Lepidopteran Pests Research Articles

Variation in parasitoid adult size is related to host egg size, maternal state and developmental time

AbstractBody size is a characteristic of many animal species that is correlated with many fitness‐related traits. Parasitoids are important biological control agents of pests; larger parasitoids often have greater fitness and enhanced performance. Host size is a major influence on parasitoid size, which has been explored in the important egg parasitoid Anastatus disparis (Hymenoptera: Eupelmidae), a potential biological control agent for several Lepidopteran pests. Here, we found that the body size of A. disparis females increased with increasing host egg size, while host egg size did not significantly affect male size. In addition, both female and male offspring sizes significantly increased with increasing maternal size, and female offspring produced by younger mothers were also larger. Furthermore, the body sizes of both females and males appeared to increase when the total development time from egg to adult eclosion decreased. Male offspring produced by larger females have shorter development times, while female offspring produced by older females have longer development times. Consequently, our results suggested that the effect of maternal status on offspring size may be mediated by influencing development time. In summary, our results indicated that the body size of A. disparis is determined by multiple factors with differing effects on females and males, and our results provide guidance for its effective mass rearing and its performance in the field.

Decoding nature's defense dance: Mechanistic insights into biochemical and metabolic shifts in Cajanus cajan and Cajanus platycarpus during combat with the lepidopteran pest Helicoverpa armigera provide evidence for non-host plant immunity

This study provides intriguing mechanistic insights into the innate immunity of the pigeonpea wild relative, Cajanus platycarpus, in comparison to the cultivated Cajanus cajan, based on biochemical and metabolic changes during Helicoverpa armigera infestation. Over a 96 h herbivory challenge, both species exhibited 164 metabolites, with 54 undergoing significant alterations. In C. platycarpus, primary carbohydrates such as sucrose, glucose, galactose, and mannose decreased, while structural carbohydrates like xylose, talose, and tagatose accumulated, indicating cell wall damage in C. cajan. Notably, C. platycarpus down-regulated most amino acids except for phenylalanine, which is crucial for the synthesis of defense compounds. The linoleic/linolenic acid pathway, which produces jasmonic acid, was exclusively identified in C. platycarpus, highlighting active defense. As herbivory continued, levels of galactinol, mannitol, and pinitol significantly increased in C. platycarpus, while sorbitol and xylitol levels rose in C. cajan. Essentially, C. platycarpus reduced nutrient availability to insects, enhanced specific defense hormones and pathways, and maintained reactive oxygen species (ROS) equilibrium. These findings offer significant potential for understanding the biology of the wild relative and developing innovative strategies for herbivore control and food sustainability.

Transgenic early japonica rice: Integration and expression characterization of stem borer resistance Bt gene

BackgroundTransgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines. MethodsThe early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages. ResultsStable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues. ConclusionsSynthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops.

CRISPR/Cas9-mediated Nap knockout affects female reproduction and egg shape in Bombyx mori.

Insect reproductive capacity can affect effective pest control and infertility studies and has become an important focus in recent molecular genetic research. Nucleosome assembly protein (Nap) is highly conserved across multiple species and is involved in forming the sperm nucleus in many species. We used clustered regularly interspaced palindromic repeats/Cas9 technology to knockout BmNap in Bombyx mori and observed that the mutations caused female infertility, whereas male fertility was not affected. BmNap mutants grew and mated normally; however, female mutants laid smaller eggs that could not be fertilised and did not hatch. In addition, female sterility produced by the mutation could be inherited stably via male mutants; therefore, Nap could be used as a potential target for lepidopteran pest control through population regulation. In the current study, we elucidated a new function of BmNap, increased the understanding of the oogenesis regulation network in Lepidoptera and promoted the development of insect sterility technologies.

