Intraguild predation of Phenacoccus manihoti parasitoid Anagyrus lopezi by two mealybug predators, Cryptolaemus montrouzieri and Plesiochrysa ramburi

Anagyrus lopezi is a specialist parasitoid was introduced to Indonesia in 2014 to control cassava mealybug, Phenacoccus manihoti. Plesiochrysa ramburi and Cryptolaemus montrouzieri are generalist predator insects associated with the mealybug. Intraguild predation is a crucial competition in an insect community. The study objective was to investigate interference of both predators on healthy mealybug (unparasitized) or parasitized mealybug by A. lopezi (mealybug which containing parasitoid larvae 1-, 3-, 8-, and 14-day old). Experiments were carried out in a non-choice test (served only one unparasitized or parasitized mealybug of a specific parasitoid age) and choice test (unparasitized and parasitized mealybug of a specific parasitoid age together) in laboratory under controlled environmental conditions. Both of predators species able to fed on both unparasitized and parasitized mealybugs of three different age (1-, 3-, and 8- day-old), but both predators discriminated against mealybug were parasitized 14-day old (hardened mummies). Interaction between parasitoid of A. lopezi and both predators showed antagonistic interaction type. We recommended A. lopezi should be released in field at the beginning of the time when the population of the mealybug is still low and could be continued with the predators release when the mealybug population is high.


Introduction
Intraguild predation (IGP) termed as a predation activity on more than one species of natural enemies that use the same resources as their host or prey.IGP is occurred symmetrically type when an animal fed on another one or asymmetry type when a species sustainably fed on the other [1].Thus, IGP may have a negative effect on biological control program.In contrast, the other studies show that the incidence have an effect on pest control system [2][3][4].Use of several predators can compete with each other, or even be as supportive as to suppress pest populations in integrated pest management [5].Some natural enemies can be used for biological control collectively [6].However, several studies confirm that the use of some natural enemies for biological control have a negative impact with each other's in an agro-ecosystem [7].The ability of complex natural enemies to limit pest populations depends on interaction type among themselves.It is possible that some predators can interact synergistically to increase suppression of pest populations together or only additive effects on pest populations [8].However, some predator species can interact with antagonistic effect where they consume each other and giving influence to agro-ecosystem [4,7,8].
The cassava mealybug, Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae) is a global important cassava pest that invaded many countries in the world including Indonesia [9][10][11][12][13][14][15][16][17].Parasitoid Anagyrus lopezi (De Santis) (Hymenoptera: Encyrtidae) has been reported to be a successfully biological agent to control this pest [2,14,18].Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) is one of the most generalist predators that used as mealybug control agents [19].Uraichuen et al. [17] and Wardani [13] report that Plesiochrisa ramburi (Schneider) (Neuroptera: Chrysopidae) is a natural enemy of P. manihoti which found in cassava plants.These predatory insects were released in the cassava field to control mealybug [20].P. ramburi and C. montrouzieri were found to be able to coexist in agricultural crops that have the same food resources [21].Therefore, multitrophic interaction among A. lopezi, C. montrouzieri, and P. ramburi become the interesting study to be investigated.Study aim was to determine the use combination of the two main predators and a parasitoid A. lopezi to control cassava mealybug, P. manihoti is synergistic, antagonistic, or neutral type.

