Immature Stages Research Articles

Monitoring of mosquito breeding sites in the municipality of Cascais, Portugal

Abstract Culex pipiens is the primary West Nile Virus vector in Portugal. Additionally, in 2017, Aedes albopictus, vector of various arboviruses, was detected in Northern Portugal. To mitigate viral transmission risks, effective surveillance is crucial, including monitoring egg, larva, and pupa stages. Located on the west of Lisbon, Cascais implemented a surveillance based on monitoring invasive species at Points of Entry and, complementarily, on detecting and intervening in breeding sites. Active searches for new breeding sites and monitoring those that were already identified are conducted weekly during the peak mosquito season (May to October) and biweekly during the colder season (November to April), following the methodology outlined below: • Upon detection, samples of immature stages are collected for submission to the National Institute of Health (INSA); • Corrective measures are requested to the managing entities of the locations where the sites are found; • Date of action, location, collection point, date of report, and status (pending or intervened) are recorded on a database; • Locations with pending status undergo continuous monitoring. In 2023, 16 artificial breeding sites were identified. These findings resulted from 23 actions conducted across 8 locations. 35 samples of immatures were collected, followed by their submission and notification to the respective managing entities. Recurrent detections were noted in 9 breeding sites. The managing entities implemented measures at 13 of the identified sites, leaving 3 with pending intervention. The identification of breeding sites demands continuous monitoring efforts and requires an effective coordination between the Public Health services and the management entities. Based on the achieved outcomes, it is evident that the implementation of corrective measures has been successful, making a significant contribution to reducing mosquito density. Key messages • Effective surveillance and intervention strategies are crucial in mitigating West Nile Virus and arbovirus transmission risks in Portugal. • Continuous monitoring and coordination between public health services and management entities are key for maintaining effective vector control measures.

Evaluation of the toxicity of translaminar insecticides against the soybean tentiform leafminer (Lepidoptera: Gracillariidae), a potential new pest of soybean.

Macrosaccus morrisella (Fitch) (Lepidoptera: Gracillariidae) is a leaf-mining microlepidopteran native to North America recently reported feeding on soybean in the United States and Canada. Control methods for the management of this pest remain unknown. The insecticides Agri-Mek SC (abamectin) and Endigo ZXC (lambda-cyhalothrin + thiamethoxam) were evaluated for: (i) effects against immature stages of M. morrisella in potted soybean plants; (ii) effects against adults of M. morrisella through different routes of exposure; and (iii) efficacy of field applications against M. morrisella. In the experiment with immature stages, when either insecticide was applied to potted plants with M. morrisella in serpentine or blotch mines, significantly fewer adults emerged compared to the untreated plants. In contrast, adult emergence from potted plants treated with either insecticide when M. morrisella was in tentiform mines did not differ from untreated plants. In the experiment with adults, the survival of M. morrisella exposed to either insecticide through oral exposure or residual contact + oral exposure was significantly lower than that for residual contact exposure alone, which in turn was lower than the untreated control. Within each exposure route, the response to the 2 insecticides did not differ. In the field experiment, at 21 days after insecticide application, the mean percentage of leaf area mined was significantly lower in plots treated with Endigo ZCX compared to plots treated with Agri-Mek SC or those left untreated. These results suggest that Endigo ZXC and to a lesser extent Agri-Mek SC may have potential for management of M. morrisella.

A new species of Coelidiana Oman (Hemiptera: Cicadellidae: Neocoelidiinae) from Brazil with key to Brazilian species, description of immature stages, and notes about parasitoids and host plant

A new species of Coelidiana Oman (Hemiptera: Cicadellidae: Neocoelidiinae) from Brazil with key to Brazilian species, description of immature stages, and notes about parasitoids and host plant

Clinical characteristics and nomogram model for predicting the risk of recurrence of complicated urinary tract infection in pediatric patients

