Blue gum chalcid, Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae), causes galling damage to Eucalyptus species (Myrtaceae) in various regions of the world, but has been controlled effectively by its primary parasitoid, Selitrichodes neseri Kelly & La Salle (Hymenoptera: Eulophidae). The objectives of this study were to evaluate the recovery of S. neseri after its initial release on Eucalyptus plants in Brazil and to provide data on its biology. Selitrichodes neseri was imported from South Africa to Brazil for the biological control of L. invasa, in Mar 2015, and recovered from Aug 2015 to Dec 2016. Successful recovery of this parasitoid shows its potential to become established in the field. Seedlings of 2 hybrids obtained from crosses between rose gum Eucalyptus grandis W. Hill. ex Maiden and flooded gum Eucalyptus urophylla S. T. Blake and between Eucalyptus sp. and (river red gum Eucalyptus camaldulensis Dehnh. × E. grandis) showed potential as hosts for culture of S. neseri on L. invasa in the laboratory because up until the adult parasitoid emergence these seedlings did not wilt. When reared at 25.1 to 26.0 °C, the total number of parasitoids and the proportion of male parasitoids were highest, relative to hosts reared at 26.1 to 27.0 °C. Peak emergence of S. neseri occurred 28 d after parasitism.
Mated Selitrichodes neseri Kelly & La Salle (Hymenoptera: Eulophidae: Tetrastichinae) females parasitize mature larvae and newly formed pupae in galls caused by the blue gum chalcid, Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae: Tetrastichinae), in eucalyptus plants, Eucalyptus (Myrtaceae) (Kelly et al. 2012). Selitrichodes neseri was collected in Australia, where it is native, in 2010 and reared in quarantine in South Africa, where it has been released for the biological control of L. invasa (Kelly et al. 2012). In South Africa, S. neseri is maintained by the Tree Protection Co-operative Programme of the Forestry and Agricultural Biotechnology Institute at the University of Pretoria in partnership with the Biological Control of Eucalypt Pests program.
Galls caused by L. invasa and bearing larvae parasitized by S. neseri were sent on 26 Mar 2015 to the “Laboratório de Quarentena Costa Lima” of the “Embrapa Meio Ambiente” in Jaguariúna, São Paulo State, Brazil, where they remained in quarantine to prevent the introduction of other exotic agents, including bacteria, fungi, mites, and other insects, into Brazil. Selitrichodes neseri was imported according to protocols of the Brazilian “Ministério da Agricultura, Pecuária e Abastecimento”, as part of the “Projeto Cooperativo de Manejo de Pragas Exóticas do Eucalipto” of the “Programa Cooperativo em Proteção Florestal” at the “Instituto de Pesquisas e Estudos Florestais.” Emerged parasitoids were sent to the “Laboratório de Controle Biológico de Pragas Florestais” of the “Universidade Estadual Paulista Júlio de Mesquita Filho” in Botucatu, São Paulo State, Brazil, where they were raised on L. invasa galls in seedlings of a cross between rose gum Eucalyptus grandis W. Hill. ex Maiden (Myrtaceae) and river red gum Eucalyptus camaldulensis Dehnh. (Myrtaceae), clone 3025, at 26 ± 2 °C, 60 ± 10% RH, and a 12:12 h L:D photoperiod.
The population dynamics of natural predators often mirrors that of their prey (Bjorksten et al. 2005; Ferreira-Filho et al. 2008). Thus, these biological agents have the greatest potential for successful biological control when released in the field at the beginning of pest outbreaks (Huber et al. 2006). Selitrichodes neseri is a specialist L. invasa parasitoid and therefore does not attack other insect species (Dittrich-Schröder et al. 2014). Mass rearing of S. neseri is important for L. invasa biological control programs, and knowledge of this parasitoid's biology could assist with large-scale production and release in the field (Kelly et al. 2012). The objectives of this study were to evaluate the recovery and establishment of S. neseri in L. invasa galls after its initial release on Eucalyptus plants in Brazil and to provide data on its biology.
