Immune response of the hibiscus mealybug, Maconellicoccus hirsutus Green (Homoptera: Pseudococcidae), to oviposition of the parasitoid Anagyrus kamali Moursi (Hymenoptera: Encyrtidae)

Journal of Insect Physiology 46 (2000) 647–653 www.elsevier.com/locate/jinsphys

Immune response of the hibiscus mealybug, Maconellicoccus hirsutus Green (Homoptera: Pseudococcidae), to oviposition of the parasitoid Anagyrus kamali Moursi (Hymenoptera: Encyrtidae) L.A. Sagarra

a, b

, D.D. Peterkin c, C. Vincent

d,*

, R.K. Stewart

a

a

d

Department of Natural Resource Sciences, Macdonald Campus of McGill University, 21,111 Lakeshore, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9 b P-R Trinidad Ltd, Orange Grove Estate, Tacarigua, Trinidad and Tobago c IIBC/CLAS, Gordon Street, Curepe, Trinidad and Tobago Horticultural Research and Development Center, Agriculture and Agri-Food Canada, 430 Gouin Blvd, Saint-Jean-sur-Richelieu, Quebec, Canada J3B 3E6 Received 29 January 1999; accepted 28 June 1999

Abstract Anagyrus kamali Moursi has been recently introduced into the Caribbean as a biological agent against the hibiscus mealybug, Maconellicoccus hirsutus Green. This host has a cellular defense reaction that involves encapsulation and melanization of the endoparasitoid egg. The impact of this immune response on the parasitoid progeny was assessed, as well as the response of the parasitoid countermeasures to overcome it. Under laboratory conditions, significant differences in the immune response were found for different developmental stages of M. hirsutus. The intensity of the immune response varied between second instar, third instar and adult mealybugs. After 30 h, the level of encapsulation was the highest for eggs oviposited in adults: 58% of eggs were encapsulated, followed by third (32%) and second (4%) instars. Three days after oviposition 23, 44 and 86% of the parasitoid eggs oviposited, respectively, in adult, third and second instars were not encapsulated. The unencapsulated parasitoid eggs could hatch and continue their development. Adult mealybugs required 30 h to encapsulate 50% of the eggs, whereas in second and third instars, 50% level encapsulation was never reached. Superparasitism had a saturating effect on the immune system; reduced levels of encapsulation occurred when more than 10 eggs were oviposited in a single mealybug. Wasp larvae were never encapsulated by M. hirsutus.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Anagyrus kamali; Maconellicoccus hirsutus; Encapsulation; Host stage; Biological control

1. Introduction Encapsulation is an active physiological defense mechanism that is an immune response of the host against the intrusion of an external element (Ratcliffe, 1993; Jervis and Copland, 1996). It consists of surrounding the parasitoid egg or larva with a multicellular sheath composed of the host hemocytes in order to asphyxiate or starve it (Waage and Greathead, 1985; Gotz and Boman, 1985; Gupta, 1985, 1986), along with the

* Corresponding author. Tel.: +1-450-346-4494, ext. 202; fax: +1450-346-7740. E-mail address: [email protected] (C. Vincent).

secretion of melanin and other cytotoxic molecules (Nappi and Sugumaran, 1993; Carton and Nappi, 1997). Host immune responses are poorly documented for encyrtid insects that parasitize mealybugs. The mealybug’s immune response to attack by parasitoids involves mainly encapsulation and melanization of eggs and juvenile stages, as described in Phenacoccus herreri Cox and Williams when attacked by Epidinocarsis diversicornis Howard (Herrera and Bellotti, 1986), E. lopezi De Santis on P. manihoti Matile-Ferrero (Sullivan and Neuenschwander, 1988), or Anagyrus pseudococci Girault on Planococcus citri Risso, P. rorae Nasonov, P. ficus Signoret, and Pseudococcus cryptus Hempel (Blumberg et al., 1995). The encyrtid endoparasitoid A. kamali Moursi was introduced into the Caribbean from China by the Inter-