Complicated urinary tract infection (cUTI) has higher incidences of antibiotic resistance, recurrence, chronicity, and progression. There exist differences in the immune functions, anatomical structures and physiological functions of urinary tract system between children and adults. In addition, the immature stage of hygiene habit development and the inability to express discomfort accurately all contribute to cUTI recurrence in pediatric patients. cUTI recurrence in children can lead to serious consequences such as growth and development delay, renal scars, and progression to end-stage kidney diseases. Despite the high incidence of cUTI recurrence, no predictive model currently exists to guide targeted intervention in pediatric patients. This study aimed to establish a nomogram to predict the risk of cUTI recurrence in children, thereby facilitating targeted prevention and treatment strategies in pediatric patients. The nomogram was developed based on a retrospective cohort that included 421 pediatric patients with cUTI at West China Second University Hospital from January 2020 to August 2023. The patients were randomly divided into a training set and a validation set in a 3:1 ratio. Logistic regression analysis was used to identify risk factors and construct the nomogram for predicting the risk of cUTI recurrence, followed by validation and performance analysis. Of the 421 children with cUTI, the recurrence rate of cUTI was 68.4% (288 cases) during an average follow-up duration of 22.9 months. The nomogram comprised female gender, history of urinary tract surgery, Escherichia coli in urine culture, renal dysfunction, and vesicoureteral reflux as predictors of cUTI recurrence in pediatric patients. The model showed favorable performance with C-index values of 0.735 and 0.750 in the training dataset and the validation dataset, respectively. The decision curve analysis revealed that the nomogram might be clinically useful. The reliable nomogram would be beneficial for clinicians to identify children with high risks of cUTI recurrence for targeted intervention.

Rearing fly larvae on various substrates: nutrient composition of larvae and frass.

Management solutions for waste in southern Thailand, such as fly larvae, are tested in a laboratory using different substrates and wastes from the Thai agricultural sector. The nutritional content of the immature stages of Musca domestica Linnaeus (Diptera: Muscidae) and Hermetia illucens (Linnaeus) (Diptera: Stratiomyidae) larvae, especially their protein and fat contents, makes them a potential animal feed. Laboratory strains of M. domestica and H. illucens were reared on selected substrates to examine how diet influenced their performance and nutritional composition. Waste from aquaculture, vegetables, and fruits in southern Thailand was examined for larval diet and showed promise as a rearing substrate for these insects. Musca domestica larvae reared on fishery waste achieved the highest larval weight and fat content, whereas H. illucens larvae reared on an aquaculture waste diet had the highest protein content. These findings imply aquaculture waste could be the best choice for large-scale fly larvae production, particularly as a protein feed additive.

Redescription of Chrysosphaerella baikalensis (Chrysophyceae, Chromulinales) with new data on the ultrastructure of its stomatocysts

An amended description of the ultrastructure of plate and spine scales is provided, along with the first description of the stomatocyst Chrysosphaerella baikalensis from Lake Baikal. C. baikalensis exhibits four types of plate scales: small and large elliptical, large rounded, and large subcircular scales. The spines can grow up to 47 μm in length. The stomatocysts of C. baikalensis are spherical or slightly oblate, with a small, regular pore surrounded by a flat pseudoanulus, lacking a collar. Based on their surface ornamentation, two forms of stomatocysts are distinguished, Forma A covered with densely arranged short needle-like outgrowths and Forma B having a smooth to psilate surface. We associate the first form with the immature stage. In this article, we report the growth of C. baikalensis in Lake Baikal during June, in the open water period, at a water temperature of 3.9–4.5 °C. During this period, whole cells and stomatocysts covered with plate scales were observed. Individual spines and plate scales were found at temperatures up to 11.5 ℃.

Morphological Characterization of Culex Quinquefasciatus (Diptera: Culicidae) Pupae in the Eastern Province of Saudi Arabia.

Over the last 30 years, several mosquito-borne diseases have spread worldwide, threatening human and animal health, including lymphatic filariasis. Lymphatic filariasis is a major public health issue in tropical regions like Saudi Arabia, caused by parasitic worms. The parasitic worms Wuchereria bancrofti and Brugia malayi are transmitted by Culex quinquefasciatus. Cx. quinquefasciatus is a potential vector for various arboviruses like encephalitis virus and West Nile virus, along with protozoa like Plasmodium relictum that cause bird malaria. With the increasing number of mosquito species, there is an increased need for additional species identification. More attention is being directed to the immature stage, specifically the pupae, due to their valuable characteristics for species identification. Cx. quinquefaciatus, one of the nine Culicine species found in Saudi Arabia, lacks a complete description of its pupal stage and only has partial descriptions of other developmental stages, making identification challenging. This study aimed to investigate the morphological characteristics of Cx. quinquefaciatus pupae, focusing on the cephalothorax, trumpet, abdomen, paddle, and genital lobes, as well as the distribution and arrangement of setal branching and chaetotaxy. The pupae were collected from Qaryat Al Ulya Governorate, Eastern region, KSA, and maintained in a Biology Laboratory at the University of Hafr Al Batin from September to October 2022. The findings of this study have provided the essential characteristics regarding the distinguishing features of Cx. quinquefaciatus pupae to distinguish it from other closely related species. This identification could assist field entomologists in enhancing their efficiency by classifying mosquitoes during virus outbreaks.

