. C. megacephala and C. putoria were not significantly different from each other across all densities and followed a trend of high survival at densities 50, 100, and 250 with a decrease in survival at density 500. Across all four species, there was an increase in LT50 from density 500 to density 1,000. At density 50 or 1,000, the LT50 of the species did not differ from each another. L. sericata and C. chloropyga were only significantly different from each other at density 100, where LT50 was reached earlier by C. chloropyga than by L. sericata. These two species had a similar response to increasing density in the cages, with a decrease in LT50 from density 50 to density 250 and from there an increase in LT50 from density 250 to 1,000. Fecundity Time elapsed (in days) until peak egg production (Fig. 2) was significantly different among species (GLZ: χ2 = 11.218, df = 3, P = 0.011) and was also affected by the interaction of species and density (GLZ: χ2 = 37.876, df = 15, P < 0.001). The main effect of density was not significant (GLZ: χ2 = 3.714, df = 5, P = 0.591), indicating the absence of a clear trend of the effect of increasing density on the day of peak egg production across all four species. The earliest day of peak egg production among the four species was achieved by C. chloropyga at density 20 while the latest day of peak egg production was achieved by C. putoria at density of 20. For C. putoria, as density increased from density 20 the time to day of peak egg production markedly decreased until density 100. Thereafter, there was no effect of density on day of peak egg production for C. putoria. L. sericata showed a different pattern from C. putoria where the density 20 reached peak egg production first followed by an increase in the time until peak egg production as density increased, with the exception of densities 250 and 1,000. These two densities were not significantly different from density 20. There were no significant differences of day of peak egg production across different species at densities 50, 250, and 1,000. Fig. 2. Open in new tabDownload slide Day of peak egg production for four calliphorid species held at different densities and number of eggs produced per female on day of peak egg production. Significant differences between different densities within a species are indicated by letters. Fig. 2. Open in new tabDownload slide Day of peak egg production for four calliphorid species held at different densities and number of eggs produced per female on day of peak egg production. Significant differences between different densities within a species are indicated by letters. The number of eggs produced per female on the day of peak egg production was significantly affected by species (GLZ: χ2 = 17.07, df = 3, P < 0.001), density (GLZ: χ2 = 20.64, df = 5, P < 0.001), and the interaction of species and density (GLZ: χ2 = 41.56, df = 15, P < 0.001). The number of eggs produced per female on day of peak egg production did not vary with density among C. megacephala and C. putoria. For L. sericata and C. chloropyga, the highest number of eggs produced per female was at density 250. As density increased or decreased from density 250, there was a decrease in the number of eggs produced per female for both species, with the exception of density 20 for L. sericata. The number of eggs produced per female per day (Fig. 3) was significantly affected by species (GLZ: χ2 = 89.34, df = 3, P < 0.0005), density (GLZ: χ2 = 52.00, df = 5, P < 0.0005), time (in days) (GLZ: χ2 = 120.474, df = 13, P < 0.0005), and the interaction of species and density (GLZ: χ2 = 100.62, df = 15, P < 0.0005). At density of 20, L. sericata was significantly different from all other species and all other densities within L. sericata, with a higher overall average number of eggs produced per female every second day between day eight and day 34. At density 250, L. sericata and C. chloropyga were significantly different from all other species and produced more eggs per day than C. putoria and C. megacephala. At density 250, C. putoria and C. megacephala were not significantly different from each other. At density 500, L. sericata was significantly different from C. megacephala and C. putoria with an overall higher egg production per female per day. There were no significant differences across species for density 50, 100, or 1,000. L. sericata at density 250 was significantly different from all other densities, with the highest average egg production per female per day. C. chloropyga was also significantly different from other species and densities at density 250, with a high average number of eggs produced per female per day. C. chloropyga at density of 20 had the lowest total number of eggs produced per female across all species. Fig. 3. Open in new tabDownload slide Average number of eggs produced per female per day across six different densities for four calliphorid species. Line style represents different species: short dash with circle markers = C. chloropyga, dots with square markers = C. megacephala, solid = C. putoria, long dash with diamond markers = L. sericata. Fig. 3. Open in new tabDownload slide Average number of eggs produced per female per day across six different densities for four calliphorid species. Line style represents different species: short dash with circle markers = C. chloropyga, dots with square markers = C. megacephala, solid = C. putoria, long dash with diamond markers = L. sericata. Fertility Effects of species and density on fertility were determined by relating the total number of eggs produced over time for each species to the total number of eggs that hatched and the initial density of females (Fig. 4). There were significant differences between the species (Logistic Regression: χ2 = 14.72, df = 3, P < 0.0005), different densities (Logistic Regression: χ2 = 32.59, df = 5, P < 0.0005), and within species by density interactions (Logistic Regression: χ2 = 34.16, df = 15, P < 0.0005). Overall, there were no strong effects of density on the overall fertility of eggs, with the exception of C. chloropyga at density of 20 which had the lowest fertility across all species and densities. C. chloropyga and C. putoria were significantly different from all other species at density 20, with C. putoria also exhibiting low fertility at density 20, while C. megacephala and L. sericata were not significantly different from each other at this density. At density 100, C. putoria and C. chloropyga were similar to each other and C. megacephala and L. sericata were not significantly different from each other. Fertility of C. chloropyga was significantly higher than L. sericata at density 500. There were no significant differences in fertility across species at densities of 50, 250, and 1,000. There were no significant differences in fertility across the different densities for C. megacephala. Fig. 4. Open in new tabDownload slide Average total number of eggs produced over time, and average total number of hatched eggs divided by the initial female density for each density for four calliphorid species. Fertility was calculated by dividing total number of hatched eggs by total number of eggs produced over time, presented