0022-1910/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 9 ) 0 0 1 5 2 - 3

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national Institute of Biological Control in 1995 for the control of the hibiscus mealybug, Maconellicoccus hirsutus. A. kamali was previously reported to be an efficient biocontrol agent of M. hirsutus in Egypt and India (Moursi, 1948; Beardsley, 1985; Mani, 1989). M. hirsutus is very prolific (384–540 eggs per female) (Mani, 1989) and completes its life cycle within 23–29 days (Ghose, 1972). It injects a toxin at the feeding point, causing severe deformation of the plants. M. hirsutus is presently found in 18 countries in the Caribbean where it attacks agricultural, ornamental and forestry crops (Matile-Ferrero and Etienne, 1996; Williams, 1996; IIE, 1997; Etienne et al., 1998). To counteract the rapid and destructive spread of hibiscus mealybug, biological control appeared to be one of the only sustainable methods due to the wide variety of host plants and habitats of the mealybug. The hibiscus mealybug exhibits a cellular immune response to eggs of A. kamali, including encapsulation and melanization (Sagarra, personal communication). Several biochemical or immunological mechanisms act within a few hours after host intrusion by a foreign body. Our objectives were to describe encapsulation of parasitoid eggs in hibiscus mealybug, to assess its temporal dynamics, to quantify its effect on oviposited eggs and adult parasitoid emergence, and to study the parasitoid’s countermeasures to avoid encapsulation (e.g. superparasitism and host selection).

2. Materials and methods 2.1. Rearing of M. hirsutus Mealybugs were reared on sprouted potatoes in nylon mesh cages (32 potatoes per cage) supported on steel wire frames (48×48×68 cm). The cultures were maintained in the dark at 27±2°C. Each week, 64 potatoes were individually infested with 20 adult female mealybugs having well-formed ovisacs. Three weeks after infestation, the potatoes supported populations of L2 (second instar) and L3 (third instar) mealybugs. Adult females with ovisacs developed 4–6 weeks after initial infestation. Size was used as the primary criterion to determine developmental stages (Ghose, 1971). 2.2. Rearing of the parasitoid Parasitoids were reared by releasing 100 mated adult females onto 3-week old populations of M. hirsutus (6000–8000 individuals) at 27±2°C, 60±10% RH, and a photoperiod of 12L:12D. Emerged parasitoids were collected 20–25 days after initial introduction. Two-day old, mated females were used for experiments. Female parasitoids were considered experienced as they had been

exposed upon emergence to different stages of mealybugs in the rearing cages for a maximum of 12 h. 2.3. Experimental procedure In preliminary tests, all M. hirsutus stages were used; however only 20% of L1 (1st instar) were parasitized and parasitoid eggs were not encapsulated. In the following experiments, only L2 (male or female), L3 female, and early adult female M. hirsutus (preovisac) were tested. Except for L2 individuals that were unsexed, mealybugs were sexed using characteristics described in Ghose (1971). M. hirsutus eggs, ovipositing females, male hosts in the pharate third and fourth nymphal stages, or the winged adult male stage were not suitable for oviposition (Moursi, 1948). All tests were conducted at 27±2°C, 60±10% RH. 2.4. Host immune response Hibiscus mealybugs were collected from potatoes and placed in groups of 100 on an hibiscus leaf (Hibiscus rosa-sinensis). The leaf was then placed inside a transparent plastic vial (10 cm diameter by 6 cm long) with a 1 cm hole covered with mesh on the screw cap. Twenty female parasitoids were introduced into each vial and fed with a drop of honey deposited on the mesh window of the cap. Parasitoids were left to forage and oviposit for 2 h. Mealybugs were then dissected in a drop of ethanol (70%) at 0, 6, 10, 14, 18, 22, 26, 30, 44, and 72 h after removal of the parasitoids to observe the dynamics of the parasitoid egg encapsulation. At each time interval, a total of 60 M. hirsutus of each stage were dissected. During preliminary tests, we distinguished four degrees of encapsulation: non-encapsulated, amber eggs, partially encapsulated and completely encapsulated. The number of eggs in each mealybug and the degree of encapsulation of each parasitoid egg were recorded. Data from dissections were also used to estimate the percentage of efficient encapsulation (hosts with all parasitoid eggs encapsulated) at each sampling interval. The percentage of parasitoid eggs encapsulated was plotted for each time interval and a logistic regression analysis was performed (Systat 7 for Windows) to determine the rate of melanization and encapsulation in the different host stages. A one-way ANOVA was performed to compare the egg encapsulation among different instars. Means were separated using Student–Neuman– Keuls tests. 2.5. Effect of encapsulation on the emergence of adult parasitoids Groups of 100 mealybugs were exposed to ovipositing A. kamali as previously described. After a 2-h exposure,

