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Effects of the microsporidian pathogen, Nosema adaliae (Nosematidae) on the seven-spotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae)
Journal of Invertebrate Pathology 168 (2019) 107253
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Effects of the microsporidian pathogen, Nosema adaliae (Nosematidae) on the seven-spotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae)
T
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S. Bjørnson , E. Elkabir Department of Biology, Saint Mary’s University, 923 Robie Street, Halifax, NS, B3H 3C3, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Adalia bipunctata Coccinella septempunctata Lady beetles Microsporidia Nosema adaliae Pathogen
Lady beetles are important predators in nature. Some species, including the two-spotted lady beetle, Adalia bipunctata L., are native to North America, whereas others, such as the seven-spotted lady beetle, Coccinella septempunctata L., have been introduced in North America for pest control on agriculture crops. Microsporidia are obligate pathogens that cause chronic disease, and these pathogens are known to infect several lady beetle species. Lady beetles are cannibalistic and, because many species share a given landscape, there is potential for microsporidia to infect susceptible coccinellids when infected eggs are eaten. The objective of this study was to examine the effects of the microsporidium Nosema adaliae isolated from A. bipunctata on C. septempunctata fitness (larval development and mortality, sex ratio, adult longevity and fecundity). Mortality was higher for C. septempunctata larvae that ate four A. bipunctata eggs (≥96% mortality) than for those that ate only one (< 63.8%), suggesting that the mortality observed was influenced by the number of eggs eaten. A. bipunctata eggs contain adaline and adalinine, two species-specific alkaloids that have been shown to be detrimental to C. septempunctata larvae. Development of larvae that consumed one uninfected or one N. adaliae-infected A. bipunctata egg, did not differ significantly (20.5 ± 0.2 d and 21.3 ± 0.4 d, respectively) and, although mortality remained high for these larvae (53.5% and 65.6% mortality, respectively), these values also did not differ significantly (p = 0.05). Over a 60-d period, mean fecundity for C. septempunctata adults that ate one uninfected A. bipunctata egg as firstinstar larvae was significantly greater (776.6 ± 122.0 eggs) than those that ate one N. adaliae-infected egg (335.6 ± 86.6 eggs, p = 0.005). Larvae from the former group also lived significantly longer (58.2 ± 1.8 d) than did those from the latter group (38.4 ± 6.4 d, p = 0.010). Sex ratios of adult beetles did not differ significantly. Because A. bipunctata and C. septempunctata share similar habitats, it is reasonable to expect these two coccinellids to encounter one another in nature. Results of this study show that the consumption of only one infected A. bipunctata egg by C. septempunctata larvae can result in high larval mortality and reduced fecundity.
1. Introduction During the late nineteenth century, the vedalia beetle, Rodolia cardinalis Mulsant, was imported to California from Australia to control the cottony cushion scale, Icerya purchasi Maskell, in California citrus (Koebele, 1890). The overwhelming success of the vedalia beetle prompted the intentional introduction of other exotic lady beetles into North America for pest control. One such introduction was the sevenspotted lady beetle, Coccinella septempunctata L., a Palearctic species that was initially released in North America for aphid control in 1956. By 1973, C. septempunctata had become established along the east coast of North America and along the coast of the Saint Lawrence River in Quebec (Gordon, 1985). The range of this coccinellid has since ⁎
increased in the United States and Canada (Schaefer et al., 1987). C. septempunctata is a rather large coccinellid with a voracious appetite. It feeds on a variety of prey, is adaptable to a wide range of environments, and tends to migrate extensively to areas where prey is plentiful (Omkar and Pervez, 2002; Hodek and Michaud, 2008; Elliott et al., 1996). These characteristics have enabled C. septempunctata to effectively invade new areas and become established throughout the Nearctic, but these same characteristics have fueled speculation that C. septempunctata may be associated with a notable decline of native coccinellids (Schaefer et al., 1987; Wheeler and Hoebeke, 1995; Elliott et al., 1996; Hodek and Michaud, 2008). The two-spotted lady beetle, Adalia bipunctata L., is a Holarctic species that was first commercialized for aphid control in Europe in 1998 (van Lenteren, 2003). In North
Corresponding author. E-mail address: [email protected] (S. Bjørnson).
https://doi.org/10.1016/j.jip.2019.107253 Received 28 June 2019; Received in revised form 29 September 2019; Accepted 2 October 2019 Available online 03 October 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 168 (2019) 107253
S. Bjørnson and E. Elkabir
America, A. bipunctata overlaps geographically with several lady beetle species, including C. septempunctata (Gordon, 1985; Hodek and Honěk, 1996; Omkar and Pervez, 2005). A. bipunctata is host to several microorganisms, including the microsporidian pathogen, Nosema adaliae, which was described from fieldcollected beetles from North America (Steele and Bjørnson, 2014). N. adaliae is transmitted vertically through the egg and horizontally when infected eggs or larvae are eaten. The pathogen prolongs the development of A. bipunctata, but has no effect on adult fecundity, longevity or sex ratios (Steele and Bjørnson, 2012). The cannibalistic tendencies of lady beetles, combined with the geographical overlap of these two coccinellid species, provide an opportunity for this pathogen to be transmitted from infected beetles to other susceptible ones when infected eggs are ingested. In some cases, microsporidia that have been described from lady beetle hosts have been shown to infect other coccinellid species (Saito and Bjørnson 2006, 2008; Steele and Bjørnson, 2012; Ellis, 2014). The aim of this study is to determine if N. adaliae can be transmitted horizontally from A. bipunctata to C. septempunctata when infected eggs are eaten by the immature larvae, and to evaluate the effects of this pathogen on larval development and mortality, adult fecundity and longevity, and sex ratios of C. septempunctata.
Table 1 Composition of uninfected and Nosema adaliae-infected Adalia bipunctata eggs provided to 48-h old Coccinella septempunctata larvae. n
Control Treatment 1 Treatment 2 Treatment 3
36 36 36 36
Uninfected
N. adaliae-infected
A. bipunctata eggs
A. bipunctata eggs
4 3 2 0
0 1 2 4
mounted in Permount (Fisher Scientific). To confirm infection status, specimens were examined for the presence of microsporidian spores by light microscopy (400 × magnification). M. persicae was also examined to ensure that the prey used were not infected with the pathogen.
2.1. Effects of Nosema adaliae – Ingestion of four A. bipunctata eggs This trial was designed to determine the effects of pathogen dose on C. septempunctata larval development and survival. Test larvae were divided into four treatment groups and individuals from each group were fed four A. bipunctata eggs consisting of various ratios of uninfected and N. adaliae-infected eggs (Table 1). Larvae fed uninfected A. bipunctata eggs served as a control. C. septempunctata test larvae (24 h old) were placed individually in clear Petri dishes (47-mm diameter, Millipore Corp., MA). Each lid had a 2-cm hole that was covered in fine, mesh screen. On the first day of the trial, water was provided on a cotton wick that was moistened daily. On the second day of the trial, a 6-mm disc of moistened filter paper with four A. bipunctata eggs was placed in the centre of each dish. Test larvae that ate all four eggs in 48 h were then provided a daily diet of aphids. Larval development and mortality were observed daily. For each treatment group, six larvae were set up daily over 6 d (36 larvae per treatment, total n = 144). With one exception, all test larvae died before eclosion. A one-way ANOVA was used to determine significance in mean survival (days) between groups. Larvae that did not eat all four eggs were excluded from the data analysis. During this study, C. septempunctata pupae eclosed in an average of 5 d; therefore, pupae that did not eclose after 7 d were considered dead. Because mortality was ≥96%, this trial was not repeated. Smear preparations were made of all individuals, and these were stained with 5% Giemsa and examined for the presence of microsporidian spores by light microscopy.