as a percentage. Significant differences between different densities within a species are indicated by letters. Solid white bar = average total number of eggs produced standard deviation, dotted bar = average total number of hatched eggs with standard deviation. Fig. 4. Open in new tabDownload slide Average total number of eggs produced over time, and average total number of hatched eggs divided by the initial female density for each density for four calliphorid species. Fertility was calculated by dividing total number of hatched eggs by total number of eggs produced over time, presented as a percentage. Significant differences between different densities within a species are indicated by letters. Solid white bar = average total number of eggs produced standard deviation, dotted bar = average total number of hatched eggs with standard deviation. Discussion Survival Survival was strongly affected by an increase in density but not in a linear manner. The effect of density is complex across the four tested species, and it may be that the stress associated with crowding is not the only factor that influences survival. All four species had a similar response to the increase in density from 500 to 1,000, with an overall but slight increase in survival and no significant differences across species. This may be an example of behavioral flexibility in response to the high density and reduced space to move around. At such a high density the flies present in these cages may have responded to the reduced space by flying less and walking more, reducing the stress and potential physical damage associated with increased crowding (Bush et al. 1976), making the response to survival similar for both densities. Both females and males experienced similar responses to density. However, males may have expended more energy or experienced more competition than females, leading to overall higher mortality in males. This trend in survival between females and males has previously been recorded in L. sericata (Moe et al. 2002, Rueda et al. 2010), but no mechanism was proposed, and little is known about the reproductive behavior or the potential effects of mating interactions on females and males in carrion-associated flies in the family Calliphoridae. In the tephritid fly, C. capitata, males exhibited higher mortality than females in response to increased density due to more male-male interactions and courting of females (Gaskin et al. 2002, Papadopoulos et al. 2010). Adult male C. homnivorax experience higher mortality than females under a number of different experimental conditions, including different initial densities, diets, and temperatures (Berkebile et al. 2006). Fecundity There was a reduction in fecundity at low densities for C. chloropyga and C. putoria. C. chloropyga is a large mammal carcass specialist, which congregates in large numbers around a suitably sized carcass (pers. obs.). It may be that at very low densities such as density 20 there are too few interactions between females and males to stimulate mating and oviposition, a response to low densities that has been observed in the field for a number of different insects (Rhainds 2010). C. putoria at low densities took a long time to reach peak egg production which may indicate that more time was required for adult flies at low densities to mate enough for eggs to be produced than at higher densities. As density increased the time to day of peak egg production was reached earlier, indicating that mating was occurring sooner, leading to females laying their eggs sooner. This also led to an increase in eggs produced per female until density 250. Thereafter there was no effect of density on the number of eggs produced per female over time or time to day of peak egg production, indicating that at higher densities there were enough opportunities for females to mate to consistently produce eggs (Ridley 1988). In comparison with C. chloropyga and C. putoria¸ L. sericata reached peak egg production and highest number of eggs produced per female at the lowest density. Time until peak egg production increased and the number of eggs produced per female tended to decline in response to increasing density. In addition, L. sericata exhibited the highest production of eggs across all densities in comparison with other species. Egg production of L. sericata in this study corresponds well with the number of eggs produced per female in other studies (Pitts and Wall 2004, Davies 2006, Rueda et al. 2010). The high egg production per female observed in L. sericata at density 20 and the early day of peak egg production may be due to a reduction in competition between females for oviposition sites (Moe et al. 2002). In a study performed by Delves and Browne (1989), it was found that new colonies of Lucilia cuprina (Wiedemann), a closely related species to L. sericata, that had not yet adapted to lab conditions had the highest egg production at very low densities. The reduction in egg production at higher densities was due to an inhibitory effect on oviposition behavior as a result of increased competition for oviposition sites. This effect disappeared as the colony was maintained over a number of generations and the flies became more adapted to the lab environment (Delves and Browne 1989). Although all species at all densities had ad libitum access to sugar, milk powder, and water, the size of the dish and the amount of liver provided did not increase with increasing density. This may have led to an increase in competition between females for sites to oviposit as density increased, leading to reduced fecundity of females with increasing density. This pattern is reflected from density 250 to density 1,000 across all species except C. megacephala. C. megacephala had a much lower egg production than the other species so it is likely that females did not encounter the strong competition for a limited resource experienced by the other species. The highest fecundity over the entire period of egg production was recorded in L. sericata and C. chloropyga at density 250. For C. chloropyga and L. sericata, this was the density where the lowest survival was observed and therefore it would indicate that a higher fecundity leads to a decrease in survival over time. It was also observed for these two species that where there was lower egg production there was higher survival, even with increasing density. This trade-off between fecundity and longevity is well documented across a number of other fly species such as A. ludens (Carey et al. 2008), B. tryoni (Fanson et al. 2012), and D. melanogaster (Gasser et al. 2000). This trade-off has been linked to nutrition, where a high protein to carbohydrate ratio leads to reduced longevity (Carey et al. 2008, Fanson et al. 2012). However, there was no observed trade-off in survival and egg production in C. putoria or in C. megacephala. The highest egg production for C. putoria was at density 100. It may be that a trade-off between survival and egg production was not observed under the experimental conditions of this study for this species. Egg production did not change at all across different densities for C. megacephala, remaining low across all densities