Effects of temperature on the development of Rachiplusia nu (Lepidoptera: Noctuidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) and implications on population growth in Brazil.

Temperature is an elementary component in mathematical models for predicting the biotic potential of insects. In this study, the objective was to evaluate the impact of different constant temperatures of 8, 10, 15, 20, 25, 30, and 32°C on the biological parameters, lower temperature thresholds (TT), and estimating the number of annual generations (NAG) of Rachiplusia nu and Chrysodeixis includens, both pests associated with the soybean crop in Brazil. There was no development of the immature stages of R. nu at 8°C, as was also found for C. includens at 8 and 10°C. However, at 10°C all stages of R. nu developed. In general, temperatures of 20 and 25°C were the most suitable for the development of R. nu and C. includens, providing egg to adult viability of over 60% and the highest total fecundities. However, the temperature of 32°C negatively affected the parameters of the fertility life table. Rachiplusia nu showed the lowest TT (eggs: 4.9°C; larvae: 10.8°C; pupa: 14.1°C; and egg to adult: 8.9°C) when compared with C. includens (eggs: 7.5°C; larvae: 15.3°C; pupa: 16.1°C; and egg to adult: 11.3°C). Based on TT values, the NAG varied from 3.9 in cold regions to 7.5 in warm regions. However, for C. includens, we can infer that the species can reach up to 8.8 generations in warm regions. The results of the present study are important for understanding the occurrence of R. nu and C. includens in field conditions and can help with the implications of management strategies.

Genomic analyses of the southern and eastern yellowjacket wasps (Hymenoptera: Vespidae) reveal evolutionary signatures of social life

Abstract Insects have evolved remarkably complex social systems. Social wasps are particularly noteworthy because they display gradations in social behaviors. Here, we sequence the genomes of two highly diverged Vespula wasps, V. squamosa and V. maculifrons Buysson (Hymenoptera: Vespidae), to gain greater insight into the evolution of sociality. Both V. squamosa and V. maculifrons are social wasps that live in large colonies characterized by distinct queen and worker castes. However, V. squamosa is a facultative social parasite, and V. maculifrons is its frequent host. We found that the genomes of both species were ~200 Mbp in size, similar to the genome sizes of congeneric species. Analyses of gene expression from members of different castes and developmental stages revealed similarities in expression patterns among immature life stages. We also found evidence of DNA methylation within the genome of both species by directly analyzing DNA sequence reads. Moreover, genes that were highly and uniformly expressed were also relatively highly methylated. We further uncovered evidence of differences in patterns of molecular evolution in the two taxa, consistent with V. squamosa exhibiting alterations in evolutionary pressures associated with its facultatively parasitic or polygyne life history. Finally, rates of gene evolution were correlated with variation in gene expression between castes and developmental stages, as expected if more highly expressed genes were subject to stronger levels of selection. Overall, this study expands our understanding of how social behavior relates to genome evolution in insects.

Abundance Trends of Immature Stages of Ticks at Different Distances from Hiking Trails from a Natural Park in North-Western Italy.

Hiking trails may act as hotspots at the wildlife-human interface, posing an acarological risk for people and their pets. Ticks that are maintained in the environment by wild animals may quest on people walking along the trails. Assessing the risk of tick bites for people involved in outdoor activities is a further step in mitigating the risk of tick-borne diseases. This work describes the variation of tick abundance along a gradient of distances from hiking trails, where wildlife passage is favored by higher accessibility. Hiking trails with dense vegetation on the sides were sampled for ticks along a 100 m dragging transect, located in a natural park in North-Western Italy. Additional transects were replicated at 1, 2 and 4 m away from the trail on both sides. After morphological identification, descriptive statistics and modeling were applied to determine the abundance patterns across distances. Larvae were most abundant near the trail, peaking at 1 m and dropping sharply at further distances. Nymphs showed a more gradual and consistent decrease at progressing distance from the trail. Few adults were collected, preventing the identification of a clear trend. With higher tick abundance, the immediate vicinity of hiking trails may represent a source of acarological risk for humans and pets.

