Egg Parasitoid Complex of Helicoverpha armigera (Hübner) and Conopomorpha cramrella Snellen in North Sulawesi

Helicoverpa  armigera (Lepidoptera: Noctuidae)  is an important pest in crops in Asia, Europe, Africa and Australia, with an estimated damage of billions of dollars per year (Tay, 2013).  Corncob borer, H. armigera  is still a major pest in corn plants in North Sulawesi. This pest caused 51.9-53.4% decrease in harvest in Sulawesi Island (Karim et al., 2013). Insecticides were inadequate in controlling H. armigera because the larvae are in corn cobs. Egg parasitoids can  be alternative control against this pest. There are nine types of egg parasitoids infecting H. armigera including Trichogrammatoidea armigera, Trichogramma  chilonis and Trichogramma ochaeae (Alba, 1988;  Reddy and Manjunatha, 2000). Trichogramma sp has shown promising results; however, H. armigera parasitoids distribution in North Sulawesi is unknown.

Indonesia is the world’s third-biggest cocoa producers after Ghana and Ivory Coast. However, the production is still low of below 1.0 ton/ha mainly due to cocoa pod borer, Conopomorpha cramerella (Lepidoptera: Gracillariidae) (Anonim, 2016). C. cramerella in cocoa plantations in North Sulawesi causes damage with an intensity of up to 40% (Kandowangko et al., 2015).  The distribution of parasitoids in North Sulawesi must be indentified prior to the initial stage of control.

In Indonesia, egg parasitoids of corn cobs and cocoa pod borer have not been reported. Hawkins et al., (1997) stated that parasitoids were studied and showed good results in pest control compared to predatory insects. Information about egg parasitoids that parasitize pest eggs on crops is still very limited. Meanwhile, agricultural fields often use synthetic pesticides that cause parasitoid extinction. Thus, it is necessary to study the parasitoids of H. armigera and C. cramerella for biological control programs.

Location description
 
The egg parasitoids survey of H. armigera of corn (Zea mays L) and C. cramerella on the cocoa plant (Theobroma cacao L) was carried out in North Sulawesi (Table 1 and 2). The survey was conducted from April to August 2018.




 
H. armigera egg sampling procedure
 
Egg collection from the female flowers of corn was carried out in a straight line along 50 m at 10 points with a 4 m distance. For egg collection, two corn plants with 80-90% of pink female flowers and the age of 45 days after planting (DAP) were taken at every point. Followed at the age of 55 DAP that generally female flowers are brownish. In each location, four plant plots were determined to obtain the eggs of H. armigera. There were two locations in each district and each location sized ± 1.0 ha. In Bolaang, Poigar and Bolaang, the collection of H. armigera  eggs was carried out to the corn plan with and without coconut shade. The study locations belong to the farmer and was not given insecticide.
 
H.armigera egg parasitoid
 
Eggs were put into test tubes (1.5 cm x 15 cm) until they hatch into parasitoids. In each test tube, 3-5 eggs were put and bred for 10 days at morning temperatures 07.00 WITA (central Indonesian time) of 26,71±0,611°C  and during the day 14.00 WITA of 28,05±0,86°C. The observations were made every day to determine changes in parasitized eggs and healthy unhatched eggs. The parasitoid obtained from the eggs were collected for identification.

 

 

 
Rearing parasitoid
 
The rearing of Trichogramma sp. parasitoid and C. cephalonica  in C. cephalonica was done by putting 30 eggs aged 0-24 hours per test tube and simultaneously putting two male individuals and three female individuals of T. armigera parasitoid and only female Trichogramma sp parasitoid. This experiment was repeated three times. Observations were made every day to find out parasitoid reproduction in the laboratory.
 
The use of C. cephalonica eggs as a trap
 
C. cephalonica eggs were irradiated with ultraviolet for 45 minutes to inhibit the development of the embryo not to hatch into larvae until they are bred in the laboratory. C. cepahlonica eggs are glued to a green plastic card (2 cm x 3 cm) to be placed on the cacao plant. Eggs are used as parasitoid traps from 0 to 24 hours.
 