in comparison to the egg production of the other species. The average number of eggs produced per female in a cage decreased over time across all densities and species. Although these flies have the potential to survive past 45 d, the females produce very low numbers of eggs after day 30 across all densities and species. This is probably due to a combination of senescence and a decrease in the density of males available for further mating. Fertility There was not as strong an effect of increased density on fertility. There was an increase in fertility in response to an increase in density for all species except C. megacephala, despite the decreased fecundity at higher densities. The increase in fertility in comparison to fecundity is likely due to an increase in opportunities for repeat mating and an increase in competition between males for females, leading to increased duration of copulation or more sperm transfer during mating to females (Kelly and Jennions 2011), which would lead to higher fertility. There was a reduction in fertility at low densities for C. chloropyga and C. putoria. This may be due to sperm depletion through reduced opportunities for remating or shorter copulations in the absence of competition between males for these two species. These hypotheses require further study with regard to these two species. When females are unable to remate, this leads to a reduction in egg fertility (Abraham et al. 2011). The lowest fertility was recorded for C. chloropyga at density 20. C. megacephala did not show any strong differences in fertility across the different densities. However, due to the observed low egg production in this species, there may not have been an effect of reduced mating opportunities as the females did not lay large numbers of eggs. Implications for Mass-Rearing Within a mass-rearing facility the most important factors for efficient production of larvae are high egg production, low mortality, and a large number of fertile adults in as little space as possible. The species that had the lowest mortality while maintaining high egg production at a high density was C. putoria, which makes it the best candidate for mass-rearing that was assessed in this study. Although L. sericata had a much higher egg production than any of the other species, their optimal density was low and not ideal for efficient use of space within a facility for mass-rearing. C. chloropyga also produced high numbers of eggs, but fecundity and fertility were more density dependent, and survival and egg production were low at higher densities. Egg production and fertility of C. megacephala did not change in response to density, but this species had low fecundity, making it less suitable for the production of large numbers of larvae for bioconversion of waste when space is limited. None of these fly species should be kept for longer than 32 d as they are no longer producing large numbers of eggs past this point. After day 32, flies should be replaced with a new cohort in order to maintain a consistent supply of eggs and larvae. Selective pressures inside a mass-rearing facility affect insect performance through adaptation to the mass-rearing environment, including indirectly from inbreeding depression and by direct effects such as crowding and artificial diets (Sørensen et al. 2012). Under natural conditions, flies are found clumped around resources such as host plants or food sources among regions of substantially lower density (Lance and McInnis 2005). These areas of higher density facilitate opportunities for breeding. However, keeping flies in permanent high-density conditions can lead to an increase in potential physical damage due to crowding, competition, and aggression which ultimately leads to an increase in mortality (Gaskin et al. 2002). This high degree of mortality is often observed at the start of the domestication process, as individuals that are better adapted to the mass-rearing environment are selected for and survive better (Itô and Yamamura 2005). For mass-rearing of insects for bioconversion, selection leads to high productivity, specifically high egg production. Once flies have been collected from the wild and are bred in cages with high densities, there is also selection for flies that are less active and less easy to startle (Weldon et al. 2010), as well as strains that are affected less by stress, leading to decreased rates of mortality in response to high densities. This intentional selection pressure can increase the performance of flies under laboratory conditions, leading to reduction in the effects of increased density (Miyatake 1998). The results of this study indicate that C. putoria is the most suited to the high-density conditions experienced in a bioconversion facility and will adapt rapidly and efficiently to mass-rearing conditions. However, the flies that were used in this study were not completely laboratory-adapted, although they had been kept for a number of generations under laboratory conditions to complete this study. Due to the high potential for laboratory adaptation in these fly species, it is necessary to continue with further studies to track changes in survival, fertility, and fecundity over successive generations in the laboratory to investigate the potential beneficial effects of laboratory adaptation. Laboratory adaptation may benefit their survival at high density and could lead to higher fecundity and decreased mortality at higher densities, especially in L. sericata, due to selection of flies that are tolerant of high-density conditions. Furthermore, the suitability of the different species for the degradation of particular waste materials and conversion into usable biomass must also be determined to assess their suitability for use in bioconversion facilities in the future. Acknowledgments Jian Du Plooy and Johan Saayman are thanked for their assistance with the maintenance of fly cultures and experiments. Kevin Malod is thanked for reviewing an earlier version of the manuscript. N. J. P. is an MSc student funded by the South African National Research Foundation (SFH150718127604) and AgriProtein Technologies. Research was funded by a University of Pretoria Research Development Programme grant awarded to C.W.W. References Cited Abd-Alla , A. M. , Bergoin M. , Parker A. G. , Maniania N. K. , Vlak J. M. , Bourtzis K. , Boucias D. G. , and Aksoy S. . 2013 . Improving sterile insect technique (SIT) for tsetse flies through research on their symbionts and pathogens . J. Invertebr. Pathol . 112 : S2 – S10 . Google Scholar Crossref Search ADS PubMed WorldCat Abraham , S. , Goane L. , Rull J. , Cladera J. , Willink E. , and Vera M.T. . 2011 . Multiple mating in Anastrepha fraterculus females and its relationship with fecundity and fertility . 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Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected]. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Economic Entomology Oxford University Press

Longevity, Fertility and Fecundity of Adult Blow Flies (Diptera: Calliphoridae) Held at Varying Densities: Implications for Use in Bioconversion of Waste

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected].
