Leptocybe invasa males and the parasitoid Quadrastichus mendeli (Hym.: Eulophidae): First report in ParaguayAbstract
Leptocybe invasa Fisher & La Salle, 2004 (Hymenoptera: Eulophidae) forms galls in tips, young branches, central veins, and petioles of Eucalyptus spp. leaves. This insect reproduces primarily through thelytokous parthenogenesis, with males being rare. The parasitism of L. invasa by Quadrastichus mendeli Kim & La Salle, 2008 (Hymenoptera: Eulophidae), with short life cycle and rapid dispersion, is high. The objective was to record the first occurrence of L. invasa males and its parasitoid Q. mendeli in galls of this pest in Paraguay. Branches of the grancam clone PL 113 (Eucalyptus grandis × Eucalyptus camaldulensis) with L. invasa galls were collected from February to April 2022 in the Maciel District, Caazapá Department, Paraguay. These branches were put in plastic bags, preserved in 70% ethanol and the insects emerged sent to the Laboratory of Biological Control of Forest Pests (LCBPF) at FCA/UNESP in Botucatu, São Paulo state, Brazil. Morphological characteristics confirmed the presence of L. invasa males at a ratio of 38:1 (females to males). Morphological and molecular analysis identified Q. mendeli and its samples deposited in GenBank (OR248672.1). The parasitism rate of L. invasa by Q. mendeli was 26.8%, marking the first record of this occurrence in Paraguay.
Keywords:
ecology; geographic distribution; population dynamics; thelytokous
Resumo
Leptocybe invasa Fisher & La Salle, 2004 (Hymenoptera: Eulophidae) forma galhas nos ponteiros, ramos jovens, nervuras centrais e pecíolos das folhas de Eucalyptus spp. Este inseto se reproduz predominantemente por partenogênese telítoca, sendo os machos raros. O parasitismo de L. invasa por Quadrastichus mendeli Kim & La Salle, 2008 (Hymenoptera: Eulophidae), que apresenta ciclo de vida curto e rápida dispersão, é elevado. O objetivo deste estudo foi registrar a primeira ocorrência de machos de L. invasa e de seu parasitoide Q. mendeli em galhas dessa praga no Paraguai. Ramos do clone grancam PL 113 (Eucalyptus grandis × Eucalyptus camaldulensis), com galhas de L. invasa, foram coletados entre fevereiro e abril de 2022, no Distrito de Maciel, Departamento de Caazapá, Paraguai. Esses ramos foram acondicionados em sacos plásticos, preservados em etanol a 70% e os insetos emergidos foram enviados ao Laboratório de Controle Biológico de Pragas Florestais (LCBPF), da FCA/UNESP, em Botucatu, estado de São Paulo, Brasil. As características morfológicas confirmaram a presença de machos de L. invasa em uma proporção de 38:1 (fêmeas: machos). Análises morfológicas e moleculares identificaram Q. mendeli, com amostras depositadas no GenBank (OR248672.1). A taxa de parasitismo de L. invasa por Q. mendeli foi de 26,8%, constituindo o primeiro registro dessa ocorrência no Paraguai.
Palavras-Chave:
ecologia; distribuição geográfica; dinâmica populacional; telítoca
1. Introduction
Galls on branches, tips, petioles, and central veins characterize damage by Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae) on Eucalyptus spp. branches (Huang et al. 2018). Larvae of this insect develop inside and enlarge these galls until the emergence of its adults (Mendel et al., 2004; Costa et al., 2008). This pest was first recorded in Paraguay in 2012, damaging clones of Eucalyptus camaldulensis and Eucalyptus grandis (Diaz et al., 2014).
Leptocybe invasa typically reproduces through thelytokous parthenogenesis and rarely sexually (Zheng et al., 2018), with males being scarce, a phenomenon that warrants further study (Liu et al., 2023). This may help to understand the genetic variability of the insect and its implications for biological control.
The body length of L. invasa females ranges from 1.1 to 1.4 mm, with the forecoxae, legs, and tarsi yellow, and the middle and hind coxae brown with a metallic blue to green sheen. The head and body are brown with a metallic blue to green sheen. The wings of females are hyaline with light brown veins, and the antennae with four anelli, three funicular segments and three club segments. The ovipositor with short sheaths, and the gaster is short and oval (Mendel et al., 2004).
