versão impressa ISSN 1519-566X
Neotrop. Entomol. v.33 n.4 Londrina jul./ago. 2004
Methodology of mass multiplication of Telenomus podisi Ash. and Trissolcus basalis (Woll.) (Hymenoptera: Scelionidae) on eggs of Euschistus heros (Fab.) (Hemiptera: Pentatomidae)
Metodologia de multiplicação massal de Telenomus podisi Ash. e Trissolcus basalis (Woll.) (Hymenoptera: Scelionidae) em ovos de Euschistus heros (Fab.) (Hemiptera: Pentatomidae)
Wilsimar A.A. PeresI; Beatriz S. Corrêa-FerreiraII
IDepto. Zoologia, Universidade Federal do Paraná, C. postal 19020, 81531-990, Curitiba, PR
IICentro Nacional de Pesquisa de Soja, Embrapa, C. postal 231, 86001-970, Londrina, PR
Laboratory studies were carried out to develop a methodology for the mass production of Telenomus podisi Ash. and Trissolcus basalis (Woll.) on eggs of Euschistus heros (Fabr.) and to compare it with the methodology presently used in the biological control program of soybean stink bugs in Brazil [T. basalis reared on Nezara viridula (L.) eggs]. In a first experiment, egg masses randomly distributed were shown the most adequate method of exposure of E. heros to parasitism. In a second experiment, egg masses of E. heros were randomly glued on cardboard cards and exposed to parasitism by T. podisi and T. basalis. The parasitoids multiplied on E. heros eggs showed parasitism (ca. 99%) similar to that observed with T. basalis reared on N. viridula eggs (99.7%). Emergence rates were higher than 80% for T. podisi and T. basalis multiplied on E. heros eggs and 95% for T. basalis reared on N. viridula eggs. Later, similar emergence rates (> 78%) were observed on cardboard cards tied on soybean plants. No differences were detected between courtship behavior of T. podisi and T. basalis emerged from E. heros eggs compared to T. basalis emerged from N. viridula eggs. The sex ratios in the offspring of T. podisi and T. basalis emerged from E. heros eggs were similar to that exhibited by the offspring of T. basalis emerged from N. viridula eggs. The results suggest that the developed methodology is simple, efficient and can be an important tool in biological control program of soybean stink bugs.
Key words: Insecta, Glycine max, egg parasitoid, mass rearing, brown stink bug
Estudos de laboratório foram conduzidos com o objetivo de desenvolver uma metodologia de multiplicação massal de Telenomus podisi Ash. e Trissolcus basalis (Woll.) em ovos de Euschistus heros (Fab.) e compará-la à metodologia utilizada no programa de controle biológico dos percevejos-pragas da soja no Brasil [T. basalis criado em ovos de Nezara viridula (L.)]. Em um primeiro experimento, foi determinado que massas de ovos aleatoriamente distribuídas foi o método mais adequado de exposição dos ovos de E. heros ao parasitismo. Em um segundo experimento, massas de ovos de E. heros foram aleatoriamente coladas em cartelas de papelão e expostas a T. podisi e T. basalis. Os parasitóides multiplicados nos ovos de E. heros apresentaram índices de parasitismo (cerca de 99%) similares ao observado para T. basalis multiplicado em ovos de N. viridula (99,7%). As taxas de emergência foram superiores a 80% para ambos parasitóides em ovos de E. heros e superior a 95% para T. basalis em ovos de N. viridula. Posteriormente, índices de emergência similares (> 78%) foram observados nas cartelas de papelão amarradas nas plantas de soja. O comportamento de cópula dos parasitóides não variou. A razão sexual da geração subsequente de T. podisi e T. basalis gerados em ovos de E. heros foi igual à de T. basalis criado em ovos de N. viridula. Os resultados deste estudo indicam que a metodologia desenvolvida é simples, eficiente e pode tornar-se importante ferramenta no programa de controle biológico dos percevejos da soja.
Palavras-chave: Insecta, Glycine max, parasitóide de ovos, criação massal, percevejo marrom
The seed suckers (Hemiptera: Pentatomidae) are the most important group of insect pests of soybean in Brazil. Nezara viridula (L.) (southern green stink bug), Euschistus heros (Fab.) (neotropical brown stink bug) and Piezodorus guildinii (West.) (small stink bug) are the more frequent species which constitute the stink bug complex. These insects cause substantial reduction in soybean yields and seed quality (Panizzi & Slansky 1985).
Chemical insecticides are the most usual control method, but its massive use increases production costs and usually results in reduction of beneficial insects, pests resurgence and leads to other environmental problems (Panizzi et al. 1977, Shepard et al. 1977). Among alternative control methods, biological control agents are potentially very useful in soybean integrated pest management programs (Orr 1988).