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0022-0493
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1938-291X
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10.1093/jee/tox251
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Abstract

Abstract Large numbers of flies are needed to produce the quantity of larvae required for insect bioconversion of waste. However, this ‘mass-rearing’ may negatively affect adult survival and reproductive output. This study assessed the suitability for mass-rearing of four blow fly species, Chrysomya chloropyga, Chrysomya chloropyga (Wiedemann), Chrysomya megacephala (F.), Chrysomya putoria (Wiedemann) and Lucilia sericata (Meigen). Flies were kept at densities of 20, 50, 100, 250, 500, and 1,000 flies per 30 × 30 × 30 cm cage with an even sex ratio. Time to 50% mortality (LT50) was recorded, and the effects of density, species, and sex on LT50, fecundity, and fertility were determined. Females survived longer than males across all species. There was evidence for a trade-off between survival and high fecundity in L. sericata and C. chloropyga at density 250. C. megacephala had low fecundity across all densities. At high densities, C. putoria had the lowest mortality and highest fecundity, making it the most suitable for mass-rearing. mass-rearing, bioconversion, Calliphoridae, density, mortality Bioconversion is a nutrient cycling process that can reduce agricultural, industrial, and household organic waste and produce value-added products. The bioconversion process can be applied to a variety of different waste types, and different organisms ranging from bacteria and yeast to insects have been used to facilitate this process (Kumar et al. 2008, Madhavan et al. 2012, van Huis 2013, Čičková et al. 2015). Flies (Insecta: Diptera) are often used in bioconversion due to their known association with decaying material, high fecundity, rapid generation times, and ease of rearing (van Huis 2013). Proteins and lipids recovered by bioconversion of organic waste can be fed to livestock (van Huis 2013) or converted into biodiesel (Li et al. 2011). Large-scale conversion of waste products by insects requires that they be mass-reared. Mass-rearing of insects has been used to produce large numbers for release in sterile insect technique (SIT; Parker 2005) and biological control programs (Sørensen et al. 2012). This has included various fly species including tephritid fruit flies such as Bactrocera tryoni (Froggatt) (Weldon et al. 2013) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (Pereira et al. 2013), as well as tsetse flies (Glossina species; Vreysen et al. 2011, Abd-Alla et al. 2013) and mosquitoes (Gilles et al. 2014). Insects that are kept in mass-rearing facilities should ideally not experience high mortality or suffer decreased egg production (fecundity) or egg hatch (fertility) when there is an increase in adult density within cages. However, different species of flies respond to the high density conditions of mass-rearing in different ways (Robinson 2005), which has implications for their suitability in large-scale bioconversion facilities. The effects of the stress associated with crowding in cages where adults are held at high density has been documented for a number of different fly species. This increased stress can lead to reduced longevity and physical injury, as well as changes in interactions between the different sexes, which then affects fecundity and fertility (Rull et al. 2012). Fecundity refers to the total number of eggs that are produced by a female, whereas fertility is the number of eggs that hatch. Male mating success and therefore male reproductive success can be reduced by increased density due to changes in mating behavior, decreased territoriality, and interrupted mating with females (Gaskin et al. 2002, Díaz-Fleischer et al. 2009). Drosophila melanogaster (Meigen) show decreased levels of territoriality at high densities, leading to fewer interactions between males (Hoffmann and Cacoyianni 1990). In C. capitata, male longevity is more negatively impacted by an increase in density than that of females, possibly due to increased aggression and increased behavioral costs to mate successfully (Gaskin et al. 2002). Some of the changes in behavioral interactions that can occur under high-density conditions have been observed in Anastrepha ludens (Loew) (Diptera: Tephritidae), where females mate sooner in high-density conditions, whereas males at lower densities mate more often (Díaz-Fleischer et al. 2009). Males of A. ludens also require a minimal area for males to establish their territories and to reduce male–male aggressive interactions that would negatively affect their reproductive success (Díaz-Fleischer et al. 2009). Egg production is one of the bottle necks in the process of mass-rearing. However, few studies have sought to maximize egg production by determining an optimal adult density for fly species that can be mass-reared in bioconversion facilities (Pastor et al. 2015). This situation demands that further research be pursued to ensure high levels of fecundity and fertility while minimizing the space, cost, and handling of mass-reared adults (Pastor et al. 2015). Limited research has been conducted on the use of blowflies (Calliphoridae) or flesh flies (Sarcophagidae) for mass-rearing (Čičková et al. 2015, Pastor et al. 2015). One exception is the screwworm fly, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), which is a major pest of cattle (Vargas-Terán et al. 2005), and has been reared successfully at large scales for release in SIT programs. In C. hominivorax, adult density does not affect survival, fecundity, or fertility (Berkebile et al. 2006). However, there was an observable difference in behavior between wild flies and those reared in the laboratory due to the selection pressure experienced during mass-rearing. These behavioral changes included an increased amount of time spent walking rather than flying and decreased competitiveness between males (Bush et al. 1976). A more sedentary phenotype may have led to the laboratory reared C. hominivorax being less susceptible to the negative effects of increased cage density that are apparent in other mass-reared fly species. Blow fly species have the potential to be used for bioconversion of a number of different waste products and should be studied in more detail to assess their relative utility. There are a number of organic waste streams that are not efficiently converted to biomass by the fly species currently used by most bioconversion facilities, predominantly black soldier flies, Hermetia illucens L. (Diptera: Stratiomyidae), and house flies, Musca domestica L. (Diptera: Muscidae) (van Huis 2013, Čičková et al. 2015), and research into finding more suitable and versatile species is required. In addition, blow fly species exhibit wandering behavior prior to pupating (Richards et al. 2009a), which can be utilized in a ‘self-harvesting’ system. This may lead to more efficient production than those using H. illucens, which require mechanical separation of larvae from the remaining waste (reviewed by Pastor et al. 2015). Potential blow fly species include Chrysomya megacephala (Fabricius), Chrysomya chloropyga (Wiedemann), Chrysomya putoria (Wiedemann), and Lucilia sericata (Meigen) (Calliphoridae). These species have not been well studied in terms of mass-rearing with little information available on the effects of density on their life history traits. Chrysmomya megacephala and C. putoria are reported to be associated with pit latrine wastes (Laurence 1986, Lindsay et al. 2013) and may be able to successfully breakdown municipal or manure waste. Additionally, C. megacephala larvae have been successfully raised on restaurant waste that contained a mixture of vegetable and meat scraps (Li et al. 2011). L. sericata