Predation Efficiency of Non-Blood Sucking Mosquito Larvae of Toxorhynchites splendens (Weidman) Determined by Kruskal–Wallis One-Way Analysis of Variance by Ranks over Immature Stages of Malaria (Anopheles stephensi), Filariasis (Culex quinquefasciatus)

Predation Efficiency of Non-Blood Sucking Mosquito Larvae of Toxorhynchites splendens (Weidman) Determined by Kruskal–Wallis One-Way Analysis of Variance by Ranks over Immature Stages of Malaria (Anopheles stephensi), Filariasis (Culex quinquefasciatus)

Radiological Evaluation of Vascular Structures in Cats Infected with Immature Worms of Dirofilaria immitis.

This study aimed to assess thoracic radiographic abnormalities in cats infected with immature stages of Dirofilaria immitis to evaluate the utility of this diagnostic technique during early infection. A total of 123 cats from a hyperendemic area were classified into three groups: asymptomatic cats seronegative to anti-D.-immitis antibodies (Group A), seropositive asymptomatic cats (Group B), and seropositive cats with clinical signs that were at high risk of heartworm-associated respiratory disease (HARD) (Group C). Radiographic measurements and lung parenchymal abnormalities were analyzed and compared across the groups. Significant differences in several parameters, including CrPA/R4, and CdPA/R9 ratios, were observed between healthy and seropositive cats, suggesting early arterial damage even in the absence of adult worms. Other parameters that showed differences between healthy and infected cats were CVC/Ao and CVC/R4 ratios, but not the VHS. Group C exhibited a marked bronchointerstitial pattern, indicating severe parenchymal alterations associated with clinical signs. The study demonstrated that thoracic radiography can detect early vascular and parenchymal changes in feline D. immitis infections, providing valuable information for diagnosing HARD. However, it also highlights the limitations of radiographic techniques, as some seropositive cats displayed no significant abnormalities. The findings underscore the importance of combining radiography with clinical and serological assessments for a more accurate diagnosis of feline heartworm disease.

Description of immature stages of Hybosa acutangula Spaeth, 1913 (Coleoptera, Chrysomelidae, Cassidinae)

The immature stages of Hybosa acutangula Spaeth, 1913, member of the tribe Cassidini Gyllenhal, 1813, are described in detail and compared with other members of Cassidini and Ischyrosonychini Chapuis, 1875 species. The eggs, oothecae and first instar larvae are described for the first time. The morphology of the immatures of H. acutangula, unusual for the species of the Cassidini tribe, was the impulse that initiated the research. The result of the comparative analysis is the conclusion that the morphological features of the immature stages of H. acutangula are a mixture of characters observed in representatives of the Cassidini and Ischyrosonychini tribes. The similarity to Ischyrosonychini may only be convergence, but it cannot be ruled out that there is a separate phylogenetic lineage within Cassidini, represented by H. acutangula. Verification of this hypothesis requires further research.

Extraction and Evaluation of the Effectiveness of Chitosan as Insecticides on the Immature Stages of the Saw-Toothed Grain Beetle Oryzaephilus Surinamensis L. (Coleoptera: Silvanidae)

This study aimed to collect samples of amber rice infected with the saw-toothed grain beetle from local markets in Kirkuk city during 13 December 2023 to 25 April 2024, and the experiments were conducted in the laboratories of the College of Education for Pure Sciences at Kirkuk University from February to April 2024. The toxic effects of chitosan were studied at three different concentrations: 0.5, 1.0, and 1.5 ppm on the immature stages of the saw-toothed grain beetle. The substance exhibited significant effects on the immature stages of the insect , recording the shortest egg incubation period with an average of 8.17 days, while chitosan treatment showed a significantly longer periods of 9.66, 11.69, and 14.00 days for concentrations of 0.5, 1.0, and 1.5 ppm respectively. The larval stage duration had a direct proportionality with the chitosan concentrations, where the 1.5 ppm concentration registered the longest larval period of 22.00 days, as opposed to the control which recorded the shortest period of 13.56 days, and the 0.5 and 1.0 ppm concentrations recorded durations of 17.19 and 19.66 days respectively. In the pupal stage, significant differences were found when the chitosan concentration was increased, with the control treatment having the shortest pupal stage duration of 4.33 days; a direct correlation was found between the concentrations and the pupal stage duration, which decreased to 6.00, 7.59, and 9.07 days for the 0.5, 1.0, and 1.5 ppm concentrations, respectively.