In Brazil, Trissolcus basalis (Woll.) was found parasitizing stink bug eggs for the first time in 1979 (Corrêa-Ferreira 1980, 1986). Nowadays, T. basalis is the most important agent used in biological control programs for soybean stink bugs (Corrêa-Ferreira 1993). The mass release of T. basalis to control the sink bug complex has been developed in large and continuos river basin areas. In Paraná State, a mean of 300,000 T. basalis adults are multiplied and released in approximately 18,020 hectares of soybean per year (Corrêa-Ferreira et al. 2000).
Eggs of N. viridula were used as hosts to multiplicate T. basalis. To obtain the host-eggs, adults of southern green stink bug are collected in the field, taken to the laboratory and maintained under controlled conditions (Corrêa-Ferreira 1985). However, changes in the dynamics of stink bug population and their parasitoids over the years (Panizzi & Corrêa-Ferreira 1997) have brought increasing difficulties in collecting N. viridula in the field, especially in low latitude regions (Cividanes & Parra 1994). As a consequence, the expansion of the biological control program for soybean stink bugs has been limited.
Presently, the brown stink bug E. heros is one of the most abundant stink bug in soybean fields in Brazil (Panizzi 1997, Costa et al. 1998). This specie can be easily reared under laboratory conditions and high rates of survivorship and fecundity can be obtained (Peres & Corrêa-Ferreira 2001). Additionally this specie is the preferential host of the important egg parasitoid Telenomus podisi Ash. (Corrêa-Ferreira 1986) and can be used as an alternative host for the multiplication of T. basalis. This study was conducted to develop a methodology for the mass multiplication of T. podisi and T. basalis on eggs of E. heros.
Material and Methods
The experiments were carried out under controlled environmental conditions (25 ± 2ºC, 65 ± 10% and 14 h L:10 h D). Two-day old T. podisi and T. basalis females previously copulated, fed on honey and without previous oviposition experience, were used in the experiments. Individualized colonies of N. viridula and E. heros were kept in a climatized room as a source of eggs, following the methodology described by Corrêa-Ferreira (1985). The eggs were collected daily and stored in the refrigerator under the temperature of 5ºC.
Methods of Exposure of E. heros Eggs to Parasitism. A preliminary test was conducted to determine the most appropriate method to expose E. heros eggs to parasitism. Approximately 40 eggs of E. heros and N. viridula, stored at 5ºC for up to 12 days were glued on a cardboard strip (3 x 1.5 cm). The cardboard strips were inserted in glass tubes (8 x 2.5 cm) containing four parasitoid females.
The stink bug eggs were exposed to parasitism in different ways: (1) E. heros egg masses with the operculum turned to the upside position (correct position); (2) E. heros egg masses with the operculum randomly turned upside, lateral side and upside down; (3) E. heros eggs separated and distributed randomly; and (4) N. viridula egg masses in correct position and only offered to T. basalis females (control).
After a parasitism period of 6h the cardboard strips were transferred to plastic petri dishes (9 x 1.5 cm) lined with moistened filter paper and kept under controlled conditions until adult emergence and parasitoid death. Those eggs that remained intact for several days after the emergence period were dissected to check their contents. Total parasitism was defined as the sum of eggs from which parasitoids emerged and parasitized eggs that did not result in parasitoid emergence.
The percentage of parasitism, adult emergence and sex ratio (number of females relatively to males and females produced) were assessed. A completely randomized design was used with four treatments, two insect species and 10 replicates.
E. heros Egg Masses Randomly Positioned on Cardboard Cards. Based on the results of a preliminary test, stink bug egg masses stored at 5ºC for up to 20 days were glued in random position on a cardboard (8 x 4 cm), resulting in eggs distributed upside, lateral side and upside down positions. The cardboard, containing ca. 1200 E. heros or N. viridula eggs, was exposed to 40 parasitoid females for 12h in celluloid tubes (20 cm x 5 cm diameter). A completely randomized design was used with three treatments and five replicates: (1) T. podisi or (2) T. basalis parasitizing E. heros egg masses randomly located; (3) T. basalis parasitizing N. viridula egg masses with the operculum turned to the upside position (control).
After parasitism, the procedure was the same as described in the previous experiment. The percentage of parasitism, adult emergence and sex ratio (number of females relatively to males and females produced) were assessed.
