Dipteran Parasitoids Research Articles

The Toll/IMD pathways mediate host protection against dipteran parasitoids

Parasitoids have utilized a variety of strategies to counteract host defense. They are in different taxonomic status and exhibit phenotypic and genetic diversity, and thus are thought to evolve distinct anti-defense mechanisms. In this study, we investigated the performance of two closely related parasitoids, Exorista japonica and Exorista sorbillans (Diptera: Tachinidae) that are biological control agents in agriculture and major insect pests in sericulture, on the host Bombyx mori. We show that the host is more susceptible to E. sorbillans infection while relatively resistant to E. japonica infection. Moreover, the expression levels of host antimicrobial peptides (AMPs) genes are repressed at early infection and induced at late infection of E. japonica, while AMPs are over-expressed at early infection and return to normal levels at late infection of E. sorbillans. In parallel, Toll and IMD pathway genes are generally induced at late infection of E. japonica, whereas these genes are up-regulated at early infection and down-regulated at late infection of E. sorbillans. Activating of host Toll/IMD pathways and AMPs expression by lipopolysaccharide (LPS) represses the larval growth of E. sorbillans. Conversely, inhibiting host Toll/IMD pathways by RNA interference significantly promotes E. japonica development. Therefore, the Toll/IMD pathways are required in the host for defense against infection of dipteran parasitoids. Overall, our study provides the new insight into the diversified host-parasitoid interactions, and offers a theoretical basis for further studies of the adaptive mechanism of dipteran parasitoids.

Population modelling for pest management: A case study using a pest land snail and its fly parasitoid in Australia

We built a DYMEX population dynamics model of the invasive land snail, Cochlicella acuta, and one of its dipteran parasitoids, Sarcophaga villeneuveana. The snail lifecycle consists of different size classes, based on shell height, as both parasitism and reproduction are influenced by snail height. The model reveals the likely role of small, cryptic snails in maintaining populations in the face of biological control and suggests that suppression of snail populations may only be possible with unrealistically high parasitism rates and with repeated annual spring releases of flies. The current model appears to describe the phenology of both the snail and fly adequately; however, construction of the model identified numerous knowledge gaps. Snail control would benefit from a thorough understanding of its basic biology and what drives its population dynamics – aspects which, surprisingly, have been largely ignored. Otherwise, any control measures will likely be unpredictable, with no real understanding of how or why management options may or may not work. We highlight the importance of modelling, preferably at an early stage, to synthesise available knowledge and data into a framework that helps target research at an early stage, and the benefits of experimentation via modelling.

Evaluation of suitable reference genes for expression analysis using quantitative real-time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae).

The parasitoid Exorista sorbillans (Diptera: Tachinidae) is a larval endoparasitoid of the silkworm Bombyx mori, causing severe damage to silkworm cocoon industry. It is also an important natural enemy resource of insect pests in agriculture and forestry. Despite their roles in biocontrol and pest status on sericulture, there has been limited research on the functional studies of dipteran parasitoids. Quantitative real-time polymerase chain reaction (qRT-PCR) is the most commonly used to address gene functions. Using qRT-PCR, stably expressed reference genes under different experimental conditions are required to normalize the expression of target genes. However, no information regarding suitable qRT-PCR reference genes in dipteran parasitoids has been reported. In this study, we evaluate the expression stability of nine commonly used reference genes in insects including eukaryotic translation elongation factor 1δ (eEF1δ), elongation factor 2, 18S ribosomal RNA (18S rRNA), tubulin 3, actin87, ribosomal protein 49 (RP49), ribosomal protein S15, glyceraldehyde-3-phosphate dehydrogenase, and TATA-box binding protein (TBP) in E. sorbillans under different treatments, including tissues, developmental stages, genders, feeding density and pesticide stress, using ∆Ct , BestKeeper, geNorm, Normfinder and RefFinder, respectively. The results showed that the genes RP49, eEF1δ and 18S rRNA were recommended as the most suitable reference genes in E. sorbillans across all experimental conditions. This finding provides the necessary foundation for future functional studies in E. sorbillans and its effective use in both sericulture and pest control.