L.A. Sagarra et al. / Journal of Insect Physiology 46 (2000) 647–653

the parasitoids were removed and the mealybugs were transferred in groups of 30 individuals onto sprouted potatoes. The mealybugs were observed daily and parasitoid emergence was recorded. L2, L3 and adult mealybugs were tested. The experiment was replicated five times. The number of parasitoids that emerged was used as a variable to determine the effect of encapsulation on parasitoid efficiency. The number of adult parasitoids that emerged from the different host stages was compared with t-tests (Systat 7 for Windows).

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mealybugs were transferred onto sprouted potatoes in groups of 30. The mealybugs were observed daily and parasitoid emergence was recorded. L3 and adult mealybugs were tested. The experiment was replicated five times. Percentage emergence of adult parasitoids was used as the criterion for measuring the effect of encapsulation on parasitoid efficiency. We used t-tests to compare the number of adults emerged among stages (Systat 7 for Windows). 2.10. Effect of superparasitism on encapsulation

2.6. Response of mealybug to wounding Fifty L3 mealybugs were collected from potatoes. Each mealybug was punctured on its back or side once using a stainless steel pin minuten (0.15 mm in diameter) in order to mimic an oviposition puncture by the parasitoid. Mealybugs were then placed on an hibiscus leaf for 48 h after which they were dissected in a drop of ethanol (70%), to observe the response of the insect to the puncture.

Groups of 25 L3 mealybugs were exposed to 25 female parasitoids for 24 h, as previously described. All mealybugs were dissected in a drop of ethanol (70%) 48 h after the removal of the parasitoid. The total number of eggs and the number of completely encapsulated eggs were recorded. Superparasitism was measured in terms of number of eggs oviposited per host. Data from dissections were also used to estimate the percentage of egg encapsulation. The experiment was replicated four times.

2.7. Physical effect of wounding Fifty L3 M. hirsutus were collected from potatoes. Small glass rods (0.5 mm long×0.15 mm diameter) were inserted through the cuticle, in order to mimic egg insertion and to determine if parasitoid egg proteins are necessary elicitors of the mealybug’s immune response. After 48 h, the 50 L3 mealybugs were dissected in a drop of ethanol (70%) and glass rods were observed for symptoms of melanization and encapsulation. 2.8. Sequential exposure to parasitism – effect on egg encapsulation Groups of 20 L3 mealybugs were initially exposed to four female A. kamali for 2 h, as previously described. Forty eight hours after removal of the parasitoids, four new female A. kamali were released onto the mealybugs for 2 h. All mealybugs were dissected in a drop of ethanol (70%) 48 h after the removal of the parasitoid. The number of eggs and the percentage of encapsulation of the parasitoid eggs were recorded. Data from dissections were also used to estimate the percentage of efficient encapsulation (hosts with all parasitoid eggs encapsulated). L3 and adult mealybugs were tested. The experiment was replicated three times. 2.9. Sequential exposure to parasitsm – effect on parasitoid emergence Groups of 50 L3 mealybugs were initially exposed to 10 female parasitoids for 2 h, as previously described. Forty eight hours after exposure, four new female A. kamali were released onto the mealybugs for 2 h. All