Materials and Methods
RECEIVING AND MAINTAINING SELITRICHODES NESERI ADULTS IN THE LABORATORY
Sixty S. neseri adults (41 females and 19 males) were brought to the “Laboratório de Proteção Florestal” of the “Bahia Specialty Cellulose/Copener Florestal Ltda.” in Alagoinhas, Bahia State, Brazil, on 1 Jul 2015. We placed 21 females and 7 males of the 60 parasitoids in woodframed cages sealed with screen on the sides and with glass on the top (45 cm length × 50 cm width × 80 cm height). Three Eucalyptus seedlings (hybrid obtained between E. grandis and Eucalyptus uropylla S. T. Blake) with L. invasa galls were placed in each cage, and pure African bee (Apis mellifera L.; Hymenoptera: Apidae) honey (principal flora: eucalyptus) was deposited inside on the top glass wall of the cages as a nutrient source to aid in parasitoid reproduction.
RELEASING SELITRICHODES NESERI ADULTS IN THE FIELD
The remaining 20 female and 12 male adults were released on 1 Jul 2015 in a 100 m2 area open to the environment with plants from an E. camaldulensis × E. grandis cross. The insects were placed in 50 mL acrylic containers with a screened lid and transported to the field where they were released at 5 equidistant points at 5 m intervals (2 male and 4 female adults per container released at 3 points, and 3 males and 4 females per container at 2 points). Insects were observed mating in the containers during transportation prior release. The containers were opened near the branches infested with galls, and the insects were released. The plants were approximately 4 m tall, 3 yr old, and highly susceptible to L. invasa galls. The release site was located in Rio Real, Bahia State, Brazil (Fig. 1) (12.37°N, 38.88°W; 280 m altitude, and 800 mm average annual rainfall). Plants were spaced 4.00 m apart within rows and rows were spaced 2.25 m apart in a total stand of 74.18 ha. Leptocybe invasa has been present at this location since 2012.
RECOVERY OF SELITRICHODES NESERI EMERGING FROM LEPTOCYBE INVASA GALLS COLLECTED IN THE FIELD
Recovery of S. neseri was evaluated monthly from Aug 2015 to Dec 2016, with 6 branches (approximately 50 cm length) of a eucalyptus tree infested by L. invasa galls bagged individually within an organza fabric bag (20 cm width × 40 cm length). Trees within the stand where S. neseri were released showed a high incidence of galling and drying of the upper third of the tree canopy due to the high level of L. invasa infestation. Pure honey was placed on the edge of the bags as food for the collected insects (S. neseri and L. invasa). The numbers of S. neseri and L. invasa individuals inside these bags were evaluated visually 2 d after being installed in the field without removal of the branches.
ADEQUACY OF EUCALYPTUS HYBRIDS FOR CULTURE OF SELITRICHODES NESERI IN THE LABORATORY, AND NOTES ON S. NESERI BIOLOGY
Three S. neseri females and 1 male were placed in each woodframed cage sealed with screen on the sides and with glass on the top (45 cm length × 50 cm width × 80 cm height) with 3 potted eucalyptus seedlings infested by L. invasa galls. Potted eucalyptus seedlings with galls exposed to parasitoids were placed in other cages after 2 d of exposure to parasitoids until the emergence of parasitoids or gall wasps. The same 3 female and 1 male parasitoids received 3 other potted eucalyptus seedlings (same hybrid) with L. invasa galls for parasitism for an additional 2 d. One assay with the hybrid E. grandis × E. uropylla and a second assay with the hybrid Eucalyptus sp. × (E. camaldulensis × E. grandis) separated by cages were used per tested temperature.