Habitats and parasitoid abundance influence spatial density dependence patterns, rendering an asilid fly as a potential biological controller of white grubs

The behavioral response of a parasitoid shows the effect on host parasitism patterns at a given host distribution. As a result, an increase or decrease in parasitism intensity according to local host densities is found. This relationship could be proportional, positive, or negative, as a consequence of host foraging.Mallophora ruficaudais a parasitoid fly that parasitizesCyclocephala signaticollisscarab beetle larvae. Females search and place egg clusters overground in open grasslands. Larvae actively search hosts underground following chemical cues arising from the host itself. The parasitism patterns are a result of this complex host-searching strategy that is shared between both stages of the fly. In this work, we carried out a study at four spatial scales in apiaries located in the Pampas region of Argentina. We aimed to assess the potential ofM. ruficaudaas a control agent of white grubs. We found that parasitism has an inverse density-dependent in relation to low female activity and a direct density-dependent in relation to high female activity at the larger spatial scale. We found an inverse density-dependent pattern at the intermediate spatial scale. Parasitism is inversely density-dependent at both smaller spatial scales, associated with oviposition substrate availability and distance. Additionally,M. ruficaudadoes not select the oviposition substrates according to the abundance ofC. signaticollisinhabiting underground. We determined thatM. ruficaudacould act as a natural biological controller ofC. signaticollis. This work shows the importance of a proper scale for the analysis of factors that influence population dynamics of entomophagous insects and for evaluating their potential as biological control agents, and how environmental characteristics mold the parasitism patterns of this dipteran parasitoid.

Optimal pit site selection in antlion larvae: the relationship between prey availability and pit maintenance costs

Traps are an efficient method of capturing prey for ambush predators, but trap building and maintenance are costly. We describe suitable hunting sites for pit-building antlion larvae living in sand dunes based on its cost–benefit relationship. In the field, antlion pits were located near natural barriers, such as cliffs, rocks and vegetation, but not closest to these barriers. Our results show that this pattern of pit location did not differ between populations; neither with or without the influence of a specific dipteran parasitoid of antlion larvae. Artificial pitfall traps deployed in their habitats revealed that invertebrates move along barriers, likely through thigmotaxis (wall hugging or wall-following), and drop sands in the traps set at barrier edges. In the laboratory, repeated artificial destruction of pits from larval antlions induced frequent pit relocation and rebuilding. This task reduces life history parameters, such as the larval growth and food conversion rates, suggesting a high cost of pit maintenance. Thus, antlion pits might shift away from barrier edges where more prey is available but pit destruction occurs more frequently due to wall-following invertebrates. Such disturbance may explain why antlions are not located closest to these barriers.

Mitogenome-wise codon usage pattern from comparative analysis of the first mitogenome of Blepharipa sp. (Muga uzifly) with other Oestroid flies

Uziflies (Family: Tachinidae) are dipteran endoparasites of sericigenous insects which cause major economic loss in the silk industry globally. Here, we are presenting the first full mitogenome of Blepharipa sp. (Acc: KY644698, 15,080 bp, A + T = 78.41%), a dipteran parasitoid of Muga silkworm (Antheraea assamensis) found in the Indian states of Assam and Meghalaya. This study has confirmed that Blepharipa sp. mitogenome gene content and arrangement is similar to other Tachinidae and Sarcophagidae flies of Oestroidea superfamily, typical of ancestral Diptera. Although, Calliphoridae and Oestridae flies have undergone tRNA translocation and insertion, forming unique intergenic spacers (IGS) and overlapping regions (OL) and a few of them (IGS, OL) have been conserved across Oestroidea flies. The Tachinidae mitogenomes exhibit more AT content and AT biased codons in their protein-coding genes (PCGs) than the Oestroidea counterpart. About 92.07% of all (3722) codons in PCGs of this new species have A/T in their 3rd codon position. The high proportion of AT and repeats in the control region (CR) affects sequence coverage, resulting in a short CR (Blepharipa sp.: 168 bp) and a smaller tachinid mitogenome. Our research unveils those genes with a high AT content had a reduced effective number of codons, leading to high codon usage bias. The neutrality test shows that natural selection has a stronger influence on codon usage bias than directed mutational pressure. This study also reveals that longer PCGs (e.g., nad5, cox1) have a higher codon usage bias than shorter PCGs (e.g., atp8, nad4l). The divergence rates increase nonlinearly as AT content at the 3rd codon position increases and higher rate of synonymous divergence than nonsynonymous divergence causes strong purifying selection. The phylogenetic analysis explains that Blepharipa sp. is well suited in the family of insectivorous tachinid maggots. It's possible that biased codon usage in the Tachinidae family reduces the effective number of codons, and purifying selection retains the core functions in their mitogenome, which could help with efficient metabolism in their endo-parasitic life style and survival strategy.