A sampling of C. cramerella eggs and egg traps
 
Survey of C. cramerella egg parasitoids was carried out in two ways, namely taking cocoa fruit with 10 cm long and C. cephalonica egg traps. In each district, one location was determined for collecting cocoa and egg traps at 10 points in a straight line with 3 m distance between points. If the length was insufficient, it was deflected back to the original distance with a distance of one line of cacao tree. Two plots of the cocoa and egg traps were determined in the research location. There were 2-3 cocoas picked per tree. C. cramerella eggs were taken with the help of a binocular microscope. Eggs were bred in the laboratory to obtain parasitoids. The cocoa plant used was 5-6 years old. The parasitoid survey was carried out 4 times at intervals of 2 weeks.

For the egg trap, 25 plastic cards containing 20 eggs of C. cephalonica were put in each location. 2-3 plastic cards were put in each point on the cocoa plant. A plastic card was stuck into the cocoa fruit with 10 cm long. The edge of the card was smeared with lime to avoid ants. After four days, the plastic card was taken back and incubated for 10 days and observed every day to find out the changes in parasitized eggs.

 

 

  
Parasitoid identification and statistical analysis
 
The identification of parasitoids was based on morphological characters using insect identification keys. The parasitism of egg parasitoid was analyzed descriptively and Chi-square test performed by SPSS version 23.
 

H.armigera egg parasitoid
 
H. armigera egg parasitoids consist of Trichogramma sp. and T. armigera. Trichogramma sp. parasitoid is a solitary parasitoid, whereas T. armigera is a solitary and gregarious one. Trichogramma sp. produces female offspring while T. armigera produces male and female ones. Trichogramma parasitoids were not found Kakas, Tomohon Timur and, Motoling. Conversely, T. armigera was found in all locations.

Trichogramma sp. was not parasitized C. cephalonica host eggs, however, the parasitoid T. armigera can parasitize C. cephalonica. Trichogramma sp. parasitoid is included in Thelytokous parthenogenesis. Huigens and Stouthamer (2003) stated that Wolbachia is common in female insects, female parasitoids infected with Wolbachia happen to the thelytoky parasitoid and female offspring. Hoffmann et al., (2001) added that females infected with Wolbachia bacteria produce fewer female offspring than uninfected parasitoids. The population of T. kaykai parasitoids infected with Wolbachia is low (Russel et al., 2018).

The population of Trichogramma sp. was 2-8 individuals in each egg sampling, while more than 10 individuals of T. armigera were taken. T. armigera parasitosoid is a reddish-brown parasite with the dark brown middle coxa. Its thorax is dark brown with a darker abdomen. Its antenna is yellow with the last club-shaped segment, the front wing has a slight trichia and long fringe setae. Its female length is 0.42 mm± 0.02 mm and 0.39 mm±0.02 mm for the male.  Trichogramma sp. yellowish color, head, thorax, yellow antenna and the last segment is club-shaped. The abdomen has a brown transverse strip and a brown abdomen tip. The front wing has many trichia and fringe setae rather long and the length of the female (0.49 mm±0.02 mm). Parasitic eggs turn black, while healthy eggs turn yellowish-white to hatch into larvae. The Chi-square analysis of parasitism for those without coconut palm shade was not significantly different from 45 DAP (÷2 =9.600; p>0,087), but 55 DAP was different (÷2 =15.222; p < 0,009) (Table 3).



Parasitism data provided a significant influence for corn cobs borer since, during the research, severe damage to the cobs had not been found. Parasitoid surveys without shading had a higher tendency of parasitism at 45 DAP. 55 DAP parasitism was low, female flowers might have 40-50 % dry and turned brownish to black. Thus, it might not be attractive for parasitoids to find the eggs. Around the study location, corn plants with red female flowers were found, which easily attracted the parasitoids. Red female flowers in H. armigera  population tends to be higher than brown female flowers.

The highest parasitism without coconut shade in Bolaang (36.46±5.18%). H.armigera population in Bolaang is relatively high compared to other locations. Thus, it is concluded from this experiment that parasitism correlated with the number of host eggs. Population density of the host is an important aspect that influences the high parasitism (Montoya et al., 2000; Jones et al., 2003). Romeis et al., (1999) said that Trichogramma sp. parasitism to H.armigera  eggs depends on the location and number of host eggs. Generalist parasitoid is higher in polyculture plantations compared to monocultures (Menalled et al., 1999). Unlike in Kakas, the number of eggs tends to be high, but the parasitism of T. armigera is not maximal. The laboratory experiment revealed that parasitoid efficiency decreases with a decrease in temperature (Mills and Getz, 1996). Besides egg density, the difference in parasitism is also influenced by microclimate, geographical location and plant cultivation practice. The Results of parasitism indicated that changes in weather factors as a result of climate change would have considerable influence on survival and development of parasitoids (Kuzhandhaivel et al., 2016).