Technological methods for protecting apple trees from dominant lepidopteran pests

Technological methods for protecting apple trees from dominant lepidopteran pests

Host plants expressing pxy-miR-34-5p demonstrate high efficacy against multiple lepidopterous pests

Host plants expressing pxy-miR-34-5p demonstrate high efficacy against multiple lepidopterous pests

A comparative approach for life history and functional response demonstrates similar survival strategies for Trichogramma evanescens and T. pintoi.

Egg parasitoids are important biological control agents of lepidopteran pests of agricultural crops. Trichogramma evanescens Westwood and T. pintoi Voegele (Hymenoptera: Trichogrammatidae) are egg parasitoids with worldwide importance. The parasitoid selection necessitates comparative assessment of the life table traits and functional response analysis to provide insights into their effectiveness in pest control. In this study, we examined theirlife table traits including survivorship and reproductivity, and functional response and associated parameters i.e., attack coefficient and handling time. Life table parameters, using age-stage, two-sex theory, revealed similar survival and reproductive strategies for both species. For example, the female longevity, oviposition days and fecundity did not differ between both species. Exceptionally, the male longevity of T. evanescens was shorter than that of T. pintoi. The population growth parameters such as gross reproductive rate (GRR), net reproductive rate (R0), intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T) did not differ between species. The polynomial logistic regression yielded a type III functional response and a non-linear least square analysis revealed different attack coefficient and similar handling time. However, their parasitism rate differed between the lowest (five eggs) and highest (80 eggs) initial host egg densities such that T. evanescens had a lower parasitism rate at the lowest density and higher parasitism rate at the highest density. The similarity in survival strategies and minor differences in host handling of both parasitoids are discussed in terms of relevance to applied biological control applications and evolutionary traits. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

Transcriptomic and proteomic elucidation of Z chromosome dosage compensation in Helicoverpa armigera.

Transcriptomic data have been used to study sex chromosome dosage compensation (SCDC) in approximately 10 Lepidoptera ZW species, yielding a consensus compensation pattern of Z It remains unclear whether this compensation pattern holds when examining more Lepidoptera ZW species and/or using proteomic data to analyse SCDC. Here we combined transcriptomic and proteomic data as well as transcriptional level of six individual Z genes to reveal the SCDC pattern in Helicoverpa armigera, a polyphagous lepidopteran pest of economic importance. Transcriptomic analysis showed that the Z chromosome expression of H. armigera was balanced between male and female but substantially reduced relative to autosome expression, exhibiting an SCDC pattern of Z . When using H. amigera midgut proteomic data, the SCDC pattern of this species changed from Z at transcriptomic level to Z = ZZ = AA at the proteomic level. RT-qPCR analysis of transcript abundance of six Z genes found that compensation for each Z gene could vary from no compensation to overcompensation, depending on the individual genes and tissues tested. These results demonstrate for the first time the existence of a translational compensation mechanism, which is operating in addition to a translational mechanism, such as has been reported in other lepidopteran species. And the transcriptional compensation mechanism functions to accomplish Z chromosome dosage balance between the sexes (M = F on the Z chromosome), whereas the translation compensation mechanism operates to achieve dosage compensation between Z chromosome and autosome (Z = AA).

High nitrogen application in maize enhances insecticide tolerance of the polyphagous herbivore Spodoptera litura by induction of detoxification enzymes and intensification of cuticle

Nitrogen (N) is one of the most intensively used fertilizers in cropping system and could exert a variety of bottom-up effects on the ecological fitness of herbivores. However, the effects of increased N inputs on insect pesticide tolerance have not been comprehensively understood. Bioassays showed that high N (HN) applied to maize plants significantly increased larval tolerance of Spodoptera litura to multiple insecticides. Activities of detoxification enzymes were significantly higher in the larvae fed on maize plants supplied with HN. RNA-seq analysis showed that numerous GST and cuticle-related genes were induced in the larvae fed on HN maize. RT-qPCR analysis further confirmed four GST genes and larval-specific cuticle gene LCP167. Furthermore, when injected with dsRNA specific to GSTe1, GSTs5, and LCP167, the mortality of larvae treated with methomyl was about 3-fold higher than that of dsGFP-injected larvae. Electron microscope observation showed that cuticle of the larvae fed on HN maize was thicker than the medium level of N. These findings suggest that increased application of N fertilizer enhances insecticide tolerance of lepidopteran pests via induction of detoxification enzymes and intensification of cuticle. Thus, overuse of N fertilizer may increase pest insecticide tolerance and usage of chemical insecticides.