The body length of L. invasa males ranges from 0.8 to 1.2 mm, with similar characteristics to its females, except for the head and mesosoma, brown with a metallic blue to green sheen. The metasoma brown with a slight metallic coloration dorsally, the legs light yellow, and the middle and hind coxae brown with a metallic sheen. The wings are hyaline with yellow veins, the antennae with a pedicel, a filiform flagellum with long spiral setae, and genitalia with a digitus with a single claw (Doğanlar, 2005).
Biological control, with the parasitoids Selitrichodes neseri Kelly & La Salle (Hymenoptera: Eulophidae) (Masson et al., 2017) and Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae) (Kim et al., 2008; Nugnes et al., 2016; Sangtongpraow & Charernsom, 2019), is the main strategy to manage L. invasa in eucalyptus plantations.
The parasitoid Q. mendeli, with length of 1.15 to 1.35 mm, rapid geographic distribution, high parasitism rates, and short life cycle, excels in managing L. invasa (Kim et al., 2008; Sangtongpraow & Charernsom, 2019; Puretz et al., 2022). This natural enemy detects volatiles emitted by galls on plants using its antennae to find its host (Huang et al., 2023). The antennae of this insect are light brown with its body predominantly yellow with dark brown markings in various parts. Its head with distinct sutures and grooves, and the antennae with three funicular segments and a large ring one. The fore wings with one seta on marginal vein. Gaster slightly longer than the head plus mesosoma, and the ovipositor slightly protruding, with a short sheath visible in dorsal view (Kim et al., 2008).
Quadrastichus mendeli was reported in Argentina, Brazil, and Mexico (Aquino et al., 2018; Luna-Cruz et al., 2020; Puretz et al., 2022) and effectively controlled L. invasa in Italy and Thailand (Nugnes et al., 2016; Sangtongpraow & Charernsom, 2019).
The identification of L. invasa males and the parasitoid Q. mendeli broadens the prospects for integrated management of this pest in Paraguay. The objective of this study is to report, for the first time, L. invasa males and Q. mendeli parasitizing this insect in galls of this pest in Paraguay.
2. Methods
2.1. Collecting L. invasa males and the parasitoid Q. mendeli
Branches 60 cm long and with L. invasa galls were collected from on plants of the grancam clone PL 113 (E. grandis × E. camaldulensis) from February to April 2022 in the Maciel District, Caazapá Department, Paraguay (26°13'12.9” S, 56°28'44.1” W). These branches were individually labeled and identified (collection date, clone, location) in plastic bags and kept at room temperature. The insects emerged were collected, preserved in 70% ethanol, and sent to the Laboratory of Biological Control of Forest Pests (LCBPF) at the Faculdade de Ciências Agronômicas, Universidade Estadual Paulista (FCA/UNESP) in Botucatu, São Paulo state, Brazil. The insect samples were cataloged at the FCA museum.
2.2. Identification of L. invasa males and Q. mendeli
Males of L. invasa and its parasitoid Q. mendeli were identified by analysing morphological features such as length and coloration of head, antennae, mesosoma, wings, and gaster of these insects (Mendel et al., 2004; Doğanlar, 2005).
DNA from 50 Q. mendeli individuals was extracted using 80 μL of 10% Chelex100® resin solution [Bio-Rad Laboratories, USA] and 8 μL of proteinase K (20 mg/ml) for molecular identification. The 28S gene was denatured through a PCR cycle with an initial denaturation step at 94 °C for 2 minutes; 38 cycles of 94 °C for 30 seconds, annealing at 58 °C for 50 seconds, and extension at 72 °C for 1 minute and 30 seconds; and a final extension at 72 °C for 10 minutes. The samples underwent electrophoresis in a 1% agarose gel and were analyzed under ultraviolet light, and amplified in a thermal cycler [Infinigen, TC-96CG, USA] using the primers 28SF3633 (5’-TACCGTGAGGGAAAGTTGAAA −3′) and 28SR4076 (5′-AGACTCCTTGTCCGTGTTT −3′) (Tiawsirisup et al., 2008). The PCR reaction products were purified with magnetic beads and sent to IBTEC at UNESP/Botucatu for Sanger sequencing. The result of this sequencing was deposited in GenBank under the accession code OR248672.1.