Life history of the Philippine endemic butterfly Faunis sappho (Nymphalidae, Satyrinae, Amathusiini) on Bohol island, with the first record of a dipteran parasitoid (Tachinidae, Exoristinae, Eryciini) for the genus and a listing of hostplant families utilized by the tribe

Life history of the Philippine endemic butterfly Faunis sappho (Nymphalidae, Satyrinae, Amathusiini) on Bohol island, with the first record of a dipteran parasitoid (Tachinidae, Exoristinae, Eryciini) for the genus and a listing of hostplant families utilized by the tribe

Parasitoids Collected from Animal Feces in Brazil

The purpose of the paper is to report the species of dipteran parasitoids in poultry feces on farms, buffalo, and cattle in the field in Brazil. The experiments were carried out from April 2006 to December 2007. The pupae were obtained by the flotation method. They were individually placed in gelatin capsules until the emergency of the adult flies or their parasitoids. The specie more frequent was S endius with 7.2%. Were obtained from bovine feces 628 pupae of dipterous in buffalo feces, 3,437 pupae were collected and from chicken feces 2,799 pupae, from which 78, 172 and 504 parasitoids emerged, respectively. The most frequent species in bovine, of buffalo and chicken feces were: Gnathopleura quadridentata Wharton (Hymenoptera: Braconidae) with 25.6%, Spalangia drosophilae Ashmead (Hymenoptera: Pteromalidae) with 21.5% and Pachycrepoideus vindemmiae (Rondani, 1875) with 46.8%, respectively.

Parasitoids Collected in Poultry Farms in Brazil

The objective of this work is to report the presence of dipteran parasitoids in poultry farms in Brazil. The experiment was carried out in two poultry farms in the Midwest Region in Brazilian territory. The pupae were removed and individualized in glass capsules for the emergence of adult dipterans or parasitoids. The specie Pachycrepoideus vindemmiae (Rondani) (Hymenoptera: Pteromalidae) presented a frequency of 46.4% and showed parasitism of 93.9%. The species Musca domestica (Diptera: Muscidae) presented a frequency of 93.3%. Of the dipterans collected, the most important species was M. domestica for causing public health problems, disturbing people and being a vector of disease-causing agents.

First detection of Megaselia scalaris (Loew) (Diptera: Phoridae) as a facultative endoparasitoid of Nezara viridula (L.) (Hemiptera: Pentatomidae)

BackgroundThe phorid fly Megaselia scalaris (Loew) (Diptera: Phoridae) is an omnivorous species, capable of exploring a large variety of environments and ecological niches. It is known as an important detritivore species with maggots feeding on a variety of food of both animal and plant origin.ResultsThe present study reports M. scalaris as an endoparasitoid attacking colonies of the southern green stink bug Nezara viridula (L.) for the first time. This case of parasitism was observed inside rearing cages of N. viridula at the Plant Protection Research Institute, Dokki, Egypt in August 2020. We firstly identified adult individuals of M. scalaris which were found moving erratically within the cages using relevant identification keys. To verify that N. viridula individuals are parasitized by the same parasitoid and they are not infected with other parasitoids, some of the parasitized bugs were transferred to a separate cage at the same laboratory conditions and the developmental stages of the dipteran parasitoid were observed until the adult emergence.ConclusionThe present investigation revealed that M. scalaris could be included to the recorded parasitoid species of N. viridula, and further studies should be carried out to assess the efficacy of this fly as a biocontrol agent.

Genomic Resources for Goniozus legneri, Aleochara bilineata and Paykullia maculata, Representing Three Independent Origins of the Parasitoid Lifestyle in Insects.