is a carrion-associated blow fly that is often used in laboratory experiments (Clark et al. 2006, Čičková et al. 2010, Blystone and Hansen 2014) and is highly prolific and successful under laboratory conditions (Blystone and Hansen 2014). C. chlorpyga is considered to be a large mammal carcass specialist and can produce high numbers of eggs at a time (Richards et al. 2009b). This species may be useful in the conversion of large quantities of waste. This study assessed the suitability for mass-rearing of four species of carrion flies, specifically assessing the effects on survival, fertility, and fecundity of holding adults at high densities. This is an important but often overlooked consideration for the successful establishment and continued high output of bioconversion facilities that use insects for organic waste valorization to usable biomass. The four species tested were: C. cholorpyga, C. megacephala, C. putoria, and L. sericata. These four calliphorid species are abundant in Gauteng province, South Africa, where this study was conducted (Parry et al. 2016). They are also present across most of South Africa (Richards et al. 2009c). C. chloropyga and C. putoria have a wide Afrotropical distribution (Picard et al. 2012), while L. sericata and C. megacephala are widely distributed across most of the world (Stevens and Wall 1996, Williams and Villet 2006). It was hypothesized that increasing adult density would negatively affect survival of all species and both sexes, but male survival would be affected more than that of females due to the energetic demands and physical interactions associated with competition for mates. At higher densities it was also predicted that fecundity and fertility per female would be reduced due to an increase in stress and fewer or interrupted interactions between females and males. Materials and Methods Colony Establishment Adult carrion flies were collected from various areas around Gauteng province, South Africa, using modified Red-Top hanging traps (Miller Methods Ltd., Pretoria). The traps were modified by opening the base of the trap and attaching a 500-ml plastic cup with several elastic bands. Adult flies were attracted to the trap by placing a bait mixture of food-grade chicken livers and fish in the plastic cup. A square piece of white voile curtain fabric was placed over the top of the cup to stop the flies from becoming fouled in the bait. Traps were hung at a height of approximately 2 m above the ground from available trees and were checked daily until the appropriate fly species and numbers were collected. Numbers of between 20 and 100 were necessary to start the initial colony, and additional flies were caught throughout the duration of the study to maintain colony fitness and genetic variability. The species that were specifically targeted for this study were C. chloropyga, C. megacephala, C. putoria, and L. sericata. Laboratory Rearing The flies that were collected live from the traps were identified and kept in single-species rearing cages. The cages were made from 13-liter transparent plastic containers (MaxiMultiBox, Hobbylife, Istanbul, Turkey) with dimensions of 0.36 × 0.25 × 0.23 m (0.013 m3). A 0.2 × 0.1 m2 section was cut from the lid of the plastic container and covered with white voile curtain fabric for ventilation. One side of the cage had a circular hole cut out with a diameter of 15 cm, fitted with a 45-cm long sleeve of white voile curtain fabric for daily feeding, removal of eggs and pupae, and cleaning of the cage. The flies were provided with unrestricted access to sugar, milk powder as a protein source, and water from soaked cotton wool. The rearing cages were maintained at approximately 25°C in a room with a window to provide a natural day:night photocycle. Food-grade chicken liver was provided in a 150-ml plastic container (WestPack Lifestyle) as a breeding medium to maintain the cultures. A larger 500-ml plastic container (WestPack Lifestyle) with sterile river sand was placed underneath the breeding medium to be used by the flies for pupation. Effects of Density on Survival, Fecundity, and Fertility Pupae were collected from the breeding cages and placed in a separate breeding cage. Pupae were kept in an incubator (LTIE, Labcon, Gauteng, South Africa) at 25°C until eclosion. Newly emerged flies were collected and sexed by measuring the occipital sclerite and placed into insect cages with dimensions of 0.3 × 0.3 × 0.3 m (0.028 m3, BugDorm 43030, MegaView Science, Taichung, Taiwan). Flies were kept at different densities with an even sex ratio (Table 1). Each density was repeated five times for each species. The density of 1,000 flies per cage was as high as or higher than the high densities that are found within bioconversion facilities based on the available cage surface area and volume per fly. Reported values from bioconversion facilities are between 2.5 and 10 cm2 internal cage surface area per fly or a volume of between 25 and 200 cm3 per fly (Charlton et al. 2015). Each cage was checked daily for fly mortality. Sex of fatalities was recorded. From 8 until 32 d after adult emergence, a Petri dish containing 30 g of liver was placed in each cage every second day. Within 36 h, the liver was removed and assessed for eggs laid in order to determine female fecundity. If eggs were present, they were carefully collected, weighed to determine the approximate number of eggs, and then placed on fresh liver where they were allowed to hatch. The approximate mass of an egg per species is 0.101 ± 0.024 mg for C. chloropyga, 0.128 ± 0.021 mg for C. megacephala, 0.124 ± 0.023 mg for C. putoria, and 0.119 ± 0.031 mg for L. sericata. The number of eggs that did not hatch into first instar larvae were counted the next day to determine egg viability. This was used to assess fertility of the females present at different densities. Recording of mortality and assessment of fecundity and fertility continued until 45 d after adult emergence or until up to 80% of females had died. Table 1. The cage surface area and cage volume that is available per fly for each initial fly density Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Open in new tab Table 1. The cage surface area and cage volume that is available per fly for each initial fly density Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Initial fly density Surface area (cm2)/fly Volume (cm3)/fly 20 270.0 1,350 50 108.0 540 100 54.0 270 250 21.6 108 500 10.8 54 1,000 5.4 27 Open in new tab Data Analysis The time elapsed, until 50% of females and males were dead (LT50), was determined for each replicate cage of each species and density. A general linear model was used to determine the effect of density, species, sex, and their interactions on LT50. Generalized linear models (GLZ) were used to analyze the effect of density, species and time, and their interactions on the number of eggs per female that were laid over time. The time (in days) until peak production and the number of eggs that were laid per female on the day of peak egg production were also analyzed in relation to density, species, and their interaction. These measures of fecundity are informative for the harvesting of eggs in bioconversion facilities. Stepwise deletion of the least significant terms was used to minimize the model. Post hoc multiple comparisons were performed using Fisher’s least significant difference tests. IBM SPSS Statistics was used to perform these analyses. A logistic regression with a quasibinomial distribution was applied to the total number of eggs produced combined with the total number of hatched eggs, both accounting for the initial density of females (included as a covariate in the model), in order to determine the effect of density and species on egg fertility. A quasibinomial distribution was applied due to over dispersion of the data when using a