2. Materials and methods Uninfected C. septempunctata adults used in this study were collected from rose bushes near Saint Mary’s University campus (44.6316° N, 63.5822° W) during the summer of 2014. Beetles were reared in the laboratory, and a sample of eggs and larvae from each fecund female was stained and examined to ensure that they were not infected with microsporidia. These beetles produced larvae that were used as mating pairs for the trials. A. bipunctata eggs that were fed to C. septempunctata larvae during the trial originated from established laboratory colonies of uninfected and N. adaliae-infected A. bipunctata. C. septempunctata and A. bipunctata were reared individually in 120mL clear polyethylene cups with tight-fitting lids. A 2.2-cm diameter hole in the side of each cup was covered with fine mesh screen to permit air circulation. Water was provided daily on a moistened cotton wick (Crosstex International, NY) and beetles were provided a diet of green peach aphids, Myzus persicae (Sulzer), and artificial diet (equal parts honey and Lacewing and Ladybug Food, Planet Natural, MT). Aphids were reared on nasturtium (Tropaeolum minus L., Dwarf Jewel Mixed; Stokes Seed Ltd., ON) and both aphids and beetles were maintained under controlled conditions (16:8 L:D; 25 °C:20 °C) in separate environmental chambers (Sanyo MLR-350H). Larvae that completed development were sexed 24 h following eclosion, and males and females were maintained in separate cups until they were mated for use in the trial. C. septempunctata mating pairs were isolated within polyethylene cups and provided a diet of aphids and artificial diet. Water was provided on a moistened wick. For each trial, 10 C. septempunctata mating pairs were used to produce uninfected larvae. Eggs were collected daily and placed in isolated cups until the eggs hatched. Larvae that were 24 h old post-hatch were used in the trials. The eggs fed to these larvae were produced by 10 uninfected and 10 N. adaliae-infected A. bipunctata mating pairs. Because C. septempunctata are known to host two species of microsporidia (Nosema tracheophila and N. coccinellae) (Cali and Briggs, 1967; Lipa, 1968; Lipa et al., 1975), the infection status of all eggs and larvae used in the trial were confirmed by smearing cohort eggs and larvae on microscope slides and examining stained specimens for microsporidian spores. Likewise, all of the mating pairs were examined at the end of the trial. Smear preparations were air-dried, fixed in methanol (10 min), stained in 5% Giemsa (pH 6.9; 2 h), rinsed in tap water (10 min), and treated to a series of ethanol in ascending concentration (70%, 3 min; 80%, 3 min; 90%, 3 min; 95%, 3 min; and absolute ethanol, 3 min). Slides were finished in xylene (10 min) and
2.2. Effects of Nosema adaliae on larval development and mortality – ingestion of one A. bipunctata egg Following the premature death of the majority of test larvae in the initial trial, a second trial was conducted whereby C. septempunctata test larvae were provided a single uninfected (control) or N. adaliae-infected (treatment) A. bipunctata egg and held for 24 h. Otherwise, the trial set up was the same as for the first trial. For each treatment group, 10 larvae were set up daily for a total of 4 d (40 larvae per treatment). This trial was repeated (total n = 80). Because larval development data did not follow a normal distribution, data were analyzed with a Mann Whitney U test. Only those individuals that completed development and emerged as adults were included in the analysis. A χ2 test (α = 0.05) was used to analyze differences in larval mortality between groups. Larvae that did not eat the A. bipunctata egg provided, and those that died before day 4 of the trials were excluded from the analyses. All individuals from the trial were smeared, stained, and examined for the presence of microsporidian spores by light microscopy.
2
Journal of Invertebrate Pathology 168 (2019) 107253
S. Bjørnson and E. Elkabir
significantly (χ2 = 2.07, df = 1, p = 0.15; Table 3).
2.3. Effects of Nosema adaliae on C. septempunctata fecundity and adult longevity
3.3. Effects on fecundity and adult longevity Adult C. septempunctata females that emerged from the second trial were used in 60-day fecundity and longevity trials. Adults were sexed 2 d following eclosion, then female beetles from the control (n = 14) and treatment (n = 14) were mated with uninfected male beetles from laboratory-reared colonies. Mating pairs were isolated within polyethylene cups, and males were removed once the females began to lay eggs (after 4–8 d). Females were provided with water daily and an abundant diet of aphids. Eggs produced were counted daily. Mating pairs were smeared and examined for microsporidian spores upon death or at the end of the 60-day trial. Three females from the control group and 7 from the treatment died without producing eggs. A t-test was used to examine differences in fecundity and a χ2 test (α = 0.05) was used to test for significance in adult longevity.
Over 60 d, C. septempunctata from the control group produced an average of 776.6 ± 122.0 eggs, more than double that of the treatment group, which produced 335.6 ± 86.6 eggs (Table 4). This difference was significant (t = 2.94, df = 16, p = 0.005). Adult longevity also differed significantly. Individuals of the control group lived an average of 58.2 ± 1.8 d, compared to 38.4 ± 6.4 d for individuals from the treatment (t = 2.98, df = 7, p = 0.010). An age-specific oviposition curve (mean eggs/day; Fig. 1) showed that egg production was initially similar for both control and treatment females at the beginning of their oviposition periods. However, fecundity decreased sharply for treated females near the 20-day mark of the trial and, for the most part, remained below that of the control for the remainder of the 60-day trial.
3. Results
3.4. Effects on sex ratios
Male and female C. septempunctata adults that were used to produce uninfected test larvae for the trials were not infected with microsporidian spores. All eggs collected and examined from these beetles were also microsporidia-free. Microscopic examination of A. bipunctata mating pairs and eggs collected from these adults, confirmed that the A. bipunctata eggs fed to C. septempunctata test larvae in this study were either uninfected (fed to control larvae) or infected with N. adaliae (fed to treatment larvae).
Sex ratios (♀: ♂) were 14:16 (control) and 15:7 (treatment), but these did not differ significantly (χ2 = 2.38, df = 1, p = 0.12). Percent infection for the control and treatment groups was 0 and 65.7%, respectively.