Drying out and mortality of eucalyptus seedlings were evaluated daily for each hybrid through visual observation and counting the number of emerged insects (S. neseri and L. invasa). The generation time from parasitism to emergence and the adult longevity of S. neseri were also evaluated. The emergence of adults and rate of parasitism were evaluated in separate rooms at 24.0 to 25.0, 25.1 to 26.0, 26.1 to 27.0, 27.1 to 28.0, 28.1 to 29.0, and 29.1 to 30 °C with a constant RH and photoperiod of 60 ± 10% and 12:12 h L:D, respectively, in the laboratory. The sex ratio of the emerged parasitoids, number of males and females (offspring) emerging per female, emergence peak after parasitism, and characteristics of the seedlings and eucalyptus galls (approximate age and coloring) were recorded to evaluate parasitism. The results presented are the average of the first and second 2 d intervals of parasitism on the 2 hybrids.
FIRST RELEASE OF SELITRICHODES NESERI IN BRAZIL AND NOTES ON ITS LIFE CYCLE
Bahia Specialty Cellulose/Copener Florestal Ltda. was the first company to receive and release S. neseri in the field in Brazil. This parasitoid was recovered from monthly samples taken from Aug 2015 to Dec 2016. When sampled 171 d after the release of 32 S. neseri adults, 2 females and 1 male of this parasitoid were recovered from the field from the 6 organza bags where S. neseri were released. Although numbers were low, this finding shows that the parasitoid was able to reproduce in the field.
GENERATION TIME, LONGEVITY, AND SEX RATIO OF SELITRICHODES NESERI ADULTS IN THE LABORATORY
Depending on the temperature, the generation time of this insect varied from 12 to 31 d in the laboratory, which suggests the insects collected were probably from the 6th or 7th field generation. The longevity of the emerged S. neseri adults varied from 8 to 15 d evaluated at temperatures ranging between 24 and 30 °C in the laboratory. At 29.1 to 30 °C, adult longevity was less than 3 d. The sex ratio of the emerged S. neseri adults was 37:63 (F:M) evaluated at temperatures ranging between 24 and 30 °C in the laboratory (Table 1).
NUMBERS OF MALE AND FEMALE SELITRICHODES NESERI ADULTS EMERGING IN THE LABORATORY
Total numbers of S. neseri males and females emerging were 155 and 94, with males and females per cage ranging from 6 to 37 and from 15 to 25, respectively, when 3 females and 1 male had been introduced per cage. A colony of S. neseri could be initiated by inoculating a cage with 3 females and 1 male for 2 d, with seedlings of susceptible hybrids and galls containing L. invasa larvae and pre-pupae. The total number of S. neseri emerging was highest at 25.1 to 26.0 °C and 60 ± 10% RH, with 73 males and 22 females, and the highest number of males emerging at this temperature range. The highest number of emerged females was observed at 26.1 to 27.0 °C, with 33 insects. A male-biased sex ratio (23:77, F:M) was obtained at 25.1 to 26.0 °C, and a female-biased sex ratio (80:20, F:M) at 29.1 to 30.0 °C (Table 1). Peak emergence occurred at 28 d, with 52 parasitoids emerging.
Sex ratio (F:M) of Selitrichodes neseri (Hymenoptera: Eulophidae) emerging under different temperature regimes (°C).
ADEQUACY OF EUCALYPTUS HYBRIDS FOR CULTURE OF SELITRICHODES NESERI
Leptocybe invasa galls on eucalyptus plants were initially green, becoming reddish at about 80 d. Mature larvae and newly formed pupae of this insect were parasitized in galls with green to red colors. Newly formed and green galls showed no sign of parasitoid emergence. Eucalyptus seedlings with early wilting of apical shoots were not suitable as hosts for rearing S. neseri because the L. invasa galls dried out and consequently prevented the development of the gall wasp larvae and the emergence of adult parasitoids.