Validation of Egg Parasitoids Multiplication Methodology on E. heros Egg Masses. Approximately 1200 eggs of E. heros and N. viridula, stored at 5ºC for up to 20 days (5ºC) were randomly glued on pieces of cardboard (8 x 4 cm) and offered to 40 parasitoid females for 24h. Three treatments were set: (1) T. podisi or (2) T. basalis parasitizing E. heros egg masses randomly positioned; (3) T. basalis parasitizing N. viridula egg masses with the operculum turned to the upside position (control). After parasitism, the procedure was the same as described in the previous experiment. A day before emergence of the adult parasitoids, the cardboards were wrapped in nylon netting and tied on the middle third of soybean plants (with immature pods) cultivated in pots and kept inside the laboratory. When emergence appeared complete, the cardboards were removed and the emerged parasitoids were counted.
To determine fertility of progeny produced, 50 females of T. podisi and T. basalis reared on E. heros eggs and 50 females of T. basalis reared on N. viridula eggs (control) were placed individually in glass tubes (8 x 2.5 cm). Honey was streaked on the inside walls of the tubes to provide food source. Two days after emergence, 20 E. heros eggs were offered to a female parasitoid for 6h. After this period the eggs were placed in petri dishes with adequate moisture and kept at laboratory environmental conditions. Emerging parasitoids were sexed.
Data Analysis. Data on parasitism, adult emergence and sex ratio, were subjected to analysis of variance. If the analysis of variance was significant at the P < 0.05% level, Tukey´s test was applied.
Results and Discussion
Methods of Exposure of E. heros Eggs to Parasitism. Egg masses of E. heros glued in upside position or random position on cardboard strips presented parasitism by T. podisi and T. basalis higher than 97.0%, similar to the control (99.7%) (Table 1). These results corroborate the high parasitism (99.7%) found by Corrêa-Ferreira & Moscardi (1994) when N. viridula eggs were exposed to T. basalis. Eggs of E. heros separated and glued in random position which were offered to T. podisi and T. basalis showed parasitism of 96.1% and 99.1% respectively. The parasitism by T. podisi in eggs separated and randomly exposed (96.1%) was similar to the parasitism found by other methods studied, but was significantly lower than in the control treatment (99.7%). The number of E. heros eggs separated and glued in random position and parasitized by T. basalis (99.1%) was statistically equal to the other treatments.
T. podisi parasitized egg masses in correct or random position on the cardboard resulted in 83.1% and 79.9% emergence rates, respectively. These treatments did not differ from the T. basalis emergence in N. viridula (control), which had the highest emergence rate (92.5%). However, emergence of T. basalis in E. heros egg masses in correct or random position (50.6% and 63.2%, respectively) was significantly lower than the control (92.5%), showing that the eggs of E. heros are moderately adequate for development of T. basalis (Table 1).
Eggs separated and parasitized in random position by T. podisi and T. basalis had the lowest emergence rates, 51.3% and 26.1%, respectively (Table 1). The separated eggs probably showed more water loss than egg masses (Capinera et al. 1981) revealing that single eggs are more susceptible to desiccation, which make them non-viable for development of the immature phases of the parasitoids. It is suggested that the single eggs naturally laid should not be used as hosts for multiplication of the parasitoids. These results agree with those of Dass & Parshad (1982), that observed significant reduction in the rates of parasitism and emergence of Telenomus remus Nixon in separated eggs of Spodoptera litura (Fabr.).
The average sex ratio in the progeny of T. podisi and T. basalis ranged from 0.7 to 0.8, suggesting that the change of host and the form of gluing the eggs on the cards did not interfere on this parameter (Table 1). Similar sex ratio was also observed in T. podisi emerged from Podisus maculiventris (Say) and E. heros eggs (Orr & Boethel 1990, Pacheco & Corrêa-Ferreira 1998) and T. basalis emerged from N. viridula eggs (Corrêa-Ferreira & Moscardi 1994).
Both T. podisi and T. basalis were capable of parasitizing egg masses of E. heros distributed at random, independently if egg masses were fixed upside, on lateral sides or upside down. The high acceptability of E. heros egg masses randomly exposed may be attributed to the size and shape of the egg masses. Usually, the egg masses of the brown stink bug, on average, seven eggs (Villas Bôas & Panizzi 1980), are arranged in small groups, so nearly all the eggs are located at the edge of the mass. This facilitates the insertion of the ovipositor in the lateral egg wall, at the tip of the operculum or even in the chorion joints of two neighboring eggs (Sales et al. 1978). Such arrangement of eggs in the mass also enables and favors the emergence of parasitoids from the eggs.
Thus in the biological control program for soybean stink bugs, E. heros egg masses randomly distributed can be successfully used as hosts for mass multiplication of T. podisi and T. basalis in the laboratory.