Preparation of host plants, mealybug, parasitoid and predator insects
Cassava stem cuttings were collected from farmers' cassava plantations in Sukaraja, West Java.Cassava cuttings were inserted in an upright position into plastic containers (d = 8 cm, h = 11.5 cm) with 200 ml of fresh water.The top of the container was covered with steroform which has been perforated according to the diameter of cassava cuttings.Cassava cuttings were kept in cages (l = 75 cm, w = 50 cm, h = 50 cm).Talinum triangulare (Jaqc.) was used as an alternative host plant for cassava melaybug.T triangulare was planted in mini pots (d = 8 cm, h = 11.5 cm).Plants that have developed with perfect leaves of eight leaves were used as experimental plant.
P. manihoti was collected from the Laboratory of Insect Bionomy and Ecology, Bogor Agricultural University.The first instar mealybug P. manihoti were transferred onto cassava plants until three weeks old days.The leaves attacked by P. manihoti were cut into pieces and infested in cassava plants that were still healthy, so that the nymphs of mealybug as soon as possible to move and multiply in other cassava plants.Two weeks later, the mealybugs were already for the mass rearing of the A. lopezi and as materials for experiments [22].
A total number of 15 cassava plants were infested ± 300 third instar nymphs P. manihoti, then put into culture cage (l = 75 cm, w = 50 cm, h = 50 cm).Furthermore, approximately 30 pairs of A. lopezi adults were put into cages.After two weeks, there were many mummies of P. manihoti on dried leaves.Mummies were collected in gelatin capsules and left until the parasitoid adults appeared for mass rearing and experiments.Mummies from A. lopezi were incubated in gelatin capsules at 25+1 °C until parasitoid emergence then use for experiments [22].
Eggs, larvae, pupae or predator adult C. montrouzieri and P. ramburi were collected from cassava fields.Specimens were taken in Bionomy and Insect Laboratory, Bogor Agricultural University.Both predators were propagated separately and maintained in insects cages (d = 30 cm, h = 20 cm) then various instars Paracoccus marginatus (as an alternative prey for predators) were provided on sprouted potato, and after one month, a number of predator larvae and adults were already to be used for experiments.Predator adults were given artificial diet with ratio of honey, yeast and water 1: 1: 1 [23].Maintenance of plants, insects and all experimental sections were done in ambient conditions 25 ± 1 o C, 90 ± 5% RH and photoperiod of 12:12 L: D.

Experimental procedure
The healthy mealybug P. manihoti were prepared by allowing 1-2 days of female A. lopezi on 5 third instar nymphs with a ratio of parasitoid and mealybug (1: 5).Provision of parasitized mealybug were carried out into a Petri dish (d = 100 mm, h = 15 mm) for 24 hours.The parasitized mealybug was immediately transferred into another petri dish on a fresh leaf of T. triangulare.Mealybugs were incubated in the laboratory with controlled environmental conditions at 25 ± 1 o C, 90 ± 5% R.H and photoperiod 12 h L:12 h D. The parasitized mealybugs were left to survive until parasitoid larvae through different ages (-1, -3, -8, and -14 day-old).Parasitoid remained as egg at one day after parasitism, and then developed into young instar larvae on the third day, and then the final instar-larvae progressed to pupa in eighth day.Mummies of mealybug were completely shaped and after the 12th day old hardened with host's cuticles and the adult parasitoid was almost emerge in 14th day old.The healthy and parasitized mealybug harboring 1-and 3-day old parasitoid larvae were mobile actively.The dead mealybug harboring 8-and 14-day old parasitoid larvae were placed on the leaves in the same position as those used in healthy mealybug.Healthy mealybugs were directly collected from mealybug massrearing cage.
No choice tests.Healthy or unparasitized mealybug (as control) and parasitized mealybug at different ages, 1-, 3-, 8-, and 14-day-old were exposed separately on the second instar and third instar larvae P. ramburi, second instar and fourth instar larvae C. montrouzieri.Before testing, predatory larvae were starved for 24 hours.A total number of 30 third instar nymphs P. manihoti were collected using a smooth brush and transferred gently on a fresh leaf of T. triangulare into a petri dish (d = 8.5 cm, h = 1.5 cm).Furthermore, a larval predator on each instar was removed from each petri dish and left to look for and consume prey which available for 24 hours.After a 24-hour period, predatory larvae were removed and the number of healthy mealybug and surviving parasitoid was recorded.
Choice tests.Preferences of predatory larvae instar-2, -3 of P. ramburi and instar-2, -4 C. montrouzieri were chosen and given the option to consume between healthy or parasitized mealybug.The parasitoid nymphs were given a black mark (0.8 mm, snowman black marker, Japan) on the abdomen to distinguish them from healthy or unparasitized mealybugs.Predator larvae was allowed to choose preys between 15 healthy or 15 parasitized mealybugs that containing parasitoid from one of the development stages A. lopezi (1-, 4-, 8-, and 14 days old).
All experiment was carried out at a temperature of 25+1 o C, 90+5% R.H., and photoperiod of 12 h L: 12 h D. After a 24-hours period, predatory larvae were removed and the number of healthy and parasitized mealybug were recorded.Each experiment was repeated 20 times.
Statistical data analysis.For all data of no-choice and choice experiments were analysed by twoproportion t-test using Minitab software version 15 (Minitab Inc., State College, PA, USA).For nochoice tests, each binary group was analyzed separately and evaluated all together and them given letters in the tables or figure.