Parasitoid insects are important model systems for a multitude of biological research topics and widely used as biological control agents against insect pests. While the parasitoid lifestyle has evolved numerous times in different insect groups, research has focused almost exclusively on Hymenoptera from the Parasitica clade. The genomes of several members of this group have been sequenced, but no genomic resources are available from any of the other, independent evolutionary origins of the parasitoid lifestyle. Our aim here was to develop genomic resources for three parasitoid insects outside the Parasitica. We present draft genome assemblies for Goniozus legneri, a parasitoid Hymenopteran more closely related to the non-parasitoid wasps and bees than to the Parasitica wasps, the Coleopteran parasitoid Aleochara bilineata and the Dipteran parasitoid Paykullia maculata. The genome assemblies are fragmented, but complete in terms of gene content. We also provide preliminary structural annotations. We anticipate that these genomic resources will be valuable for testing the generality of findings obtained from Parasitica wasps in future comparative studies.

Can plantings of partridge pea (Chamaecrista fasciculata) enhance beneficial arthropod communities in neighboring soybeans?

Grass buffer strips are often deployed along crop borders as part of conservation programs to improve water quality, filter sediments from runoff, and foster wildlife biodiversity. Addition of wildflowers in these strips can augment natural enemies by provisioning nectar and pollen resources. Here we investigated the impact of partridge pea [Chamaecrista fasciculata (Michx.) Greene] as an insectary plant to enhance natural enemy abundance in adjacent soybeans. In 2005, sticky card counts of hymenopteran and dipteran parasitoids were 72.7% higher in buffers of partridge pea and partridge pea mixed with purple tansy (Phacelia tanacetifolia Benth.) compared to purple tansy grown alone or proso millet (Panicum miliaceum L.), whereas predator abundance was greater in purple tansy and millet compared to buffers with partridge pea. Similarly, communities of beneficial arthropods captured in 2006 by sticky card and pitfall traps were 79.8–72.3% higher, respectively, in partridge pea buffers compared to neighboring soybeans. Buffer effects on populations of parasitoids and predators in adjoining soybean fields were mixed and varied among taxon groups. Partridge pea was a source for dipteran parasitoids and saprovores, but density effects were generally not discernible beyond 6 m within the adjoining soybean. More significantly, partridge pea acted as a natural enemy sink for mymarid parasitoids, dipteran predators, and ground-dwelling ants, and had a neutral effect on other arthropod guilds. Implications of these findings are discussed.

Anchored phylogenomics unravels the evolution of spider flies (Diptera, Acroceridae) and reveals discordance between nucleotides and amino acids

The onset of phylogenomics has contributed to the resolution of numerous challenging evolutionary questions while offering new perspectives regarding biodiversity. However, in some instances, analyses of large genomic datasets can also result in conflicting estimates of phylogeny. Here, we present the first phylogenomic scale study of a dipteran parasitoid family, built upon anchored hybrid enrichment and transcriptomic data of 240 loci of 43 ingroup acrocerid taxa. A new hypothesis for the timing of spider fly evolution is proposed, wielding recent advances in divergence time dating, including the fossilized birth-death process to show that the origin of Acroceridae is younger than previously proposed. To test the robustness of our phylogenetic inferences, we analyzed our datasets using different phylogenetic estimation criteria, including supermatrix and coalescent-based approaches, maximum-likelihood and Bayesian methods, combined with other approaches such as permutations of the data, homogeneous versus heterogeneous models, and alternative data and taxon sets. Resulting topologies based on amino acids and nucleotides are both strongly supported but critically discordant, primarily in terms of the monophyly of Panopinae. Conflict was not resolved by controlling for compositional heterogeneity and saturation in third codon positions, which highlights the need for a better understanding of how different biases affect different data sources. In our study, results based on nucleotides were both more robust to alterations of the data and different analytical methods and more compatible with our current understanding of acrocerid morphology and patterns of host usage.