binomial distribution. In all cases, the minimal adequate model was obtained by applying a model simplification procedure based on Akaike’s information criterion (Crawley 2007). Differences between groups were identified by inspecting model coefficients. These analyses were performed using RStudio version 0.99.903 running R version 3.3.1 (R Development Core Team, 2016). Results Survival Survival for each species at each density was determined by looking at the time in days until 50% of females and males had died (LT50) for each cage. LT50 differed significantly between females and males (Fig. 1; F1, 217 = 24.669, P < 0.001), with females reaching 50% mortality later than males. Significant differences in LT50 were also found between species (F3, 217 = 17.871, P < 0.001), between densities (F5, 217 = 9.261, P < 0.001), and within species by density interactions (F15, 217 = 4.763, P < 0.001). Fig. 1. Open in new tabDownload slide Time until 50% mortality (LT50) of females and males of four calliphorid species held at different initial densities. Significant differences between different densities within a species are indicated by letters. Solid white bar = male, dotted bar = female. Significant differences between different densities within a species are indicated by letters with homogenous groups sharing the same letters. Fig. 1. Open in new tabDownload slide Time until 50% mortality (LT50) of females and males of four calliphorid species held at different initial densities. Significant differences between different densities within a species are indicated by letters. Solid white bar = male, dotted bar = female. Significant differences between different densities within a species are indicated by letters with homogenous groups sharing the same letters. Due to differences in the effect of density within different species, there was no clear trend for the overall effect of increasing density on LT50 (Fig. 1). At density 250, C. chloropyga and L. sericata were significantly different from all other species, with LT50 reached before all other species and densities. LT50 was reached last by C. putoria females at density 250 and 100. C. megacephala and C. putoria were not significantly different from each other across all densities and followed a trend of high survival at densities 50, 100, and 250 with a decrease in survival at density 500. Across all four species, there was an increase in LT50 from density 500 to density 1,000. At density 50 or 1,000, the LT50 of the species did not differ from each another. L. sericata and C. chloropyga were only significantly different from each other at density 100, where LT50 was reached earlier by C. chloropyga than by L. sericata. These two species had a similar response to increasing density in the cages, with a decrease in LT50 from density 50 to density 250 and from there an increase in LT50 from density 250 to 1,000. Fecundity Time elapsed (in days) until peak egg production (Fig. 2) was significantly different among species (GLZ: χ2 = 11.218, df = 3, P = 0.011) and was also affected by the interaction of species and density (GLZ: χ2 = 37.876, df = 15, P < 0.001). The main effect of density was not significant (GLZ: χ2 = 3.714, df = 5, P = 0.591), indicating the absence of a clear trend of the effect of increasing density on the day of peak egg production across all four species. The earliest day of peak egg production among the four species was achieved by C. chloropyga at density 20 while the latest day of peak egg production was achieved by C. putoria at density of 20. For C. putoria, as density increased from density 20 the time to day of peak egg production markedly decreased until density 100. Thereafter, there was no effect of density on day of peak egg production for C. putoria. L. sericata showed a different pattern from C. putoria where the density 20 reached peak egg production first followed by an increase in the time until peak egg production as density increased, with the exception of densities 250 and 1,000. These two densities were not significantly different from density 20. There were no significant differences of day of peak egg production across different species at densities 50, 250, and 1,000. Fig. 2. Open in new tabDownload slide Day of peak egg production for four calliphorid species held at different densities and number of eggs produced per female on day of peak egg production. Significant differences between different densities within a species are indicated by letters. Fig. 2. Open in new tabDownload slide Day of peak egg production for four calliphorid species held at different densities and number of eggs produced per female on day of peak egg production. Significant differences between different densities within a species are indicated by letters. The number of eggs produced per female on the day of peak egg production was significantly affected by species (GLZ: χ2 = 17.07, df = 3, P < 0.001), density (GLZ: χ2 = 20.64, df = 5, P < 0.001), and the interaction of species and density (GLZ: χ2 = 41.56, df = 15, P < 0.001). The number of eggs produced per female on day of peak egg production did not vary with density among C. megacephala and C. putoria. For L. sericata and C. chloropyga, the highest number of eggs produced per female was at density 250. As density increased or decreased from density 250, there was a decrease in the number of eggs produced per female for both species, with the exception of density 20 for L. sericata. The number of eggs produced per female per day (Fig. 3) was significantly affected by species (GLZ: χ2 = 89.34, df = 3, P < 0.0005), density (GLZ: χ2 = 52.00, df = 5, P < 0.0005), time (in days) (GLZ: χ2 = 120.474, df = 13, P < 0.0005), and the interaction of species and density (GLZ: χ2 = 100.62, df = 15, P < 0.0005). At density of 20, L. sericata was significantly different from all other species and all other densities within L. sericata, with a higher overall average number of eggs produced per female every second day between day eight and day 34. At density 250, L. sericata and C. chloropyga were significantly different from all other species and produced more eggs per day than C. putoria and C. megacephala. At density 250, C. putoria and C. megacephala were not significantly different from each other. At density 500, L. sericata was significantly different from C. megacephala and C. putoria with an overall higher egg production per female per day. There were no significant differences across species for density 50, 100, or 1,000. L. sericata at density 250 was significantly different from all other densities, with the highest average egg production per female per day. C. chloropyga was also significantly different from other species and densities at density 250, with a high average number of eggs produced per female per day. C. chloropyga at density of 20 had the lowest total number of eggs produced per female across all species. Fig. 3. Open in new tabDownload slide Average number of eggs produced per female per day across six different densities for four calliphorid species. Line style represents different species: short dash with circle markers = C. chloropyga, dots with square markers = C. megacephala, solid = C. putoria, long dash with diamond markers = L. sericata. Fig. 3. Open in new tabDownload slide Average number of eggs produced per female per day across six different densities for four calliphorid species. Line style represents different species: short dash with circle markers = C. chloropyga, dots with square markers = C. megacephala, solid = C. putoria, long dash with diamond markers = L. sericata. Fertility Effects of species and density on fertility were determined by relating the total number of eggs produced over time for each species to the total number of eggs that hatched and the initial density of females (Fig. 4). There were significant