The hybrids E. grandis × E. urophylla and Eucalyptus sp. × (E. camaldulensis × E. grandis) both were highly susceptible to galls in the nursery and field. When infested, they displayed early wilt and drying of apical tissues when the roots were not wetted, and late drying even when kept watered. The E. grandis × E. urophylla hybrid had 3 or more pairs of leaves per branch, whereas Eucalyptus sp. × (E. camaldulensis × E. grandis) generally had only 2 pairs per branch. Leaves with galls were green during early stages of L. invasa development (L. invasa egg and larval stages) and then turned reddish (beginning of the L. invasa pupal stage). Leptocybe invasa galls occurred on the petiole and midrib of leaves.
Selitrichodes neseri adults mated in cages in this study, but also in 50 mL glass or acrylic tubes with pure honey and a screened lid (F. de A. L., personal observation), allowing the introduction of mated females into the cages for parasitism. The process of mating S. neseri in small containers is disadvantageous and may not be necessary because this insect could mate quickly in larger cages avoiding the loss of 1 d of the females' life.
In this study, the S. neseri generation time evaluated at temperatures ranging between 24 and 30 °C in the laboratory in Brazil was close to that reported previously from 2 South African laboratories: Kelly et al. (2012) reported 12 to 31 d and Dittrich-Schröder et al. (2014) reported approximately 19 d required for S. neseri to complete its life cycle at 26 °C.
In this study, S. neseri adult longevity and male sex ratio in the laboratory in Brazil were lower than those for this parasitoid in a South African laboratory, where adult longevity and sex ratio for this insect were 26 d and 67:33 (M:F), respectively, at 26 °C (Dittrich-Schröder et al. 2014). The number of S. neseri adults emerging in our study was similar to that seen in a South African laboratory, where a single female produced 39 offspring, with a maximum emergence of 10 individuals per d at 26 °C (Dittrich-Schröder et al. 2014).
Parasitoid reproduction in the laboratory in our study was adequate with both hybrids, but the rate of reproduction was higher with the E. grandis × E. urophylla hybrid. In a Brazilian laboratory, Eucalyptus hybrids were previously tested, and their suitability as hosts for Thaumastocoris peregrinus Carpintero and Dellape (Hemiptera: Thaumastocoridae: Thaumastocorinae) rearing showed that E. grandis and E. urophylla were the most suitable candidates for the development and reproduction of this insect. Other suitable hosts included E. camaldulensis, E. grandis, E. urophylla, clone 1277 (hybrid E. grandis × E. camaldulensis), clone VM-1 (hybrid E. urophylla × E. camaldulensis), and clone H-13 (hybrid E. urophylla × E. grandis) (Soliman et al. 2012).
In summary, this is the first study demonstrating successful release and establishment of the parasitoid S. neseri in a Eucalyptus plantation in Brazil. Seedlings of the E. grandis × E. urophylla and Eucalyptus sp. × (E. camaldulensis × E. grandis) hybrids have potential as hosts for rearing S. neseri in the laboratory. These plants did not wilt or show drying of upper parts and therefore favored the development of L. invasa and its parasitoid. The total number of parasitoids emerging per cage was highest when cultured at 25.1 to 26.0 °C and 60 ± 10% RH. However, the number of females emerging was highest when cultured at 26.1 to 27.0 °C. The female sex ratio for S. neseri was highest at 29.1 to 30.0 °C, with 80:20 (F:M). Peak emergence occurred 28 d after parasitism.
The authors would like to thank Jacyr M. Alves for the logistical support with equipment, information, and resources; John La Salle for confirming the parasitoid species and Michael J. Wingfield for sending the parasitized galls to Brazil; Dilmara Carvalho, Evany Velozo, Selma Valadares, Deivide Pereira, and William Matos for supporting the rearing of the parasitoid in the laboratory and the releasing in the field; Luis R. Junqueira for helping in the release of the parasitoid in the field. This study was funded by the following Brazilian institutions: “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),” “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),” “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),” and “Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)”. Phillip J. Villani revised and corrected the English language used in an early version of this manuscript.