E. heros Egg Masses Randomly Positioned on Cardboard Cards. An average (± SEM) of 161.9 ± 6.33 E. heros egg masses were randomly glued per cardboard, corresponding to a total of 1196.8 ± 27.50 eggs exposed to parasitism. Of these, a mean (± SEM) of 437.1 ± 15.52 (36.6%), 457.1 ± 22.29 (38.0%) and 302.6 ± 9.92 (25.4%) eggs were positioned upside, on the lateral sides and upside down, respectively. In the control, a mean of 20.1 ± 0.41 N. viridula egg masses were glued upside on each cardboard, corresponding to a total of 1365.9 ± 61.3 eggs, which were offered to T. basalis females.
Examination of E. heros eggs showed that about 28.7% (342.8 ± 30.6) of those eggs had lateral deformations. These deformed eggs were not suitable for parasitoid larval development and, therefore, they were excluded from calculation of the adult emergence.
The E. heros eggs were parasitized by T. podisi and T. basalis at the rate of 99.1% and 99.2%, respectively. These values were statistically equal between each other or the control (99.7%) (Table 2).
In general, adult emergence rates were close to 80% (Table 2), which is the value accepted by the soybean stink bug biological control program, using egg parasitoids. Sex ratio of T. podisi developed in E. heros eggs (0.8) did not differ from the sex ratio found for T. basalis developed in N. viridula eggs (control). However, T. basalis reared in E. heros eggs showed a significant reduction in its sex ratio, compared to the other treatments (Table 2). This reduction is probably due to the oviposition sequence followed by T. basalis females, and to the smaller size of E. heros egg masses compared to those of N. viridula. As females of T. basalis lay proportionally more male eggs early in the oviposition sequence (Strand 1988, Colazza & Wajnberg 1998), the decrease in the number of eggs offered resulted in a higher proportion of males in the offspring (Strand 1988, Weber et al. 1996, Colazza & Wajnberg 1998).
Validation of Egg Parasitoids Multiplication Methodology on E. heros Egg Masses. Parasitism of 98.0% for T. podisi and 99.7% for T. basalis were found in the validation study on E. heros eggs. These values were statistically equal between each other and to the 99.8% parasitism by T. basalis in N. viridula eggs (control) (Table 3).
T. podisi and T. basalis emerged successfully from egg masses of E. heros randomly distributed on cardboard cards tied on soybean plants. The emergence rates were higher than 78% (Table 3) and close to the emergence rate of 80%, which is considered acceptable in parasitoid multiplication for field releases (Corrêa Ferreira 2002) Similarly, as in the previous study, a mean of 26.3% eggs offered had lateral deformation, and were non-viable for parasitoid multiplication and, therefore, were not considered in the emergence rate calculation.
No differences were detected in courtship behavior of T. podisi and T. basalis emerged from egg masses of E. heros, comparatively to T. basalis emerged from egg masses of N. viridula. Males were the first parasitoids to emerge from stink bug eggs. They remained on the egg masses waiting for female emergence to copulate, which occurred immediately after female emergence, a behavior already described to Telenomus heliothidis Ash. (Strand 1988) and T. basalis (Colazza & Wajnberg 1998). After mating, the females remained on the soybean plant leaves, flying to search for hosts and start the new life cycle.
The average (±SEM) sex ratios in the offsprings of T. podisi (0.8 ± 0.02) and T. basalis (0.9 ± 0.01) emerged from E. heros eggs were similar to that exhibited by the offspring of T. basalis emerged from N. viridula eggs (0.9 ± 0.02). Of the fifty female samples, only one T. podisi female, (2%) which multiplied on E. heros eggs, had its subsequent generation composed only of males, indicating no copulation after emergence.
The results indicate that the methodology developed allows easy and efficient multiplication of a great number of parasitoids to be released in the field and can be very useful in biological control programs of soybean stink bugs. The lateral deformation observed on E. heros egg is probably related to the storage temperature used (5ºC) which may not have completely blocked eggs metabolism reducing host suitability for the parasitoids (Gautam 1987). Further researches on the cause of lateral deformation and on the storage techniques of E. heros eggs are necessary to improve production of the egg parasitoids.
We thank Dr. F. Moscardi, Dr. C.B. Hoffmann-Campo and Dr. D.R. Sosa-Gomez for their contributions on this manuscript. This study was sponsored by the Universidade Federal do Paraná (UFPR) and Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Centro Nacional de Pesquisa de Soja. W.A.A. Peres was supported by a scholarship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Received 13/05/04. Accepted 15/06/04.