3.1. Effects when four A. bipunctata eggs were eaten
Mean development for individuals that ate one uninfected A. bipunctata egg as first-instar larvae, and later eclosed as adults, was 20.5 d (46.5% survival; Table 3), whereas those that ate a single N. adaliaeinfected egg took 21.3 d to develop. Although mean development did not differ significantly, these results differed substantially from those reported earlier by Omkar and Srivastava (2003). During their study, the duration from first-instar to adult was 15.24 d on a diet of M. persicae (66.6% survival; 25 °C). In another study, mean preimaginal development for C. septempunctata reared on pea aphids, Acyrthosiphon pisum (Harris) was between 14.3 and 14.9 d for individuals reared at 26 °C, (79–93% survival; Phoofolo and Obrycki, 1995). However, the larvae in our study ingested a single A. bipunctata egg before being provided an abundant diet of aphids, and it is possible that alkaloids within the eggs were responsible for the prolonged development period observed. A. bipunctata eggs and developmental stages contain adaline and adalinine (Tursch et al. 1973; King and Meinwald, 1996; Lognay et al. 1996), two species-specific alkaloids that are thought to provide a means for developing larvae to distinguish between conspecific and heterospecific eggs. Consumption of the former provides adequate nutrition for developing larvae when prey is scarce (Agarwala and Dixon, 1992; Hemptinne et al., 2000a), whereas consumption of the latter can cause deleterious effects. Agarwala and Dixon (1992) observed that C. septempunctata larvae were more likely to die after eating three A. bipunctata eggs than after eating the same quantity of their own. C. septempunctata larvae are also reluctant to eat their own eggs when they
4. Discussion 4.1. Effects on larval development and mortality
Mortality of C. septempunctata larvae that ate four uninfected or infected A. bipunctata eggs was ≥96% (Table 2). The high mortality of the test larvae during this trial made it impossible to assess the effect of the pathogen, N. adaliae, on the host. Percent infection was between 80.8 and 87.0% for larvae that ate at least one N. adaliae-infected egg. With one exception, all test larvae died, the majority before pupation. One pupa (Treatment 3) eclosed successfully in 5 d. Mean survival for larvae that ate four uninfected A. bipunctata eggs (control) was 26.0 ± 1.3 d, which was 0.5 to 1.7 d less than the larvae from the other treatments. Mean survival did not differ significantly (F = 0.46, df = 3, p = 0.71). 3.2. Effects on larval development and mortality Development time of larvae that ate one A. bipunctata egg and eclosed as adults was 20.5 ± 0.2 d (control) and 21.3 ± 0.8 d (treatment). These values did not differ significantly (U = 248.5, p = 0.14; Table 3). In the control group, 14 larvae died before they pupated, another 20 died during the pupal stage before eclosion, and a total of 30 beetles emerged. For the treatment group, 24 larvae and 18 pupae died, and a total of 22 adults emerged. Premature death (combined larval and pupal mortality) was 53.5 and 65.6% for the control and treatment groups, respectively, but mortality did not differ
Table 2 Mean survival (days), mortality (life stage and %), and infection (%) of Coccinella septempunctata that ate four uninfected or Nosema adaliae-infected eggs as 24 h-old larvae and thereafter provided Myzus persicae as food. Treatment
Survival n
Control Treatment 1 Treatment 2 Treatment 3
21 22 26 25
Mortality
Mean Days ± SE 26.0 26.5 27.7 26.6
± ± ± ±
1.3a 1.0a 1.1a 1.8a
Larval
Pupal
Total (%)
Eclosed Adults
Infection (%)
18 18 23 21
3 4 3 3
100 100 100 96
0 0 0 1
0 86.4 80.8 87.0
Means with the same letters do not differ significantly, ANOVA (F = 0.46, df = 3, p = 0.71). 3
Journal of Invertebrate Pathology 168 (2019) 107253
S. Bjørnson and E. Elkabir
Table 3 Mean development (days), mortality (life stage and %), sex ratio and infection (%) of Coccinella septempunctata that ate one uninfected or Nosema adaliae-infected egg as 24 h-old larvae and thereafter provided Myzus persicae as food. Treatment
Development
Control Treatment
Mortality
n
Mean Days ± SE
Larval
Pupal
Total (%)
Eclosed Adults
Sex Ratio (♀: ♂)
Infection (%)
68 59
20.5 ± 0.2a 21.3 ± 0.4a
14 24
20 18
53.5b 65.6b
30 22
14:16c 15:7c
0 65.7
Means with the same letters do not differ significantly, a, Mann-Whitney (U = 248.5, p = 0.14); b, Chi-square (χ2 = 2.07, df = 1, p = 0.15); c, Chi-square (χ2 = 2.38, df = 1, p = 0.12).
4.2. Effects on fecundity and adult longevity
Table 4 Mean fecundity and adult longevity (60 days) of Coccinella septempunctata that ate one uninfected or Nosema adaliae-infected egg as 24 h-old larvae and thereafter provided Myzus persicae as food. Treatment
Control Treatment
Fecundity
Longevity
n
Mean eggs ± SE
Total
Days ± SE
11 7
776.6 ± 122.0a 335.6 ± 86.6b
2984 1290
58.2 ± 1.8a 38.4 ± 6.4b
Although N. adaliae had no effect on the development or mortality of C. septempunctata larvae, the pathogen had a profound effect on fecundity and longevity. Over 60 d, C. septempunctata females that had consumed a single uninfected A. bipunctata egg as first-instar larvae produced twice the number of eggs on average (mean: 776.6 ± 122.0 eggs) and lived longer (58.2 ± 1.8 d) than their cohorts that had eaten one N. adaliae-infected egg (335.6 ± 86.6 eggs, 38.4 ± 6.4 d survival). Although this difference was significant (p = 0.005), the fecundity of uninfected C. septempunctata (776.6 ± 122.0 eggs over 60 d) was far lower than what has been reported for C. septempunctata in previous studies. According to Omkar and Srivastava (2003), C. septempunctata produce an average of 1198.5 eggs over their oviposition period (58 d) when reared on a diet of M. persicae at 25 °C. However, when fed a diet of Aphis gossypii at 25 °C, mean fecundity for C. septempunctata was 1660.5 eggs (66-day ovipositional period; Kawauchi, 1985). Such variations in fecundity are often attributed to prey quality and availability, but it is unlikely that these factors were responsible for the lower fecundity observed during our study because larvae were fed an abundant diet of M. persicae, an aphid that provides adequate nutrition for larval development and oviposition (Hodek and Honěk, 1996; Kalushkov and Hodek, 2004). With this in mind, the low fecundity observed may have been caused by the consumption of adaline or other alkaloids that are present in A. bipunctata eggs.
Means with different letters differ significantly, fecundity (t = 2.94, df = 16, p = 0.005), longevity (t = 2.98, df = 7, p = 0.010).
4.3. Effects on sex ratio Some microsporidia alter the sex ratio of arthropods (Dunn et al., 1993; Terry et al., 1998; Bjørnson and Keddie, 1999), but this was not the case for C. septempunctata. The sex ratio was ~1:1 for C. septempunctata in this study and was consistent with the sex ratio of beetles reported by Phoofolo and Obrycki (1995). In another study, the microsporidium Tubulinosema hippodamiae did not alter sex ratios of A. bipunctata, C. septempunctata, or H. axyridis when they were fed microsporidia-infected Hippodamia convergens eggs as larvae (Saito and Bjørnson, 2008).