3. Results 3.1. Host immune response Encapsulated eggs were easily observed through the host cuticle. Unencapsulated eggs were transluscent. The first symptom of egg encapsulation was the formation of an orange/amber corolla surrounding the egg whose color changed from transparent to amber. This was followed by aggregates of melanized material forming black spots (partial encapsulation) which gradually spread over the entire egg surface, finally covering it completely. The thickness of the melanized capsule increased over time. All tested stages of M. hirsutus were parasitized by A. kamali. The number of eggs oviposited in L1, L2, L3 immatures and adults was 1.14±0.35, 1.48±0.57, 2.21±1.10, and 2.52±1.32 eggs/host, respectively. Significantly more eggs were laid in older stages (Adult M. hirsutus and L3) than in L1 and L2 (t-test, p⬍0.05) (Table 1). A parasitized mealybug could contain both encapsulated and non-encapsulated eggs. Mealybugs were completely immune to successful parasitism when they contained only encapsulated eggs. Parasitoid egg encapsulation was host stage-dependent. As the eggs of A. kamali usually hatched within 3 days (Moursi, 1948), it was possible to observe the effect of encapsulation on egg hatching after this time has elapsed. After 72 h, only 4.4% of parasitoid eggs oviposited in L2 M. hirsutus were completely encapsulated. In L3 and adult hosts, 32.6 and 58.3% of parasitoid eggs were completely encapsulated. In L2, L3 and adults, this resulted in an

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Table 1 Effect of host stages and number of oviposition periods on the oviposition, encapsulation and emergence of A. kamali Number of oviposition periods

Host stage (n=60 individuals/stage) L2

L3

Adult

Average no. of eggs/host

1 2

1.48±0.57aa N/A

2.21±1.10b 3.45±1.48a

2.52±1.32b 2.85±1.42b

Average no. of encapsulated eggs/host

1 2

0.04±0.11a N/A

0.66±0.89b 0.77±1.14a

1.38±1.41c 1.33±1.57c

Efficient encapsulation (%)b

1 2

0.7a N/A

17.5b 10.0a

24.7c 24.1c

Progeny emergence (%)

1 2

47.7a N/A

16.7b 24.2a

18.0b 18.3a

Sex ratio of progeny (M/T)

1

0.91a

0.41b

0.44b

a b

Within rows, pairs of means followed by the same letters are not significantly different (p⬍0.05) (SNK tests). Hosts with all parasitoid eggs encapsulated.

encapsulation level of 0.04±0.11, 0.66±0.89 and 1.38±1.41 eggs encapsulated/M. hirsutus, respectively. Levels of encapsulation were significantly different among host stages (ANOVA, F=105.2, p⬍0.05). There was also a difference in the time taken for the onset of the melanization and encapsulation (i.e. the “amber egg” stage) which was initially observed 6 h after oviposition in L3 and adult mealybugs, and 10 h in L2. The time taken for 50% of eggs to be melanized and completely encapsulated was estimated using a regression analysis between the time after oviposition and the percentage of complete encapsulation. The estimated time for 50% of eggs to be completely encapsulated in early adult mealybug was estimated at 30 h. L3 and L2 never reached 50% of complete encapsulation (Fig. 1). The percentages of efficient encapsulation (i.e. hosts with all parasitoid eggs encapsulated) were also significantly different among host stages (Mann-Whitney Utests, p⬍0.05). Seventy-two hours after oviposition, 24.7% of adult M. hirsutus had encapsulated all parasitoid eggs oviposited in their body. In L3 and L2, 17.5 and 0.7% of the mealybug encapsulated all the oviposited eggs. The maximum percentage of efficient encapsulation was achieved at 30 h, and after that, egg encapsulation levels plateaued at 17.5% in L3 and 24.7% in adults (Table 1). Encapsulation of parasitoid larvae was never observed. 3.2. Effect of encapsulation on the emergence of adult parasitoids A. kamali developed and emerged from parasitized L2, L3 and preovisac adult females. The emergence of the parasitoids was not significantly (t-test, p⬎0.05) different for L3 and the adult females (5.0±1.59 and

Fig. 1. Percentage of encapsulation of A. kamali eggs as a function of time and mealybug stages (n=60 individuals/stages/time interval).