Whole transcriptome analysis identifies differentially expressed mRNA, miRNA and lncRNA associated with male sterility in the silkworm, Bombyx mori

Insect sterility technology is gradually being applied to the control of lepidoptera pests, and the target gene for male sterility is the core of this technology. JMS is a mutant silkworm that exhibits male sterility, and to elucidate its formation mechanism, this study conducted a full transcriptome analysis of the testes of JMS and its wild-type silkworms 48 h after pupation, identifying 205 DElncRNAs, 913 mRNAs, and 92 DEmiRNAs. The KEGG pathway enrichment analysis of the DEmRNAs revealed that they were involved in the biosynthesis of amino acids and ECM-receptor interactions. Combined with ceRNA regulatory network KEGG analysis suggests that pathways from amino acid biosynthesis to hydrolytic processes of protein synthesis may play a crucial role in the formation of JMS mutant variants. Our study deepens our understanding of the regulatory network of male sterility genes in silkworms; it also provides a new perspective for insect sterility technology.

Diversity of Insect Pest on Various Crops from Sakoli Tahshil, Bhandara, Maharashtra

The field survey in Sakoli Tahshil, Bhandara District, Maharashtra State, during 2023-24 discovered 41 insect pests from diverse crops. Lepidoptera was the dominant order, with 18 species, followed by Hemiptera, Orthoptera, Coleoptera, and Thysanoptera, which had 17, 2, and 1 species, respectively. Hemipteran and Lepidopteran insect pests cause significant harm to all crops. The current study provides valuable information on many insect pests and their associated insects in agriculture fields.

Plant characterization of insect-protected soybean

Insect-protected soybean (SIP) that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal proteins has been developed to provide protection from feeding damage caused by targeted lepidopteran insect pests. Typically, as part of environmental risk assessment (ERA), plant characterization is conducted, and the data submitted to regulatory agencies prior to commercialization of genetically modified (GM) crops. The objectives of this research were to: (a) compare soybean with and without the SIP trait in plant characterization field trials designed to fulfill requirements for submissions to global regulatory agencies and address China-specific considerations and (b) compare risk assessment conclusions across regions and the methodologies used in the field trials. The soybean with and without the SIP trait in temperate, tropical, and subtropical germplasm were planted in replicated multi-location trials in the USA (in 2012 and 2018) and Brazil (in 2013/2014 and 2017/2018). Agronomic, phenotypic, plant competitiveness, and survival characteristics were assessed for soybean entries with and without the SIP trait. Regardless of genetic background, growing region, season, or testing methodology, the risk assessment conclusions were the same: the evaluated insect-protected soybean did not differ from conventional soybean in evaluated agronomic, phenotypic, competitiveness, and survival characteristics indicating no change in plant pest/weed potential. These results reinforce the concept of data transportability across global regions, different seasons, germplasm, and methodologies that should be considered when assessing environmental risks of GM crops.

Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region.Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage.cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls.Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.

Genome-wide identification and phylogenetic analysis of the tetraspanin gene family in lepidopteran insects and expression profiling analysis in Helicoverpa armigera.