Fig. 1. Age-specific oviposition curves (60 days) for Coccinella septempunctata that ate one uninfected or Nosema adaliae-infected egg as 24 h-old larvae and thereafter provided Myzus persicae as food.
are painted with water extracts made from crushed, A. bipunctata eggs (Hemptinne et al., 2000b). However, the effects of these alkaloids are dependent on the predator that consumes them. For example, larval mortality for Harmonia axyridis Pallas larvae that ate four A. bipunctata eggs was only 15% (Ellis, 2014), and adaline causes no ill effects when beetles are consumed by blue tit nestlings, Parus caeruleus L. (Marples et al. 1989). Although the results and observations from these previous studies suggest that alkaloids can influence larval development and/or mortality, this conclusion cannot be made in our study with certainty because an adequate control (with larvae fed a diet of aphids only) was not included. Mortality was higher for C. septempunctata larvae that ate four uninfected or infected A. bipunctata eggs (≥96% mortality) than for those that ate only one egg (< 63.8%), suggesting that the mortality observed was influenced by the number of eggs eaten. For individuals that consumed only one uninfected or infected egg, premature death (combined larval and pupal mortality) was 53.5 and 65.6%, respectively. Although mortality was relatively high, these values did not differ significantly and suggested that the pathogen had no effect on mortality.
4.4. Summary Microsporidia are common pathogens that cause chronic, debilitating disease; however, the effects that microsporidia cause are unpredictable and may vary, depending on the host species that is infected. In the case of N. adaliae, mean development for C. septempunctata larvae that consumed a single, infected A. bipunctata egg was prolonged by an average of 0.8 d compared to larvae that ate an uninfected egg (Table 3), but this difference was not significant. However, mean fecundity of adults infected by eating N. adaliae-infected eggs was about half that of their uninfected cohorts. In earlier studies, the microsporidian pathogen T. hippodamiae had the opposite effect on C. septempunctata. This pathogen prolonged the development of individuals that ate one infected H. convergens egg as first-instar larvae (Saito and Bjørnson, 2006; 2008), but the pathogen had no effect on fecundity (Saito and Bjørnson, 2008). These results show that the 4
Journal of Invertebrate Pathology 168 (2019) 107253
S. Bjørnson and E. Elkabir
effects of a given pathogen on a particular host cannot be predicted, even if the hosts are closely related. Several microsporidian pathogens have been described from lady beetles (Lipa and Steinhaus, 1959; Cali and Briggs, 1967; Lipa, 1968; Bjørnson et al. 2011; Steele and Bjørnson, 2014), and studies have shown that at least two of these pathogens, N. adaliae and T. hippodamiae, are transmitted horizontally among several coccinellid host species (Saito and Bjørnson, 2006, 2008; Steele and Bjørnson, 2012). Because A. bipunctata and C. septempunctata share overlapping habitats (Gordon, 1985; de Jong et al., 1991), it is reasonable to expect that these two coccinellids will encounter one another in nature. N. adaliae is transmitted vertically, and the results from the current study suggest that C. septempunctata larvae that consume a single infected A. bipunctata egg in nature are likely to become infected. Although the pathogen has no effect on larval development, infection is likely to result in high larval mortality or reduced fecundity of those infected larvae that survive to adulthood. Because infected female beetles produce half as many eggs as uninfected ones, the pathogen may reduce the intrinsic rate of increase of C. septempunctata in nature. It is also interesting that intraguild predation on a native species (A. bipunctata) by an invasive species (C. septempunctata) may have consequences for the invasive species. The result appears to be a form of ‘biotic resistance’, whereby the consumption of infected A. bipunctata may limit the invasion ability of C. septempunctata within a localized community.
on eggs of Coccinella septempunctata and Adalia bipunctata (Coleoptera: Coccinellidae). Eur. J. Entomol. 97, 559–562. Hemptinne, J.-L., Lognay, G., Gauthier, C., Dixon, A.F., 2000b. Role of surface chemical signals in egg cannibalism and intraguild predation in ladybirds (Coleoptera: Coccinellidae). Chemoecology 10, 123–128. Hodek, I., Honěk, A., 1996. Ecology of Cocinnellidae. Klewer Academic Publishers, Dordrecht, The Netherlands. Hodek, I., Michaud, J.P., 2008. Why is Coccinella septempunctata so successful? Eur. J. Entomol. 105, 1–12. Kalushkov, P., Hodek, I., 2004. The effects of thirteen species of aphids on some life history parameters of the ladybird Coccinella septempunctata. Biocontrol 49, 21–32. Kawauchi, S., 1985. Comparative studies on the fecundity of three aphidophagous coccinellids (Coleoptera: Coccinellidae). Japanese J. Appl. Entomol. Zool. 29, 203–209. King, A.G., Meinwald, J., 1996. Review of the defensive chemistry of coccinellids. Chem. Rev. 96, 1105–1122. Koebele, A., 1890. Report of a trip to Australia made under direction of the entomologist to investigate the natural enemies of the fluted scale. In: US Department of Agriculture, Division of Entomology, Bulletin 21, pp. 32. Lognay, G., Hemptinne, J.L., Chan, F.Y., Gaspar, C.H., Marlier, M., Braekman, J.C., Daloze, D., Pasteels, J.M., 1996. Adalinine, a new piperdine alkaloid from the ladybird beetles Adalia bipunctata and Adalia decimpunctata. J. Nat. Prod. 59, 510–511. Lipa, J.J., 1968. Nosema coccinellae sp. n., a new microsporidian parasite of Coccinella septempunctata, Hippodamia tredecimpunctata, and Myrrha octodecimguttata. Acta Protozoologica 5, 369–374. Lipa, J.J., Pruszynski, S., Bartkowski, J., 1975. The parasites and survival of the lady bird beetles (Coccinellidae) during winter. Acta Parasitologica Polonica 23, 453–461. Lipa, J.J., Steinhaus, E.A., 1959. Nosema hippodamiae n. sp., a microsporidian parasite of Hippodamia convergens Guérin (Coleoptera, Coccinellidae). J. Insect Pathol. 1, 304–308. Marples, N.M., Brakefield, P., Cowie, R.J., 1989. Differences between the 7-spot and 2spot ladybird beetles (Coccinellidae) in their toxic effects on a bird predator. Ecol. Entomol. 14, 79–84. Omkar, Pervez, A., 2002. Ecology of aphidophagous lady beetle, Coccinella septempunctata (Coleoptera: Coccinellidae): A review. J. Aphidol. 16, 175–201. Omkar, Pervez, A., 2005. Ecology of two-spotted ladybird Adalia bipunctata: A review. J. Appl. Entomol. 129, 465–474. Omkar, Srivastava, S., 2003. Influence of six aphid prey species on development and reproduction of a ladybird beetle, Coccinella septempunctata. BioControl 48, 379–393. Phoofolo, M., Obrycki, J.J., 1995. Comparative life-history studies of Nearctic and Palearctic populations of Coccinella septempunctata (Coleoptera: Coccinellidae). Environ. Entomol. 24, 581–587. Saito, T., Bjørnson, S., 2006. Horizontal transmission of a microsporidium from the convergent lady beetle, Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), on three non-target coccinellids. J. Invertebr. Pathol. 99, 294–301. Saito, T., Bjørnson, S., 2008. Effects of a microsporidium from the convergent lady beetle, Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), to three coccinellid species of Nova Scotia. Biol. Control 39, 427–433. Schaefer, P.W., Dysart, R.J., Specht, H.B., 1987. North American distribution of Coccinella septempunctata (Coleoptera: Coccinellidae) and its mass appearance in coastal Delaware. Environ. Entomol. 16, 368–373. Steele, T., Bjørnson, S., 2012. The effects of two microsporidian pathogens on the twospotted lady beetle, Adalia bipunctata L. (Coleoptera: Coccinellidae). J. Invertebr. Pathol. 109, 223–228. Steele, T., Bjørnson, S., 2014. Nosema adaliae sp. nov., a new microsporidian pathogen from the two-spotted lady beetle, Adalia bipunctata L. (Coleoptera: Coccinellidae) and its relationship to microsporidia that infect other coccinellids. J. Invertebr. Pathol. 115, 108–115. Terry, R.S., Smith, J.E., Dunn, A.M., 1998. Impact of a novel, feminising microsporidium on its crustacean host. J. Eukaryot. Microbiol. 45, 497–501. Tursch, B., Braekman, J.C., Daloze, D., Hootele, C., Losman, D., Karlsson, R., Pasteels, J.M., 1973. Adaline, a novel alkaloid from Adalia bipunctata L. (Coleoptera, Coccinellidae). Tetrahedron Lett. 3, 201–202. van Lenteren, J.C., 2003. Commercial availability of biological control agents. In: van Lenteren, J.C. (Ed.), Quality Control and Production of Biological Control Agents: Theory and Testing Procedures. CAB International, pp. 167–179. Wheeler Jr., A.G., Hoebeke, E.R., 1995. Coccinella novemnotata in northeastern North America: Historical occurrence and current status (Coleoptera: Coccinellidae). In: Proceedings of the Entomological Society of Washington, pp. 701–716.