5.4±1.82). This represented an average percentage of emergence of 16.7% for L3 and 18.0% for the adult M. hirsutus, with an average sex ratio of 0.4 in both cases. Emergence from L2 was significantly higher (p⬍0.05) than in L3 and adults, with a production of 14.3±2.58 offspring, which represents 47.7% emergence with a mean sex ratio of 0.9. 3.3. Response of mealybug to wounding – specificity of the immune response All M. hirsutus hosts that were wounded with a pin showed only a slight darkening at the puncture hole. In

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contrast, 43.75% of the glass rods introduced in the mealybug body showed signs of encapsulation after 48 h in the insect body. Similar to egg encapsulation, the glass rods were surrounded by a black melanized capsule. The thickness of this capsule was on average 56 µm with a maximum of 70 µm after 48 h. The immune response is therefore not specific to the proteins of the parasitoid egg coating, but a generalized encapsulation response against any foreign body introduced into the haemocel. 3.4. Saturation of the immune system – effect on egg encapsulation and emergence When parasitized M. hirsutus (L3 and adult females) were reexposed to ovipositing parasitoids, the average number of parasitoid eggs per mealybug was significantly (t-test, p⬍0.05) different with 3.45±1.48 and 2.89±1.41 eggs per mealybug, respectively. In L3, the percentage of efficient encapsulation was 10.0% and in adult females, 24.1%. The percentage of egg encapsulation was 32.6% in L3 and 58.3% in adult females, which represented an average of 0.77±1.14 and 1.33±1.57 eggs encapsulated per host for each respective stage, showing no significant difference (t-test, p⬍0.05). Most of the adult females formed an ovisac. The emergence from L3 and adult females was, respectively, 10.6±2.2 and 6.3±1.6 parasitoids per infested group of 30 M. hirsutus. This represents an average percentage of emergence of 24.2% for L3 and 18.3% for the adult M. hirsutus. This difference between the two stages was not significantly (t-test, p⬎0.05) different. 3.5. Saturation of the immune system – effect of egg number With increasing numbers of eggs per M. hirsutus (Fig. 2), the number of eggs successfully encapsulated gradually decreased and no eggs were successfully encapsulated in a host with more than 14 eggs per mealybug.

4. Discussion Many endoparasitoid species develop successfully within a range of different host instars (Van Alphen and Jervis, 1996). In A. kamali all host stages were parasitized; however, parasitoid eggs were mostly encapsulated in L3 and adult mealybugs. Encapsulation in L2 was rare. The immune response is not specific to the parasitoid egg, since slender glass rods were also encapsulated, suggesting a general cellular response of the phagocytosis type (Bess, 1939). For A. kamali to develop successfully, it must overcome the immune response of its host. Suppression of the host immune response is essential for the survival of endoparasitic Hymenoptera and other endoparasites (Webb and Luckhart, 1996).

Fig. 2. Effect of A. kamali egg density on encapsulation by mealybug third instar nymphs.

A. kamali can utilize two methods for evading encapsulation of juvenile stages. Parasitoids may oviposit in early stages of hosts in which the immune system is not yet mature (Salt, 1968). Parasitoid survival is highest due to age-related changes in physiological host defenses. This strategy is utilized by numerous species of parasitoids, with a variation depending on the attacked stage. Older stages of the hemispherical scale, Saissetia coffeae Olivier, are able to encapsulate more eggs of the encyrtid parasitoid Metaphycus swirskii Annecke and Mynhardt and Encyrtus infelix (Embleton) (Blumberg, 1988; Blumberg and Goldenburg, 1991). A similar phenomenon was observed with Metaphycus helvolus (Compere) and Coccus hesperidium L. (Visser and van Alphen, 1988). This mechanism for evading the host immune response has also been described in the Lepidoptera (Davies and Vinson, 1988; Harvey et al., 1996; Webb and Luckhart, 1996). In other mealybugs, the opposite has been observed. For example, in Phenacoccus herreri (Cox and Williams) parasitized by Epidiniocarsis diversicornis (Howard), 9.2% of eggs were encapsulated in L2, compared to 5.2% in L3 and adults (Herrera and Bellotti, 1986; Van Driesche et al., 1986). Although all M. hirsutus stages are parasitized by A. kamali, only L3 and adult mealybugs have a sufficiently developed immune system to effectively encapsulate the parasitoid eggs. The parasitoid is able to avoid the host immune response by ovipositing in younger stages (L1 and L2) that are unable to elaborate a significant defense. This may be explained by the small amount of blood, blood cells and the smaller proportion of reactive cells (hemocytes) available in the host at those early stages