Result and discussion
The results of the no choice test showed that the 2nd instar larvae of P. ramburi consumed mostly on non-parasitized mealybugs (F19.79=15.1369,P=0.0001; figure 1a).Meanwhile, the 3rd instar larvae of P. ramburi consumed more on the unparasitized mealybug (F19.79=7.324,P=0.0001; figure 1b).The 2nd and 3rd instar larvae of P. ramburi were able to discriminate between the mummified mealybugs containing parasitoid pupae (aged 14-days after parasitization).Both instar-2 and instar-3 P. ramburi fed on mummified mealybugs with lowest predation.The difference in the level of predation ability of various types of predators of different species against parasitized mealybugs might be related to the differences in prey's morphology, changes in physical and chemical characteristics of parasitized mealybug [24].The preference of predators on to different parasitoid immature ages might be depend on physiological conditions and morphological changes associated with parasitoid larval developmental status [3].
Case study by Mustu and Kilincer [25] mentioned that N. kreissli fed on 6-day-old vine mealybugs parasitized by L. dactylopii but not occurred on the same mealybug parasitized by A. pseudococci.The choice test confirmed that there was discrimination against the mummified mealybug by the 2nd and 3rd instar larvae of P. ramburi (table 1).The 2nd and 3rd instar larvae of P. ramburi showed almost the same predation levels for both parasitized and unparasitized mealybugs (containing parasitoid larvae from 1-, 3-, and 8-days-old) with predation levels ranging from 10.85-13.90mealybugs.In the 14-dayold combination of unparasitized and mummified mealybugs containing parasitoid larvae, the 2nd instar larvae of P. ramburi showed significantly different levels of predation (t19.79=-21.19,P=0.0001).In general, present study found that the 2nd instar larvae of P. ramburi consumed on 11.90 unparasitized mealybugs and only 0.10 mummified mealybugs were available.
Predation of 3rd instar larvae of P. ramburi showed no significant difference to unparasitized and parasitized mealybugs at 1, 3, and 8 days of age with predation rates ranging between 11.30 and 13.95 mealybugs.However, the 3rd instar larvae of P. ramburi showed significant differences in unparasitized compared to parasitized mealybugs at 14 days (t19.79 =-24.43,P=0.0001).The 3rd instar larvae P. ramburi consumed on 13.90 unparasitized mealybugs.This was clearly far more than the mummies which only consumed on 0.15 mealybugs.In contrast to Coccinella septempunctata L. which continued to prey on both when exposed to mummified or unparasitized mealybugs [27].
Mustu and Kilincer [25] showed that N. kreissli fed on 6-day old vine mealybugs parasitised by L. dactylopii but not on vine mealybugs parasitised by A. pseudococci.The reason is the mealybugs parasitized by L. dactylopii do not completely harden at six days of parasitism.The onset of the mummification of P. ficus parasitized by A. pseudococci is roughly one day shorter than the onset of the mummification of P. ficus parasitized by L. dactylopii.Şengonca and Yanuwiadi [28] reported that C. montrouzieri feeding on 2-and 4-day-old parasitised by L. dactylopii, had the same rates with unparasitized mealybugs, but the prey consumption rates of the predator decreased in 6-day-old parasitised mealybugs compared with those which were unparasitized.In another study, it was reported that C. montrouzieri indiscriminately fed on 1-, 4-and 7-day-old P. citri parasitised by L. dactylopii [26].

Conclusion
Parasitism level of parasitoid A. lopezi was significantly affected by both predator of Plesiochrysa ramburi and Cryptolaemus montrouzieri.Interference of predators showed antagonistic interaction type and disrupted to the performance of parasitoid A. lopezi.Both predators discriminated between mummies and unparasitized mealybug.Implementation of both predators was recommended only when the mealybug population P. manihoti is high in the field or after mummification of mealybug containing A. lopezi larvae is fully hardened about 14 days after release.

Figure 1 .
Figure 1.Consumption of unparasitized mealybug (control) and parasitized mealybug by instar-2 larvae (left) and instar-3 larvae (right) P. ramburi in a 24 h using choice test.Within bar chart means followed by the different letter shows the significantly different statistically (P < 0.05).

Figure 2 .
Figure 2. Consumption of unparasitized mealybug (control) and parasitized mealybug by instar-2 larvae (left) and instar-4 larvae (right) C. montrouzieri in a 24 h using choice test.Within bar chart means followed by the different letter shows the significantly different statistically (P < 0.05).

Table 1 .
Consumption preference of unparasitized dan parasitized mealybug by different stage of two predators in a 24 h choice test.
*Within table means followed by the different letter shows the significantly different statistically (P < 0.05).