Acknowledgements Research funding was provided by NSERC (Discovery Grant, SB), the Canadian Foundation for Innovation (CFI New Opportunities Fund) and Saint Mary’s University. References Agarwala, B.K., Dixon, A.F., 1992. Laboratory study of cannibalism and interspecific predation in ladybirds. Ecolog. Entomol. 17, 303–309. Bjørnson, S., Keddie, B.A., 1999. Effects of Microsporidium phytoseiuli (Microsporidia) on the performance of the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae). Biol. Control 15, 153–161. Bjørnson, S., Le, J., Saito, T., Wang, H., 2011. Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guérin-Méneville. J. Invertebr. Pathol. 106, 280–288. Cali, A., Briggs, J.D., 1967. The biology and life history of Nosema tracheophila sp. n. (Protozoa: Cnidospora: Microsporidea) found in Coccinella septempunctata Linnaeus (Coleoptera: Coccinellidae). J. Invertebr. Pathol. 9, 515–522. de Jong, P.W., Holloway, G.J., Brakefield, P.M., de Vos, H., 1991. Chemical defence in ladybird beetles (Coccinellidae). II. Amount of reflex fluid the alkaloid adaline and individual variation in defence in 2-spot ladybirds (Adalia bipunctata). Chemoecology 2, 15–19. Dunn, A.M., Adams, J., Smith, J.E., 1993. Transovarial transmission and sex ratio distortion by a microsporidian parasite in shrimp. J. Invertebr. Pathol. 61, 248–252. Elliott, N., Kieckhefer, R., Kauffman, W., 1996. Effects of an invading coccinellid on native coccinellids in an agricultural landscape. Oecologia 105, 537–544. Ellis, B., 2014. Effects of the microsporidium Nosema adaliae from Adalia bipunctata L. on the multicoloured Asian lady beetle Harmonia axyridis Pallas. Honours Thesis, Saint Mary’s University. Gordon, R.D., 1985. The Coccinellidae (Coleoptera) of America north of Mexico. J. New York Entomolog. Soc. 93, 1–912. Hemptinne, J.-L., Dixon, A.F., Gauthier, C., 2000a. Nutritive cost of intraguild predation
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Effects of the microsporidian pathogen, Nosema adaliae (Nosematidae) on the seven-spotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae)
T
⁎
S. Bjørnson , E. Elkabir Department of Biology, Saint Mary’s University, 923 Robie Street, Halifax, NS, B3H 3C3, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Adalia bipunctata Coccinella septempunctata Lady beetles Microsporidia Nosema adaliae Pathogen
Lady beetles are important predators in nature. Some species, including the two-spotted lady beetle, Adalia bipunctata L., are native to North America, whereas others, such as the seven-spotted lady beetle, Coccinella septempunctata L., have been introduced in North America for pest control on agriculture crops. Microsporidia are obligate pathogens that cause chronic disease, and these pathogens are known to infect several lady beetle species. Lady beetles are cannibalistic and, because many species share a given landscape, there is potential for microsporidia to infect susceptible coccinellids when infected eggs are eaten. The objective of this study was to examine the effects of the microsporidium Nosema adaliae isolated from A. bipunctata on C. septempunctata fitness (larval development and mortality, sex ratio, adult longevity and fecundity). Mortality was higher for C. septempunctata larvae that ate four A. bipunctata eggs (≥96% mortality) than for those that ate only one (< 63.8%), suggesting that the mortality observed was influenced by the number of eggs eaten. A. bipunctata eggs contain adaline and adalinine, two species-specific alkaloids that have been shown to be detrimental to C. septempunctata larvae. Development of larvae that consumed one uninfected or one N. adaliae-infected A. bipunctata egg, did not differ significantly (20.5 ± 0.2 d and 21.3 ± 0.4 d, respectively) and, although mortality remained high for these larvae (53.5% and 65.6% mortality, respectively), these values also did not differ significantly (p = 0.05). Over a 60-d period, mean fecundity for C. septempunctata adults that ate one uninfected A. bipunctata egg as firstinstar larvae was significantly greater (776.6 ± 122.0 eggs) than those that ate one N. adaliae-infected egg (335.6 ± 86.6 eggs, p = 0.005). Larvae from the former group also lived significantly longer (58.2 ± 1.8 d) than did those from the latter group (38.4 ± 6.4 d, p = 0.010). Sex ratios of adult beetles did not differ significantly. Because A. bipunctata and C. septempunctata share similar habitats, it is reasonable to expect these two coccinellids to encounter one another in nature. Results of this study show that the consumption of only one infected A. bipunctata egg by C. septempunctata larvae can result in high larval mortality and reduced fecundity.