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(Gotz and Boman, 1985). Adult female mealybug showed the highest encapsulation level due to the fact that her immune system is fully mature. This stage is able to encapsulate up to eight eggs in a single host. The sex ratio of the progeny from L1 and L2 mainly consists of males due to insufficient nutrient reserves required for female development (Sagarra and Vincent, 1999). Parasitizing only those stages to overcome encapsulation is not an evolutionary stable strategy, as it would lead to the extinction of the parasitoid species. However, female parasitoids could maximize their fitness by preferential host selection for male and female eggs. Oviposition in L3 gave a slightly superior emergence rate, with a lower number of eggs encapsulated per M. hirsutus, and the same sex ratio of progeny as for the adults. Oviposition in this mealybug stage is advantageous, compared to fully mature stages. A number of authors have also noted that the probability of at least one egg surviving encapsulation in solitary endoparasitoids is greater when the host contains several eggs (e.g. Askew, 1968; Blumberg et al., 1990). Superparasitism may increase the probabilities of survival of the parasitoid progeny. Several eggs may exhaust cellular the cellular immune response of a host. This was observed in A. kamali. Three to five eggs were often encountered in adults, a stage which shows a high level of egg encapsulation. In contrast, superparasitism was rare in L2. Thirty hours after oviposition, the percentage of encapsulation plateaued, indicating a saturation of the host immune system (Fig. 1). When mealybugs were exposed to a second parasitization period, there was no corresponding increase in encapsulation of newly oviposited eggs. This strongly suggests that the immune system is exhausted by high levels of superparasitism. Excessively high levels (i.e. 11–14 eggs per host) of superparasitim completely suppressed the encapsulation response (Fig. 2). Overcoming of host immune system by this strategy is termed the “Multiple Target Hypothesis” (Salt, 1968). Thus, A. kamali is able to overcome egg encapsulation by a combination of two strategies: avoidance of the host immune response by ovipositing in L2 or L3 hosts and saturation of the immune system of older hosts through superparasitism. However, the size of emerging female parasitoids was determined by host size, and the maximum number of eggs a parasitoid could lay in 2 h was correlated with its size (Sagarra, unpublished data). Male eggs were laid in small hosts. Female progeny were rarely obtained from parasitized L2 (Table 1). A. kamali maximizes the fitness of its female progeny by ovipositing in third instars and early adults, but it has to superparasitize these hosts to ensure complete development of its offspring and maximize its own fitness. Oviposition in third instar hosts should be the most evolutionarily stable strategy as it ensures the development of the progeny and the production of numerous females.

It allows the parasitoid to lay fewer eggs per mealybug to ensure the saturation of the immune response and the development of one of the eggs into an adult. This behavioral adaptation suggests that A. kamali may have developed through its co-evolution with the hibiscus mealybug a strategy to counteract its host’s defenses and improve the survival rate of its progeny.

Acknowledgements We thank N. Casanova for technical assistance, the International Institute of Biological Control/Caribbean and Latin America Station (IIBC/CLAS) for their technical support. We thank R. Van Driesche, University of Massachusetts at Amherst, and J.C. Wissocq, Universite´ de Picardie–Jules Verne at Amiens, France, for commenting on an early version of the manuscript. This is contribution no 335/99.06.01R of Horticultural Research and Development Center, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Qc, Canada.

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