Influence of Prey Consumption on the Predatory Potential and Biological Parameters of Exochomus nigripennis (Erichson) Against Phenacoccus solenopsis Tinsley and Icerya seychellarum (Westwood)

Phenacoccus solenopsis Tinsley and Icerya seychellarum (Westwood) are recent invasive sap-sucking insects in Egypt. Exochomus nigripennis (Erichson) being a viable option for conventional biological control. In the laboratory of the Plant Protection Research Institute, Agricultural Research Centre, Egypt, the biological parameters and predatory potential of E. nigripennis were assessed using P. solenopsis and I. seychellarum as preys. Fourth instar E. nigripennis larvae was recorded the highest average total consumption of 455 individuals, with a feeding capacity of 43.10 when reared on P. solenopsis. While the third larval instar of E. nigripennis consumed the maximum number of I. Seychellarum 43.11 with a feeding capacity of 45.18 compared to other instars. E. nigripennis females had a lifespan of 30.9 ± 1.21 days, and consuming 3255 preys on P. solenopsis, while males had a feeding capacity of 2198.64. The immature stages of E. nigripennis varied significantly based on the prey species. Larvae developed faster on I. Seychellarum, while pupal periods were shortest on I. Seychellarum and longest on P. solenopsis. The shortest development time was on I. Seychellarum (22.8 days), while the longest was on P. solenopsis (30.37 days). The longevity and reproduction of E. nigripennis were significantly influenced by the preys they were reared on. Males and females fed on P. solenopsis exhibited longer survival and females had an extended oviposition period. The fecundity was highest when females were fed P. solenopsis supplemented with sugar solution. The study suggests that an integrated pest management program can effectively manage P. solenopsis and I. seychellarum, by utilizing E. nigripennis as a biological control agent, making it suitable for mass rearing in agricultural ecosystems.

Determination of adipogenesis stages of human umbilical cord-derived mesenchymal stem cells using three-dimensional label-free holotomography

Adipogenesis involves complex changes in gene expression, morphology, and cytoskeletal organization. However, the quantitative analysis of live cell images to identify their stages through morphological markers is limited. Distinct adipogenesis markers on human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were identified through holotomography, a label-free live cell imaging technique. In the MSC-to-preadipocyte transition, the nucleus-to-cytoplasm ratio (0.080 vs. 0.052) and lipid droplet (LD) refractive index variation decreased (0.149 % vs. 0.061 %), whereas the LD number (20 vs. 65) increased. This event was also accompanied by the downregulation and upregulation of THY1 and Preadipocyte Factor-1 (PREF-1), respectively. In the preadipocyte to immature adipocyte shift, cell sphericity (0.20 vs. 0.43) and LD number (65 vs. 200) surged, large LDs (>10 μm3) appeared, and the major axis of the cell was reduced (143.7 μm vs. 83.12 μm). These findings indicate features of preadipocyte and immature adipocyte stages, alongside the downregulation of PREF-1 and upregulation of Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In adipocyte maturation, along with PPARγ and Fatty Acid-Binding Protein 4 upregulation, cell compactness (0.15 vs. 0.29) and sphericity (0.43 vs. 0.59) increased, and larger LDs (>30 μm3) formed, marking immature and mature adipocyte stages. The study highlights the distinct adipogenic morphological biomarkers of adipogenesis stages in UC-MSCs, providing potential applications in biomedical and clinical settings, such as fostering innovative medical strategies for treating metabolic disease.

Investigation on the mosquitocidal property of cis-13-Octadecenoic acid isolated from Andrographis paniculata against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae)

Mosquitoes are a critical global public health concern, causing millions of deaths each year due to the viral infections and diseases they spread. In recent decades, synthetic insecticides and repellents have been used to control mosquito populations and virus transmission, but they often harm the environment and non-target organisms, including humans. This research investigates the isolation, characterization, and structural elucidation of cis-13-Octadecenoic acid derived from Andrographis paniculata, and its effectiveness against the immature stages of Aedes aegypti and Culex quinquefasciatus. The extracts from A. paniculata underwent fractionation, with the most bioactive fractions being further purified and analyzed. Spectroscopic techniques were employed to confirm the structure of cis-13-Octadecenoic acid. Biological assays were then performed to evaluate its ovicidal, larvicidal, and pupicidal activities. cis-13-Octadecenoic acid exhibited notable ovicidal activity, causing 25 % mortality in Ae. aegypti eggs and 29 % mortality in Cx. quinquefasciatus eggs at a concentration of 2 ppm. Additionally, it showed strong larvicidal and pupicidal activities, with LC50 values of 2.67 ppm and 3.39 ppm for Ae. aegypti larvae and pupae, and 3.10 ppm and 2.93 ppm for Cx. quinquefasciatus larvae and pupae, respectively. These findings demonstrate the potential of cis-13-Octadecenoic acid as an effective natural compound for mosquito control, highlighting the broader importance of natural products as alternatives to synthetic insecticides in the management of vector-borne diseases.