Parasitism for those with coconut palm shade is higher than without those shade. Parasitism with the highest shade is in Bolaang (60.91±2.52%) (Table 4). 



In Bolaang, in addition to coconut plants, banana and mango trees are found in maize plantations that also affect microclimate. Liu et al., (2016) stated that heterogeneous landscapes could effectively increase H. armigera parasitoids in cotton plants. Data obtained suggest that the ecological influence for biological control through conservation is needed for non-corn plants. In Kakas, Tomohon and Motoling, corn was planted without shade, because of the lack of coconut plantations. The Chi-square analysis showed significant differences in parasitism of the 45 DAP (÷2 =20.397; p< 0,000, 55 DAP ÷2 =10.047; p<0,007) (Table 4).

The shade density of coconut palm plants in Bolaang is irregular (3 m x 3 m and 3 m x 4 m), while in Poigar it was 6 m x 6 m, in East Dumoga Timur was 5 m x 5 m and 5 m x 6 m. Shade density in Bolaang is denser, thus affecting the microclimate to be cooler and temperatures to be lower, this has an impact on parasitism being high. Microclimate change causes the temperature of maize polyculture to be lower, while without shade the temperature becomes high. Change in microclimate is shown in tea plants, air temperature of tea plants without a shade is 27°C and with the shade of 24°C, humidity is 48% to 74% (Widayat and Rayati, 2011). Geetha and Balakrishnan (2010) stated that cooler microclimates affect T. chilonis parasitism and better spread.
 
C. cramerella egg parasitoids
 
C. cramerella egg parasitoids are Trichogrammatoidea sp, obtained through C. cramerella and C. cephalonica egg trap. However, there were only two C. cramerella eggs we parasitized from 4 times of cacao sampling. The parasitoid morphology of Trichogrammatoidea sp. on C. cramerella eggs is the same as C. cephalonica egg traps. The parasitoid is brownish yellow, the head is brownish yellow and the abdomen has dark patches, the antenna is with a long tassel and the middle coxa is dark brown. Female antennas are in the form of a club, whereas males are not big, but have long hair. The front wing with fringe setae, long tornus section and little trichia on a remigium. The C. cramerella egg parasites are Trichogrammatoidea bactrae fumata, Trichogrammatoidea cojuangcoi and Trichogramma chiolonis. (Alias et al., 2004). 

Parasitism shows that the population of Trichogrammatoidea sp. in cocoa plantations is relatively low. The effective monitoring of these parasitoids is the C. cephalonica egg traps. In Passi and Poigar, the Trichogrammatoidea sp. parasitoid through C. cramerella eggs have not been found. It was found in Tombariri. C. cramerella ‘s parasitized eggs turn from orange to black (Table 5).



C. cephalonica egg trap is an active trap that uses an agent to attract parasitoids in yellowish-white color. Traps are active with chemical and physical stimuli in the forms of light, color, or chemical compounds making insects are attracted to the trap (Yi et al., 2012). The reported parasitism on the card containing C. cephalonica eggs is very low. Low levels of C. cephalonica eggs parasitized reported by Kandowangko et al., (2015). All parasitized C. cephalonica eggs that are bred can become imago parasitoids. However, the dominant population is male as an indication of low parasitism since the parasitoid that parasitizes the egg is female. Dolichodorus sp, Oecophyla sp., Iridomyrmex sp and spiders which are predators of C. cephalonica eggs were found in cocoa plant ecosystems. Thus, it consequently affected the low parasitism.

C. cephalonica eggs can be used to monitor C. cramerella parasitoids. The cost for C. cephalonica egg traps is relatively cheaper than cocoa taking. Besides that, the parasitized eggs of C. cephalonica are more easily detected. In Malaysia, to obtain information on T. bactrae fumata, yellow and green cylindrical contacts are used. Green is more attractive than yellow (Azhar and Long, 2004). The use of the cylindrical trap method is inappropriate since the trapped parasitoids cannot be bred as they have died.

All authors declare that they have no conflict of interest.