2.3. Parasitism of L. invasa by Q. mendeli
The parasitism rate of L. invasa by Q. mendeli on eucalyptus branches in Paraguay was determined by dividing the total number of parasitoids by that of insects emerged (parasitoid + host) (Kim et al., 2008; Nugnes et al., 2016).
3. Results
3.1. Leptocybe invasa males
Sensilla on the antenna and gaster confirmed the identification of the 10 L. invasa males (Figure 1) collected, with a ratio of 38 females to each male.
Antenna (A), gaster (B), and body (C) of male Leptocybe invasa (Hymenoptera: Eulophidae) emerged from galls of this pest collected in the field from eucalyptus plants. Due to possible morphological distortion after specimen drying, segment proportions in (A) may not precisely reflect those described by Doğanlar (2005).
3.2. Quadrastichus mendeli parasitising L. invasa
Quadrastichus mendeli was identified based on the morphological characteristics of adults emerged from the galls (Figure 2), including the length and coloration of head, antennae, mesosoma, wings, and gaster. This identification was confirmed by sequencing the 28S gene of the DNA of this insect.
Adults of Quadrastichus mendeli (Hymenoptera: Eulophidae) (A and B) emerged from Leptocybe invasa (Hymenoptera: Eulophidae) galls collected in the field.
Quadrastichus mendeli parasitized 26.8% of L. invasa larvae in their galls in the field, with no other parasitoid observed.
4. Discussion
4.1. Leptocybe invasa males
The detection of males in eucalyptus plantations infested by L. invasa could increase the genetic variability of this pest, which predominantly reproduces through thelytokous parthenogenesis, with males being rare (Doğanlar, 2005; Zheng et al., 2018). This could be an adaptive response of L. invasa to factors such as edaphoclimatic conditions and resistance of different Eucalyptus species and clones (Liu et al., 2023), highlighting the importance of monitoring and tracking the detection of males to understand the impact of their occurrence on the ecology and management of L. invasa in eucalyptus plantations (Zhang et al., 2021).
4.2. Quadrastichus mendeli parasitising L. invasa
The identification of Q. mendeli based on the morphological characteristics of its adults is similar to that reported for this parasitoid in Israel (Kim et al., 2008). The natural biological control of L. invasa by the parasitoid Q. mendeli in Paraguay is important, as it has been reported with high efficiency against this pest after being introduced in China, Italy, and Tanzania (Garonna et al., 2018; Lazaro et al., 2023; Li & Li, 2023). Quadrastichus mendeli, with a longer lifespan and shorter life cycle than its host, parasitizes young and mature larvae of L. invasa (Sangtongpraow & Charernsom, 2019). These characteristics help to reduce pest populations in the field and, consequently, decrease their damage (Nugnes et al., 2016).
The parasitism rate of 26.8% of L. invasa larvae in their galls in the field by Q. mendeli is lower than the rates reported in Rome and San Giorgio a Cremano, Italy, and Gallipoli, Turkey of 30.2%, 38.0%, and 50.5%, respectively, and higher than that in China, from 2.96% to 19.53% (Nugnes et al., 2016; Huang et al., 2018). However, the high search efficiency and rapid dispersal of Q. mendeli facilitate the increase of its populations, as reported in India, Israel and South Africa (Kim et al., 2008; Huang et al., 2018). The parasitism of L. invasa larvae in eucalyptus galls by Q. mendeli is crucial for the biological control of this pest in Paraguay.
5. Conclusion
This is the first report of L. invasa males and of its parasitoid Q. mendeli in Paraguay. The detection of L. invasa males is relevant because it suggests possible sexual reproduction in local populations, which may affect the pest’s adaptability and population dynamics. The presence of the parasitoid Q. mendeli in Paraguay is promising for biological control programs targeting L. invasa.
Acknowledgements
To “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES- Finance Code 001), “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” and “Programa Cooperativo sobre Proteção Florestal (PROTEF) do Instituto de Pesquisas e Estudos Florestais (IPEF)” for financial support.
Data Availability Statement
The entire data set that supports the results of this study was published in the article itself.
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Editor:
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