1. Introduction During the late nineteenth century, the vedalia beetle, Rodolia cardinalis Mulsant, was imported to California from Australia to control the cottony cushion scale, Icerya purchasi Maskell, in California citrus (Koebele, 1890). The overwhelming success of the vedalia beetle prompted the intentional introduction of other exotic lady beetles into North America for pest control. One such introduction was the sevenspotted lady beetle, Coccinella septempunctata L., a Palearctic species that was initially released in North America for aphid control in 1956. By 1973, C. septempunctata had become established along the east coast of North America and along the coast of the Saint Lawrence River in Quebec (Gordon, 1985). The range of this coccinellid has since ⁎
increased in the United States and Canada (Schaefer et al., 1987). C. septempunctata is a rather large coccinellid with a voracious appetite. It feeds on a variety of prey, is adaptable to a wide range of environments, and tends to migrate extensively to areas where prey is plentiful (Omkar and Pervez, 2002; Hodek and Michaud, 2008; Elliott et al., 1996). These characteristics have enabled C. septempunctata to effectively invade new areas and become established throughout the Nearctic, but these same characteristics have fueled speculation that C. septempunctata may be associated with a notable decline of native coccinellids (Schaefer et al., 1987; Wheeler and Hoebeke, 1995; Elliott et al., 1996; Hodek and Michaud, 2008). The two-spotted lady beetle, Adalia bipunctata L., is a Holarctic species that was first commercialized for aphid control in Europe in 1998 (van Lenteren, 2003). In North
Corresponding author. E-mail address: [email protected] (S. Bjørnson).
https://doi.org/10.1016/j.jip.2019.107253 Received 28 June 2019; Received in revised form 29 September 2019; Accepted 2 October 2019 Available online 03 October 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 168 (2019) 107253
S. Bjørnson and E. Elkabir
America, A. bipunctata overlaps geographically with several lady beetle species, including C. septempunctata (Gordon, 1985; Hodek and Honěk, 1996; Omkar and Pervez, 2005). A. bipunctata is host to several microorganisms, including the microsporidian pathogen, Nosema adaliae, which was described from fieldcollected beetles from North America (Steele and Bjørnson, 2014). N. adaliae is transmitted vertically through the egg and horizontally when infected eggs or larvae are eaten. The pathogen prolongs the development of A. bipunctata, but has no effect on adult fecundity, longevity or sex ratios (Steele and Bjørnson, 2012). The cannibalistic tendencies of lady beetles, combined with the geographical overlap of these two coccinellid species, provide an opportunity for this pathogen to be transmitted from infected beetles to other susceptible ones when infected eggs are ingested. In some cases, microsporidia that have been described from lady beetle hosts have been shown to infect other coccinellid species (Saito and Bjørnson 2006, 2008; Steele and Bjørnson, 2012; Ellis, 2014). The aim of this study is to determine if N. adaliae can be transmitted horizontally from A. bipunctata to C. septempunctata when infected eggs are eaten by the immature larvae, and to evaluate the effects of this pathogen on larval development and mortality, adult fecundity and longevity, and sex ratios of C. septempunctata.
Table 1 Composition of uninfected and Nosema adaliae-infected Adalia bipunctata eggs provided to 48-h old Coccinella septempunctata larvae. n
Control Treatment 1 Treatment 2 Treatment 3
36 36 36 36
Uninfected
N. adaliae-infected
A. bipunctata eggs
A. bipunctata eggs
4 3 2 0
0 1 2 4
mounted in Permount (Fisher Scientific). To confirm infection status, specimens were examined for the presence of microsporidian spores by light microscopy (400 × magnification). M. persicae was also examined to ensure that the prey used were not infected with the pathogen.
2.1. Effects of Nosema adaliae – Ingestion of four A. bipunctata eggs This trial was designed to determine the effects of pathogen dose on C. septempunctata larval development and survival. Test larvae were divided into four treatment groups and individuals from each group were fed four A. bipunctata eggs consisting of various ratios of uninfected and N. adaliae-infected eggs (Table 1). Larvae fed uninfected A. bipunctata eggs served as a control. C. septempunctata test larvae (24 h old) were placed individually in clear Petri dishes (47-mm diameter, Millipore Corp., MA). Each lid had a 2-cm hole that was covered in fine, mesh screen. On the first day of the trial, water was provided on a cotton wick that was moistened daily. On the second day of the trial, a 6-mm disc of moistened filter paper with four A. bipunctata eggs was placed in the centre of each dish. Test larvae that ate all four eggs in 48 h were then provided a daily diet of aphids. Larval development and mortality were observed daily. For each treatment group, six larvae were set up daily over 6 d (36 larvae per treatment, total n = 144). With one exception, all test larvae died before eclosion. A one-way ANOVA was used to determine significance in mean survival (days) between groups. Larvae that did not eat all four eggs were excluded from the data analysis. During this study, C. septempunctata pupae eclosed in an average of 5 d; therefore, pupae that did not eclose after 7 d were considered dead. Because mortality was ≥96%, this trial was not repeated. Smear preparations were made of all individuals, and these were stained with 5% Giemsa and examined for the presence of microsporidian spores by light microscopy.
2. Materials and methods Uninfected C. septempunctata adults used in this study were collected from rose bushes near Saint Mary’s University campus (44.6316° N, 63.5822° W) during the summer of 2014. Beetles were reared in the laboratory, and a sample of eggs and larvae from each fecund female was stained and examined to ensure that they were not infected with microsporidia. These beetles produced larvae that were used as mating pairs for the trials. A. bipunctata eggs that were fed to C. septempunctata larvae during the trial originated from established laboratory colonies of uninfected and N. adaliae-infected A. bipunctata. C. septempunctata and A. bipunctata were reared individually in 120mL clear polyethylene cups with tight-fitting lids. A 2.2-cm diameter hole in the side of each cup was covered with fine mesh screen to permit air circulation. Water was provided daily on a moistened cotton wick (Crosstex International, NY) and beetles were provided a diet of green peach aphids, Myzus persicae (Sulzer), and artificial diet (equal parts honey and Lacewing and Ladybug Food, Planet Natural, MT). Aphids were reared on nasturtium (Tropaeolum minus L., Dwarf Jewel Mixed; Stokes Seed Ltd., ON) and both aphids and beetles were maintained under controlled conditions (16:8 L:D; 25 °C:20 °C) in separate environmental chambers (Sanyo MLR-350H). Larvae that completed development were sexed 24 h following eclosion, and males and females were maintained in separate cups until they were mated for use in the trial. C. septempunctata mating pairs were isolated within polyethylene cups and provided a diet of aphids and artificial diet. Water was provided on a moistened wick. For each trial, 10 C. septempunctata mating pairs were used to produce uninfected larvae. Eggs were collected daily and placed in isolated cups until the eggs hatched. Larvae that were 24 h old post-hatch were used in the trials. The eggs fed to these larvae were produced by 10 uninfected and 10 N. adaliae-infected A. bipunctata mating pairs. Because C. septempunctata are known to host two species of microsporidia (Nosema tracheophila and N. coccinellae) (Cali and Briggs, 1967; Lipa, 1968; Lipa et al., 1975), the infection status of all eggs and larvae used in the trial were confirmed by smearing cohort eggs and larvae on microscope slides and examining stained specimens for microsporidian spores. Likewise, all of the mating pairs were examined at the end of the trial. Smear preparations were air-dried, fixed in methanol (10 min), stained in 5% Giemsa (pH 6.9; 2 h), rinsed in tap water (10 min), and treated to a series of ethanol in ascending concentration (70%, 3 min; 80%, 3 min; 90%, 3 min; 95%, 3 min; and absolute ethanol, 3 min). Slides were finished in xylene (10 min) and
2.2. Effects of Nosema adaliae on larval development and mortality – ingestion of one A. bipunctata egg Following the premature death of the majority of test larvae in the initial trial, a second trial was conducted whereby C. septempunctata test larvae were provided a single uninfected (control) or N. adaliae-infected (treatment) A. bipunctata egg and held for 24 h. Otherwise, the trial set up was the same as for the first trial. For each treatment group, 10 larvae were set up daily for a total of 4 d (40 larvae per treatment). This trial was repeated (total n = 80). Because larval development data did not follow a normal distribution, data were analyzed with a Mann Whitney U test. Only those individuals that completed development and emerged as adults were included in the analysis. A χ2 test (α = 0.05) was used to analyze differences in larval mortality between groups. Larvae that did not eat the A. bipunctata egg provided, and those that died before day 4 of the trials were excluded from the analyses. All individuals from the trial were smeared, stained, and examined for the presence of microsporidian spores by light microscopy.