Soft soap and linalool as potential management tools for Philaenus spumarius (Hemiptera: Aphrophoridae), vector of Xylellafastidiosa

Philaenus spumarius (Hemiptera: Aphrophoridae) is one of the most polyphagous xylem-feeding pests. It recently acquired bad fame when it was identified as the main vector of the bacterium Xylella fastidiosa, the causal agent of the Olive Quick Decline Syndrome that, within ten years, has affected and led to the death of more than 21 million olive trees in the Southern Italian region Apulia alone. Despite the agricultural practices and synthetic insecticides available, as not sufficiently effective, more strategies to target all the developmental instars of the pest and eco-friendly formulations, especially in organic orchards, are urgently needed. Therefore, the main goals of this work were to test the toxicity of a solution of potassium salts of fatty acids (soft soap) on P. spumarius juveniles and assess the attractiveness exerted by linalool on adults in a planar olfactometer. According to our results, the average neanids/nymphs’ mortality was 82.2 ± 10.4% 24h after spraying the soft soap on them at the labelled dose. Regarding the adults, linalool was significantly attractive to both sexes in a concentrations range between 0.01 and 0.12 μL linalool L−1 air. Our purpose is to potentially suggest a dual, integrated control approach against P. spumarius, with the soft soap as a biopesticide to reduce the immature stages of the pest and attractive, linalool-activated traps to numerically cull the population of the meadow spittlebug as part of a broader management system.

Fecundity without nectar is insufficient for the persistence of a blue butterfly.

Organisms with complex life cycles undergo ecological transitions between life stages, often resulting in stage-specific resource use. The relative contribution of each stage-specific resource to vital rates influences population dynamics and subsequently whether habitats can support viable populations. In lepidopterans, survival to reproduction requires sufficient resources for immature life stages, but the extent to which resources for adults are critical to population persistence is variable. We studied Boisduval's blue butterflies (Icaricia icarioides), in a greenhouse experiment, to quantify the effect of the adult diet, nectar, on vital rates. Butterflies fed ad libitum produced 3.4 times more eggs, on average, over their lifetime and lived 6 more days relative to those which only had access to water. We used these experimental data to parameterize a population model to test if vital rates with and without nectar result in viable population growth rates. Despite individual females laying 68 eggs without nectar, we found that Boisduval's blue butterfly populations will not persist without the improved fecundity associated with nectar resources (λ<1). In this species, although amino acids in the adult diet contributed to various improvements in fecundity, these improvements did not translate to improvements in population growth rates. Incorporating our experimental vital rates into a population model indicates that the relative abundance and quality of nectar can alter at what threshold other resource(s) are limiting the population.

Identification of morphologically similar genera based on reproductive systems and immature stages: a case study of Anastatus and Mesocomys (Hymenoptera: Eupelmidae)

Abstract Parallel or convergent evolution may result in phenotypically closely similar species. Many studies have reported that using solely traditional external morphological features can result in misidentification. Here, we discuss using the reproductive systems and immature developmental stages for scientific classification in Eupelmidae (Hymenoptera: Chalcidoidea), exemplifying with Anastatus Motschulsky and Mesocomys Cameron (Eupelmidae, Eupelminae). Results show that there are at least 6 distinct differences in the immature stages and the reproductive systems of the adults between species of the 2 genera: (1) the terminal ends of the left and right ovarioles are connected together in the studied species of Anastatus but not in Mesocomys, (2) the base of the lateral oviduct of Anastatus has a diverticulum that is lobate-shaped, whereas this diverticulum is spherical in Mesocomys, (3) the peduncle of the egg of Anastatus is significantly longer than that of Mesocomys, (4) the head capsule of the terminal instar larvae of Anastatus is uniformly translucent, whereas in Mesocomys it has a brown pattern, (5) the ocelli of the Anastatus pupa are smooth and slightly raised whereas the ocelli of Mesocomys each have a long filament each, and (6) the phallobase of adult male Anastatus is sharp basally but rounded in Mesocomys. Our findings not only provide novel insights for the differentiation of similar genera that can be difficult to distinguish in the immature stages but also provide a more comprehensive theoretical basis for clarifying the evolutionary and phylogenic status of Anastatus and Mesocomys.