differences between the species (Logistic Regression: χ2 = 14.72, df = 3, P < 0.0005), different densities (Logistic Regression: χ2 = 32.59, df = 5, P < 0.0005), and within species by density interactions (Logistic Regression: χ2 = 34.16, df = 15, P < 0.0005). Overall, there were no strong effects of density on the overall fertility of eggs, with the exception of C. chloropyga at density of 20 which had the lowest fertility across all species and densities. C. chloropyga and C. putoria were significantly different from all other species at density 20, with C. putoria also exhibiting low fertility at density 20, while C. megacephala and L. sericata were not significantly different from each other at this density. At density 100, C. putoria and C. chloropyga were similar to each other and C. megacephala and L. sericata were not significantly different from each other. Fertility of C. chloropyga was significantly higher than L. sericata at density 500. There were no significant differences in fertility across species at densities of 50, 250, and 1,000. There were no significant differences in fertility across the different densities for C. megacephala. Fig. 4. Open in new tabDownload slide Average total number of eggs produced over time, and average total number of hatched eggs divided by the initial female density for each density for four calliphorid species. Fertility was calculated by dividing total number of hatched eggs by total number of eggs produced over time, presented as a percentage. Significant differences between different densities within a species are indicated by letters. Solid white bar = average total number of eggs produced standard deviation, dotted bar = average total number of hatched eggs with standard deviation. Fig. 4. Open in new tabDownload slide Average total number of eggs produced over time, and average total number of hatched eggs divided by the initial female density for each density for four calliphorid species. Fertility was calculated by dividing total number of hatched eggs by total number of eggs produced over time, presented as a percentage. Significant differences between different densities within a species are indicated by letters. Solid white bar = average total number of eggs produced standard deviation, dotted bar = average total number of hatched eggs with standard deviation. Discussion Survival Survival was strongly affected by an increase in density but not in a linear manner. The effect of density is complex across the four tested species, and it may be that the stress associated with crowding is not the only factor that influences survival. All four species had a similar response to the increase in density from 500 to 1,000, with an overall but slight increase in survival and no significant differences across species. This may be an example of behavioral flexibility in response to the high density and reduced space to move around. At such a high density the flies present in these cages may have responded to the reduced space by flying less and walking more, reducing the stress and potential physical damage associated with increased crowding (Bush et al. 1976), making the response to survival similar for both densities. Both females and males experienced similar responses to density. However, males may have expended more energy or experienced more competition than females, leading to overall higher mortality in males. This trend in survival between females and males has previously been recorded in L. sericata (Moe et al. 2002, Rueda et al. 2010), but no mechanism was proposed, and little is known about the reproductive behavior or the potential effects of mating interactions on females and males in carrion-associated flies in the family Calliphoridae. In the tephritid fly, C. capitata, males exhibited higher mortality than females in response to increased density due to more male-male interactions and courting of females (Gaskin et al. 2002, Papadopoulos et al. 2010). Adult male C. homnivorax experience higher mortality than females under a number of different experimental conditions, including different initial densities, diets, and temperatures (Berkebile et al. 2006). Fecundity There was a reduction in fecundity at low densities for C. chloropyga and C. putoria. C. chloropyga is a large mammal carcass specialist, which congregates in large numbers around a suitably sized carcass (pers. obs.). It may be that at very low densities such as density 20 there are too few interactions between females and males to stimulate mating and oviposition, a response to low densities that has been observed in the field for a number of different insects (Rhainds 2010). C. putoria at low densities took a long time to reach peak egg production which may indicate that more time was required for adult flies at low densities to mate enough for eggs to be produced than at higher densities. As density increased the time to day of peak egg production was reached earlier, indicating that mating was occurring sooner, leading to females laying their eggs sooner. This also led to an increase in eggs produced per female until density 250. Thereafter there was no effect of density on the number of eggs produced per female over time or time to day of peak egg production, indicating that at higher densities there were enough opportunities for females to mate to consistently produce eggs (Ridley 1988). In comparison with C. chloropyga and C. putoria¸ L. sericata reached peak egg production and highest number of eggs produced per female at the lowest density. Time until peak egg production increased and the number of eggs produced per female tended to decline in response to increasing density. In addition, L. sericata exhibited the highest production of eggs across all densities in comparison with other species. Egg production of L. sericata in this study corresponds well with the number of eggs produced per female in other studies (Pitts and Wall 2004, Davies 2006, Rueda et al. 2010). The high egg production per female observed in L. sericata at density 20 and the early day of peak egg production may be due to a reduction in competition between females for oviposition sites (Moe et al. 2002). In a study performed by Delves and Browne (1989), it was found that new colonies of Lucilia cuprina (Wiedemann), a closely related species to L. sericata, that had not yet adapted to lab conditions had the highest egg production at very low densities. The reduction in egg production at higher densities was due to an inhibitory effect on oviposition behavior as a result of increased competition for oviposition sites. This effect disappeared as the colony was maintained over a number of generations and the flies became more adapted to the lab environment (Delves and Browne 1989). Although all species at all densities had ad libitum access to sugar, milk powder, and water, the size of the dish and the amount of liver provided did not increase with increasing density. This may have led to an increase in competition between females for sites to oviposit as density increased, leading to reduced fecundity of females with increasing density. This pattern is reflected from density 250 to density 1,000 across all species except C. megacephala. C. megacephala had a much lower egg production than the other species so it is likely that females did not encounter the strong competition for a limited resource experienced by the other species. The highest fecundity over the entire period of egg production was recorded in L. sericata and C. chloropyga at density 250. For C. chloropyga and L. sericata, this was the density where the lowest survival was observed and therefore it would indicate that a higher fecundity leads to a decrease in survival over time. It was also observed for these two species that where there was lower egg production there was higher survival, even