Mechanisms underlying spruce budworm outbreak processes as elucidated by a 14‐year study in New Brunswick, Canada

AbstractWe conducted a 14‐yr intensive study of spruce budworm (Choristoneura fumiferana (Clem.)) survivorship at three study plots in largely balsam fir (Abies balsamea (L.) Mill.) stands in New Brunswick, Canada, to elucidate certain key mechanisms underlying spruce budworm outbreak cycles. The study covered a peak‐to‐declining phase (from 1981 and 1994) of the budworm outbreak cycle that had started in the early 1960s. Frequent sampling was carried out in each plot‐year to construct a practically continuous survivorship curve, and the annual variation in population density was estimated. We found a high level of correlation between the studied phase of the outbreak cycle and annual variations in the survivorship over the postdiapause period, suggesting that postdiapause survivorship was the chief determinant of the cycle. We found the annual changes in population density in the present study to be closely similar in pattern to those from the provincial budworm surveys conducted in much larger areas. This implies that the mechanism underlying the population process found in the few study plots in largely balsam fir stands also applies to the process in much larger areas of diverse stand types. The main source of postdiapause mortality is found to be natural enemies. The impacts of parasitoids and disease are evaluated by rearing budworm samples in the laboratory. Hymenopteran and dipteran parasitoids are by far the major sources of mortality, and microsporidians are the most prevalent pathogen. Occurrences of other entomopathogenic fungi and viruses were insignificant throughout the study. Seasonal changes in laboratory survivorship are compared with the corresponding field survivorship to estimate the effect of predation. No major mortality factor is found to singly play a predominant role in determining the outbreak cycle. Conversely, some minor factors are shown to have played significant roles. Thus, the importance of recognizing the action of natural enemies as a complex is emphasized for understanding the budworm outbreak cycle. Finally, centered on the roles played by the chronological succession of natural enemies in the present study, the results of budworm research in New Brunswick since the mid‐1940s are synthesized to outline basic mechanisms underlying the outbreak processes as a guide for further studies.

Functional sensorial complementation during host orientation in an Asilidae parasitoid larva.

Changes in environmental conditions influence the performance of organisms in every aspect of their life. Being capable of accurately sensing these changes allow organisms to better adapt. The detection of environmental conditions involves different sensory modalities. There are many studies on the morphology of different sensory structures but not so many studies showing their function. Here we studied the morphology of different sensory structures in the larva of a dipteran parasitoid. We occluded the putative sensory structures coupling the morphology with their function. First, we could develop a non-invasive method in which we occluded the putative sensorial structures annulling their function temporarily. Regarding their functionality, we found that larvae of Mallophora ruficauda require simultaneously of the sensilla found both in the antennae and those of the maxillary palps in order to orient to its host. When either both antennae or both maxillary palps were occluded, no orientation to the host was observed. We also found that these structures are not involved in the acceptance of the host because high and similar proportion of parasitized hosts was found in host acceptance experiments. We propose that other sensilla could be involved in host acceptance and discuss how the different sensilla in the antennae and maxillary palps complement each other to provide larvae with the information for locating its host.

The impact of adult diet on parasitoid reproductive performance

Diet is one of the most common influences on parasitoid reproductive traits. The life span, mating ability, fecundity, fertility and sex ratio of parasitoids can be affected by the quality of the adult diet. In the field, parasitoids can rely on different hosts and non-host nutrient sources, such as floral and extrafloral nectar, hemipteran honeydew and pollen, and various artificial diets have been used in mass rearing. In addition, some parasitoid species obtain nutrients by feeding on their host while adult (host feeding). In this review, we summarize current knowledge on the impact of the adult diet on the reproductive behavior of hymenopteran and dipteran parasitoids, with a particular focus on longevity, offspring production and host searching traits. First, we focus on food preferences and learning abilities of parasitoids to discriminate high-quality diets. Second, we analyze the impact of the adult diet on longevity, examining different natural and artificial food sources as well as the effect of their concentration and frequency. Third, we highlight the impact of the adult diet on host foraging. Fourth, we review the impact of adult diet on parasitoid offspring with special reference to (1) egg load, maturation and resorption, (2) parasitism and (3) progeny production and sex ratio. Finally, a number of implications for biological control and integrated pest management are discussed.