The tetraspanin gene family encodes cell-surface proteins that span the membrane 4 times and play critical roles in a wide range of biological processes across numerous organisms. Recent findings highlight the involvement of a tetraspanin of the lepidopteran pest Helicoverpa armigera in resistance to Bacillus thuringiensis Cry insecticidal proteins, which are extensively used in transgenic crops. Thus, a better understanding of lepidopteran tetraspanins is urgently needed. In the current study, genome scanning in 10 lepidopteran species identified a total of 283 sequences encoding potential tetraspanins. Based on conserved cysteine patterns in the large extracellular loop and their phylogenetic relationships, these tetraspanins were classified into 8 subfamilies (TspA to TspH). Six ancestral introns were identified within lepidopteran tetraspanin genes. Tetraspanins in TspA, TspB, TspC, and TspD subfamilies exhibit highly similar gene organization, while tetraspanins in the remaining 4 subfamilies exhibited variation in intron loss and/or gain during evolution. Analysis of chromosomal distribution revealed a lepidopteran-specific cluster of 10 to 11 tetraspanins, likely formed by tandem duplication events. Selective pressure analysis indicated negative selection across all orthologous groups, with ω values ranging between 0.004 and 0.362. However, positive selection was identified at 18 sites within TspB5, TspC5, TspE3, and TspF10. Furthermore, spatiotemporal expression analysis of H. armigera tetraspanins demonstrated variable expression levels across different developmental stages and tissues, suggesting diverse functions of tetraspanin members in this globally important insect pest. Our findings establish a solid foundation for subsequent functional investigations of tetraspanins in lepidopteran species.

BpAFP, a Broussonetia papyrifera latex chitinase, exhibits a dual role in resisting to both Verticillium wilt disease and lepidopterous pests, Plutella xylostella and Prodenia litura

Paper mulberry (Broussonetia papyrifera) is a fast-growing tree known for its tolerance to diverse biotic and abiotic stresses. To explore genes combating Verticillium wilt, a devasting and formidable disease damage to cotton and many economically significant crops, we purified an antifungal protein, named BpAFP, from the latex of paper mulberry. Based on peptide fingerprint, we cloned the full cDNA sequence of BpAFP and revealed that BpAFP belongs to Class I chitinases, sharing 74 % identity with B. papyrifera leaf chitinase, PMAPII. We further introduced BpAFP into Arabidopsis, tobacco, and cotton. Transgenic plants exhibited significant resistance to Verticillium wilt. Importantly, BpAFP also demonstrated insecticidal activity against herbivorous pests, Plutella xylostella, and Prodenia litura, when feeding the larvae with transgenic leaves. Our finding unveils a dual role of BpAFP in conferring resistance to both plant diseases and lepidopterous pests.

Lethal and Sublethal Effects of Cyantraniliprole on the Biology and Metabolic Enzyme Activities of Two Lepidopteran Pests, Spodoptera littoralis and Agrotis ipsilon, and A Generalist Predator, Chrysoperla carnea (Neuroptera: Chrysopidae).

Cyantraniliprole is a novel anthranilic diamide insecticide registered for controlling chewing and sucking insect pests. Here, the lethal and sublethal effects of this insecticide on two destructive lepidopteran pests, Spodoptera littoralis Boisduval and Agrotis ipsilon Hufnagel, were evaluated. Because the effects of novel insecticides on beneficial and non-target arthropods must be considered, the impact of cyantraniliprole on a generalist biological control agent, Chrysoperla carnea [Stephens 1836], were also examined. Overall, our study revealed that cyantraniliprole was more toxic to A. ipsilon than to S. littoralis. Moreover, the LC15 and LC50 of the insecticide significantly prolonged the duration of the larval and pupal stages and induced enzymatic detoxification activity in both species. Treatment of the second-instar larvae of C. carnea with the recommended concentration of cyantraniliprole (0.75 mg/L) doubled the mortality rates and resulted in a slight negative effect on the biology and detoxification enzymes of C. carnea. Our results indicate that both sublethal and lethal concentrations of cyantraniliprole can successfully suppress S. littoralis and A. ipsilon populations. They also suggest that C. carnea, as a generalist predator, is compatible with cyantraniliprole under the modelled realistic field conditions. In future investigations, insights into the effects of cyantraniliprole on S. littoralis, A. ipsilon, and C. carnea under field conditions will be required to appropriately validate our results.