2
Journal of Invertebrate Pathology 168 (2019) 107253
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significantly (χ2 = 2.07, df = 1, p = 0.15; Table 3).
2.3. Effects of Nosema adaliae on C. septempunctata fecundity and adult longevity
3.3. Effects on fecundity and adult longevity Adult C. septempunctata females that emerged from the second trial were used in 60-day fecundity and longevity trials. Adults were sexed 2 d following eclosion, then female beetles from the control (n = 14) and treatment (n = 14) were mated with uninfected male beetles from laboratory-reared colonies. Mating pairs were isolated within polyethylene cups, and males were removed once the females began to lay eggs (after 4–8 d). Females were provided with water daily and an abundant diet of aphids. Eggs produced were counted daily. Mating pairs were smeared and examined for microsporidian spores upon death or at the end of the 60-day trial. Three females from the control group and 7 from the treatment died without producing eggs. A t-test was used to examine differences in fecundity and a χ2 test (α = 0.05) was used to test for significance in adult longevity.
Over 60 d, C. septempunctata from the control group produced an average of 776.6 ± 122.0 eggs, more than double that of the treatment group, which produced 335.6 ± 86.6 eggs (Table 4). This difference was significant (t = 2.94, df = 16, p = 0.005). Adult longevity also differed significantly. Individuals of the control group lived an average of 58.2 ± 1.8 d, compared to 38.4 ± 6.4 d for individuals from the treatment (t = 2.98, df = 7, p = 0.010). An age-specific oviposition curve (mean eggs/day; Fig. 1) showed that egg production was initially similar for both control and treatment females at the beginning of their oviposition periods. However, fecundity decreased sharply for treated females near the 20-day mark of the trial and, for the most part, remained below that of the control for the remainder of the 60-day trial.
3. Results
3.4. Effects on sex ratios
Male and female C. septempunctata adults that were used to produce uninfected test larvae for the trials were not infected with microsporidian spores. All eggs collected and examined from these beetles were also microsporidia-free. Microscopic examination of A. bipunctata mating pairs and eggs collected from these adults, confirmed that the A. bipunctata eggs fed to C. septempunctata test larvae in this study were either uninfected (fed to control larvae) or infected with N. adaliae (fed to treatment larvae).
Sex ratios (♀: ♂) were 14:16 (control) and 15:7 (treatment), but these did not differ significantly (χ2 = 2.38, df = 1, p = 0.12). Percent infection for the control and treatment groups was 0 and 65.7%, respectively.
3.1. Effects when four A. bipunctata eggs were eaten
Mean development for individuals that ate one uninfected A. bipunctata egg as first-instar larvae, and later eclosed as adults, was 20.5 d (46.5% survival; Table 3), whereas those that ate a single N. adaliaeinfected egg took 21.3 d to develop. Although mean development did not differ significantly, these results differed substantially from those reported earlier by Omkar and Srivastava (2003). During their study, the duration from first-instar to adult was 15.24 d on a diet of M. persicae (66.6% survival; 25 °C). In another study, mean preimaginal development for C. septempunctata reared on pea aphids, Acyrthosiphon pisum (Harris) was between 14.3 and 14.9 d for individuals reared at 26 °C, (79–93% survival; Phoofolo and Obrycki, 1995). However, the larvae in our study ingested a single A. bipunctata egg before being provided an abundant diet of aphids, and it is possible that alkaloids within the eggs were responsible for the prolonged development period observed. A. bipunctata eggs and developmental stages contain adaline and adalinine (Tursch et al. 1973; King and Meinwald, 1996; Lognay et al. 1996), two species-specific alkaloids that are thought to provide a means for developing larvae to distinguish between conspecific and heterospecific eggs. Consumption of the former provides adequate nutrition for developing larvae when prey is scarce (Agarwala and Dixon, 1992; Hemptinne et al., 2000a), whereas consumption of the latter can cause deleterious effects. Agarwala and Dixon (1992) observed that C. septempunctata larvae were more likely to die after eating three A. bipunctata eggs than after eating the same quantity of their own. C. septempunctata larvae are also reluctant to eat their own eggs when they
4. Discussion 4.1. Effects on larval development and mortality
Mortality of C. septempunctata larvae that ate four uninfected or infected A. bipunctata eggs was ≥96% (Table 2). The high mortality of the test larvae during this trial made it impossible to assess the effect of the pathogen, N. adaliae, on the host. Percent infection was between 80.8 and 87.0% for larvae that ate at least one N. adaliae-infected egg. With one exception, all test larvae died, the majority before pupation. One pupa (Treatment 3) eclosed successfully in 5 d. Mean survival for larvae that ate four uninfected A. bipunctata eggs (control) was 26.0 ± 1.3 d, which was 0.5 to 1.7 d less than the larvae from the other treatments. Mean survival did not differ significantly (F = 0.46, df = 3, p = 0.71). 3.2. Effects on larval development and mortality Development time of larvae that ate one A. bipunctata egg and eclosed as adults was 20.5 ± 0.2 d (control) and 21.3 ± 0.8 d (treatment). These values did not differ significantly (U = 248.5, p = 0.14; Table 3). In the control group, 14 larvae died before they pupated, another 20 died during the pupal stage before eclosion, and a total of 30 beetles emerged. For the treatment group, 24 larvae and 18 pupae died, and a total of 22 adults emerged. Premature death (combined larval and pupal mortality) was 53.5 and 65.6% for the control and treatment groups, respectively, but mortality did not differ
Table 2 Mean survival (days), mortality (life stage and %), and infection (%) of Coccinella septempunctata that ate four uninfected or Nosema adaliae-infected eggs as 24 h-old larvae and thereafter provided Myzus persicae as food. Treatment
Survival n
Control Treatment 1 Treatment 2 Treatment 3
21 22 26 25
Mortality
Mean Days ± SE 26.0 26.5 27.7 26.6
± ± ± ±
1.3a 1.0a 1.1a 1.8a
Larval
Pupal
Total (%)
Eclosed Adults
Infection (%)
18 18 23 21
3 4 3 3
100 100 100 96
0 0 0 1
0 86.4 80.8 87.0
Means with the same letters do not differ significantly, ANOVA (F = 0.46, df = 3, p = 0.71). 3
Journal of Invertebrate Pathology 168 (2019) 107253
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Table 3 Mean development (days), mortality (life stage and %), sex ratio and infection (%) of Coccinella septempunctata that ate one uninfected or Nosema adaliae-infected egg as 24 h-old larvae and thereafter provided Myzus persicae as food. Treatment
Development
Control Treatment
Mortality
n
Mean Days ± SE
Larval
Pupal
Total (%)
Eclosed Adults
Sex Ratio (♀: ♂)
Infection (%)
68 59
20.5 ± 0.2a 21.3 ± 0.4a
14 24
20 18
53.5b 65.6b
30 22
14:16c 15:7c
0 65.7
Means with the same letters do not differ significantly, a, Mann-Whitney (U = 248.5, p = 0.14); b, Chi-square (χ2 = 2.07, df = 1, p = 0.15); c, Chi-square (χ2 = 2.38, df = 1, p = 0.12).