with increasing density. This trade-off between fecundity and longevity is well documented across a number of other fly species such as A. ludens (Carey et al. 2008), B. tryoni (Fanson et al. 2012), and D. melanogaster (Gasser et al. 2000). This trade-off has been linked to nutrition, where a high protein to carbohydrate ratio leads to reduced longevity (Carey et al. 2008, Fanson et al. 2012). However, there was no observed trade-off in survival and egg production in C. putoria or in C. megacephala. The highest egg production for C. putoria was at density 100. It may be that a trade-off between survival and egg production was not observed under the experimental conditions of this study for this species. Egg production did not change at all across different densities for C. megacephala, remaining low across all densities in comparison to the egg production of the other species. The average number of eggs produced per female in a cage decreased over time across all densities and species. Although these flies have the potential to survive past 45 d, the females produce very low numbers of eggs after day 30 across all densities and species. This is probably due to a combination of senescence and a decrease in the density of males available for further mating. Fertility There was not as strong an effect of increased density on fertility. There was an increase in fertility in response to an increase in density for all species except C. megacephala, despite the decreased fecundity at higher densities. The increase in fertility in comparison to fecundity is likely due to an increase in opportunities for repeat mating and an increase in competition between males for females, leading to increased duration of copulation or more sperm transfer during mating to females (Kelly and Jennions 2011), which would lead to higher fertility. There was a reduction in fertility at low densities for C. chloropyga and C. putoria. This may be due to sperm depletion through reduced opportunities for remating or shorter copulations in the absence of competition between males for these two species. These hypotheses require further study with regard to these two species. When females are unable to remate, this leads to a reduction in egg fertility (Abraham et al. 2011). The lowest fertility was recorded for C. chloropyga at density 20. C. megacephala did not show any strong differences in fertility across the different densities. However, due to the observed low egg production in this species, there may not have been an effect of reduced mating opportunities as the females did not lay large numbers of eggs. Implications for Mass-Rearing Within a mass-rearing facility the most important factors for efficient production of larvae are high egg production, low mortality, and a large number of fertile adults in as little space as possible. The species that had the lowest mortality while maintaining high egg production at a high density was C. putoria, which makes it the best candidate for mass-rearing that was assessed in this study. Although L. sericata had a much higher egg production than any of the other species, their optimal density was low and not ideal for efficient use of space within a facility for mass-rearing. C. chloropyga also produced high numbers of eggs, but fecundity and fertility were more density dependent, and survival and egg production were low at higher densities. Egg production and fertility of C. megacephala did not change in response to density, but this species had low fecundity, making it less suitable for the production of large numbers of larvae for bioconversion of waste when space is limited. None of these fly species should be kept for longer than 32 d as they are no longer producing large numbers of eggs past this point. After day 32, flies should be replaced with a new cohort in order to maintain a consistent supply of eggs and larvae. Selective pressures inside a mass-rearing facility affect insect performance through adaptation to the mass-rearing environment, including indirectly from inbreeding depression and by direct effects such as crowding and artificial diets (Sørensen et al. 2012). Under natural conditions, flies are found clumped around resources such as host plants or food sources among regions of substantially lower density (Lance and McInnis 2005). These areas of higher density facilitate opportunities for breeding. However, keeping flies in permanent high-density conditions can lead to an increase in potential physical damage due to crowding, competition, and aggression which ultimately leads to an increase in mortality (Gaskin et al. 2002). This high degree of mortality is often observed at the start of the domestication process, as individuals that are better adapted to the mass-rearing environment are selected for and survive better (Itô and Yamamura 2005). For mass-rearing of insects for bioconversion, selection leads to high productivity, specifically high egg production. Once flies have been collected from the wild and are bred in cages with high densities, there is also selection for flies that are less active and less easy to startle (Weldon et al. 2010), as well as strains that are affected less by stress, leading to decreased rates of mortality in response to high densities. This intentional selection pressure can increase the performance of flies under laboratory conditions, leading to reduction in the effects of increased density (Miyatake 1998). The results of this study indicate that C. putoria is the most suited to the high-density conditions experienced in a bioconversion facility and will adapt rapidly and efficiently to mass-rearing conditions. However, the flies that were used in this study were not completely laboratory-adapted, although they had been kept for a number of generations under laboratory conditions to complete this study. Due to the high potential for laboratory adaptation in these fly species, it is necessary to continue with further studies to track changes in survival, fertility, and fecundity over successive generations in the laboratory to investigate the potential beneficial effects of laboratory adaptation. Laboratory adaptation may benefit their survival at high density and could lead to higher fecundity and decreased mortality at higher densities, especially in L. sericata, due to selection of flies that are tolerant of high-density conditions. Furthermore, the suitability of the different species for the degradation of particular waste materials and conversion into usable biomass must also be determined to assess their suitability for use in bioconversion facilities in the future. Acknowledgments Jian Du Plooy and Johan Saayman are thanked for their assistance with the maintenance of fly cultures and experiments. Kevin Malod is thanked for reviewing an earlier version of the manuscript. N. J. P. is an MSc student funded by the South African National Research Foundation (SFH150718127604) and AgriProtein Technologies. Research was funded by a University of Pretoria Research Development Programme grant awarded to C.W.W. References Cited Abd-Alla , A. M. , Bergoin M. , Parker A. G. , Maniania N. K. , Vlak J. M. , Bourtzis K. , Boucias D. G. , and Aksoy S. . 2013 . Improving sterile insect technique (SIT) for tsetse flies through research on their symbionts and pathogens . J. Invertebr. Pathol . 112 : S2 – S10 . Google Scholar Crossref Search ADS PubMed WorldCat Abraham , S. , Goane L. , Rull J. , Cladera J. , Willink E. , and Vera M.T. . 2011 . Multiple mating in Anastrepha fraterculus females and its relationship with fecundity and fertility . 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Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected].

Journal

Journal of Economic Entomology – Oxford University Press

Published: Dec 5, 2017

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