Comparative analysis of testicular fusion in Spodoptera litura (cutworm) and Bombyx mori (silkworm): Histological and transcriptomic insights

Spodoptera litura commonly known as the cutworm, is among the most destructive lepidopteran pests affecting over 120 plants species. The powerful destructive nature of this lepidopteran is attributable to its high reproductive capacity. The testicular fusion that occurs during metamorphosis from larvae to pupa in S.litura positively influences the reproductive success of the offspring. In contrast, Bombyx mori, the silkworm, retains separate testes throughout its life and does not undergo this fusion process. Microscopic examination reveals that during testicular fusion in S.litura, the peritoneal sheath becomes thinner and more translucent, whereas in B.mori, the analogous region thickens. The outer basement membrane in S.litura exhibits fractures, discontinuity, and uneven thickness accompanied by a significant presence of cellular secretions, large cell size, increased vesicles, liquid droplets, and a proliferation of rough endoplasmic reticulum and mitochondria. In contrast, the testicular peritoneal sheath of B.mori at comparable developmental stage exhibits minimal change. Comparative transcriptomic analysis of the testicular peritoneal sheath reveals a substantial difference in gene expression between the two species. The disparity in differential expressed genes (DEGs) is linked to an enrichment of numerous transcription factors, intracellular signaling pathways involving Ca2+ and GTPase, as well as intracellular protein transport and signaling pathways. Meanwhile, structural proteins including actin, chitin-binding proteins, membrane protein fractions, cell adhesion, extracellular matrix proteins are predominantly identified. Moreover, the study highlights the enrichment of endopeptidases, serine proteases, proteolytic enzymes and matrix metalloproteins, which may play a role in the degradation of the outer membrane. Five transcription factors-Slforkhead, Slproline, Slcyclic, Slsilk, and SlD-ETS were identified, and their expression pattern were confirmed by qRT-PCR. they are candidates for participating in the regulation of testicular fusion. Our findings underscore significant morphological and trancriptomic variation in the testicular peritoneal sheath of S.litura compared to the silkworm, with substantial changes at the transcriptomic level coinciding with testicular fusion. The research provides valuable clues for understanding the complex mechanisms underlying this unique phenomenon in insects.

A new species of Diolcogaster (Hymenoptera: Braconidae) from Brazil of potential use in the biological control of the pest Hypercompe brasiliensis (Stoll) (Lepidoptera: Erebidae).

Species of Diolcogaster parasitize Lepidoptera pests of commercial plants. The diversity of this genus is high, but few species of Diolcogaster have been described. The description of a new Diolcogaster species provides information for the biological control using this insect. This study presents the description and key notes on the biology of a new Diolcogaster parasitoid wasp. This species was reared from a caterpillar of Hypercompe brasiliensis collected after feeding on a Gloxinia perennis plant important to floriculture. Two complementary identification analyzes were performed on Diolcogaster adult bodies. The first was the analyses of its external morphology and the second its molecular analysis (mitochondrial DNA). The morphological analysis defined the insect as a new species of Diolcogaster, named Diolcogaster joanesi sp. nov. A maximum-likelihood (ML) analysis partially confirmed the morphological analysis, placing D. joanesi within a cluster including a previously identified species (Diolcogaster choi) and seven other morphospecies. The proximity of D. joanesi to D. choi is discussed and an updated key for all New World species of the xanthaspis group is provided. Twenty-eight adult wasps were obtained (22 females and six males) out of 50 cocoons which larvae emerged from the caterpillar host. The findings contribute to the broader understanding of Diolcogaster in the Neotropics and its potential for the biological control of lepidopteran defoliators.