4.2. Effects on fecundity and adult longevity
Table 4 Mean fecundity and adult longevity (60 days) of Coccinella septempunctata that ate one uninfected or Nosema adaliae-infected egg as 24 h-old larvae and thereafter provided Myzus persicae as food. Treatment
Control Treatment
Fecundity
Longevity
n
Mean eggs ± SE
Total
Days ± SE
11 7
776.6 ± 122.0a 335.6 ± 86.6b
2984 1290
58.2 ± 1.8a 38.4 ± 6.4b
Although N. adaliae had no effect on the development or mortality of C. septempunctata larvae, the pathogen had a profound effect on fecundity and longevity. Over 60 d, C. septempunctata females that had consumed a single uninfected A. bipunctata egg as first-instar larvae produced twice the number of eggs on average (mean: 776.6 ± 122.0 eggs) and lived longer (58.2 ± 1.8 d) than their cohorts that had eaten one N. adaliae-infected egg (335.6 ± 86.6 eggs, 38.4 ± 6.4 d survival). Although this difference was significant (p = 0.005), the fecundity of uninfected C. septempunctata (776.6 ± 122.0 eggs over 60 d) was far lower than what has been reported for C. septempunctata in previous studies. According to Omkar and Srivastava (2003), C. septempunctata produce an average of 1198.5 eggs over their oviposition period (58 d) when reared on a diet of M. persicae at 25 °C. However, when fed a diet of Aphis gossypii at 25 °C, mean fecundity for C. septempunctata was 1660.5 eggs (66-day ovipositional period; Kawauchi, 1985). Such variations in fecundity are often attributed to prey quality and availability, but it is unlikely that these factors were responsible for the lower fecundity observed during our study because larvae were fed an abundant diet of M. persicae, an aphid that provides adequate nutrition for larval development and oviposition (Hodek and Honěk, 1996; Kalushkov and Hodek, 2004). With this in mind, the low fecundity observed may have been caused by the consumption of adaline or other alkaloids that are present in A. bipunctata eggs.
Means with different letters differ significantly, fecundity (t = 2.94, df = 16, p = 0.005), longevity (t = 2.98, df = 7, p = 0.010).
4.3. Effects on sex ratio Some microsporidia alter the sex ratio of arthropods (Dunn et al., 1993; Terry et al., 1998; Bjørnson and Keddie, 1999), but this was not the case for C. septempunctata. The sex ratio was ~1:1 for C. septempunctata in this study and was consistent with the sex ratio of beetles reported by Phoofolo and Obrycki (1995). In another study, the microsporidium Tubulinosema hippodamiae did not alter sex ratios of A. bipunctata, C. septempunctata, or H. axyridis when they were fed microsporidia-infected Hippodamia convergens eggs as larvae (Saito and Bjørnson, 2008).
Fig. 1. Age-specific oviposition curves (60 days) for Coccinella septempunctata that ate one uninfected or Nosema adaliae-infected egg as 24 h-old larvae and thereafter provided Myzus persicae as food.
are painted with water extracts made from crushed, A. bipunctata eggs (Hemptinne et al., 2000b). However, the effects of these alkaloids are dependent on the predator that consumes them. For example, larval mortality for Harmonia axyridis Pallas larvae that ate four A. bipunctata eggs was only 15% (Ellis, 2014), and adaline causes no ill effects when beetles are consumed by blue tit nestlings, Parus caeruleus L. (Marples et al. 1989). Although the results and observations from these previous studies suggest that alkaloids can influence larval development and/or mortality, this conclusion cannot be made in our study with certainty because an adequate control (with larvae fed a diet of aphids only) was not included. Mortality was higher for C. septempunctata larvae that ate four uninfected or infected A. bipunctata eggs (≥96% mortality) than for those that ate only one egg (< 63.8%), suggesting that the mortality observed was influenced by the number of eggs eaten. For individuals that consumed only one uninfected or infected egg, premature death (combined larval and pupal mortality) was 53.5 and 65.6%, respectively. Although mortality was relatively high, these values did not differ significantly and suggested that the pathogen had no effect on mortality.
4.4. Summary Microsporidia are common pathogens that cause chronic, debilitating disease; however, the effects that microsporidia cause are unpredictable and may vary, depending on the host species that is infected. In the case of N. adaliae, mean development for C. septempunctata larvae that consumed a single, infected A. bipunctata egg was prolonged by an average of 0.8 d compared to larvae that ate an uninfected egg (Table 3), but this difference was not significant. However, mean fecundity of adults infected by eating N. adaliae-infected eggs was about half that of their uninfected cohorts. In earlier studies, the microsporidian pathogen T. hippodamiae had the opposite effect on C. septempunctata. This pathogen prolonged the development of individuals that ate one infected H. convergens egg as first-instar larvae (Saito and Bjørnson, 2006; 2008), but the pathogen had no effect on fecundity (Saito and Bjørnson, 2008). These results show that the 4
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effects of a given pathogen on a particular host cannot be predicted, even if the hosts are closely related. Several microsporidian pathogens have been described from lady beetles (Lipa and Steinhaus, 1959; Cali and Briggs, 1967; Lipa, 1968; Bjørnson et al. 2011; Steele and Bjørnson, 2014), and studies have shown that at least two of these pathogens, N. adaliae and T. hippodamiae, are transmitted horizontally among several coccinellid host species (Saito and Bjørnson, 2006, 2008; Steele and Bjørnson, 2012). Because A. bipunctata and C. septempunctata share overlapping habitats (Gordon, 1985; de Jong et al., 1991), it is reasonable to expect that these two coccinellids will encounter one another in nature. N. adaliae is transmitted vertically, and the results from the current study suggest that C. septempunctata larvae that consume a single infected A. bipunctata egg in nature are likely to become infected. Although the pathogen has no effect on larval development, infection is likely to result in high larval mortality or reduced fecundity of those infected larvae that survive to adulthood. Because infected female beetles produce half as many eggs as uninfected ones, the pathogen may reduce the intrinsic rate of increase of C. septempunctata in nature. It is also interesting that intraguild predation on a native species (A. bipunctata) by an invasive species (C. septempunctata) may have consequences for the invasive species. The result appears to be a form of ‘biotic resistance’, whereby the consumption of infected A. bipunctata may limit the invasion ability of C. septempunctata within a localized community.
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