Oxidative stress and cytotoxicity elicited lipid peroxidation in hemocytes of Bombyx mori larva infested with dipteran parasitoid, Exorista bombycis

Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (P<0.01) high release into extracellular medium in vitro after exposure of hemocytes to parasitoid larval tissue protein compared with control revealing membrane permeability and loss of cell integrity. At five minutes after exposure, cytotoxicity was 43% and was increased to 99% at 3h. The cytotoxicity is signalled by increased content of hydrogen peroxide (H2O2) causing lipid peroxidation followed by porosity in plasma membrane. A test for lipid peroxidation by measurement of lipid peroxidation breakdown product, malondialdehyde (MDA) revealed significant increase in peroxidation from one to 24 h post-invasion, with maximum at 12 h (P<0.008). Level of reactive oxygen species measured as H2O2 production increased from 6 to 12 h post-invasion and continued to increase significantly (P<0.03) reaching maximum at 48 h. These observations reveal that dipteran endoparasitoid invasion induced H2O2 production in the hemocytes causing cytotoxicity, lipid peroxidation and membrane porosity that suppressed both humoral- and cell-mediated immune responses of hemocytes in B. mori.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.

Eavesdropping to Find Mates: The Function of Male Hearing for a Cicada-Hunting Parasitoid Fly, Emblemasoma erro (Diptera: Sarcophagidae).

Females of several species of dipteran parasitoids use long-range hearing to locate hosts for their offspring by eavesdropping on the acoustic mating calls of other insects. Males of these acoustic eavesdropping parasitoids also have physiologically functional ears, but so far, no adaptive function for male hearing has been discovered. I investigated the function of male hearing for the sarcophagid fly Emblemasoma erro Aldrich, an acoustic parasitoid of cicadas, by testing the hypothesis that both male and female E. erro use hearing to locate potential mates. I found that both male and nongravid female E. erro perform phonotaxis to the sounds of calling cicadas, that male flies engage in short-range, mate-finding behavior once they arrive at a sound source, and that encounters between females and males at a sound source can lead to copulation. Thus, cicada calling songs appear to serve as a mate-finding cue for both sexes of E. erro. Emblemasoma erro’s mate-finding behavior is compared to that of other sarcophagid flies, other acoustic parasitoids, and nonacoustic eavesdropping parasitoids.

Larvae and Nests of Six Aculeate Hymenoptera (Hymenoptera: Aculeata) Nesting in Reed Galls Induced by Lipara spp. (Diptera: Chloropidae) with a Review of Species Recorded.

Wetland species of aculeate Hymenoptera are poorly known, even though many of them may serve as diagnostic or flagship species in nature conservation. Here we examined 6,018 galls induced ≥1 year prior their collection by the chloropid flies Lipara spp. The galls were collected at 34 sites in Central Europe. We examined 1,389 nests (4,513 individuals) of nine species, part of which were parasitized by one dipteran and two chrysidid parasitoid species. We describe the nests of seven dominant species and larvae of four species (Pemphredon fabricii, Trypoxylon deceptorium, Hoplitis leucomelana and Hylaeus pectoralis) and two parasitoids (Trichrysis cyanea and Thyridanthrax fenestratus, both in nests of Pemphredon fabricii and Trypoxylon deceptorium). All the species, but H. pectoralis, preferred robust galls at very thin stalks (induced typically by Lipara lucens) over the narrow galls on thick stalks. The larvae of P. fabricii and T. deceptorium resembled strongly their sibling species (Pemphredon lethifer and Trypoxylon attenuatum sensu lato, respectively). The larvae of T. fenestratus showed features different from those previously described. By hatching set of another 10,583 galls induced by Lipara spp. ≥1 year prior their collection, we obtained 4,469 individuals of 14 nesting hymenopteran species, two cleptoparasites, three chrysidid and one dipteran parasitoid. Of these species, four new nesting species have been recorded for the first time in galls induced by Lipara spp.: Chelostoma campanularum, Heriades rubicola, Pseudoanthidium lituratum and Hylaeus incongruus. We also provide first records of their nest cleptoparasites Stelis breviuscula and Stelis ornatula, and the parasitoid Holopyga fastuosa generosa. Thyridanthrax fenestratus formed strong populations in nests of Pemphredon fabricii and Trypoxylon deceptorium, which are both newly recorded hosts for T. fenestratus. The descriptions provided here allow for the first time to identify the larvae of the most widespread central European aculeate hymenopteran reed gall specialists.