Larvae Research Articles

Insights from common buzzard broods on the interaction between Leucocytozoon infection, watercourse habitats and simuliid blackfly vectors

Blood parasites of the genus Leucocytozoon commonly infect many bird species worldwide and are particularly prevalent in birds of prey. As a vector-borne parasitic disease, the infection occurrence overlaps with that of the dominant vectors: blackflies (Diptera, Simuliidae). These blood-sucking insects are dependent on habitats with flowing freshwaters for the development of their larval stages. We investigated the correlation between the proximity to flowing waters and Leucocytozoon infection probability in common buzzard (Buteo buteo) broods, as well as the occurrence of adult blackflies directly at the nests. In addition, we investigated the survival of captured simuliids in relation to host infection intensity. In total in 2019, we examined 112 different nests, including 297 common buzzard nestlings, with a Leucocytozoon prevalence of 56.6% among the nestlings and of 80.3% at brood level. We found no significant association of Leucocytozoon infection probability with nestling age, the distance to the nearest stream and the sum of the length of streams within a radius of 200 and 1000 meters around each nest. The number of blackflies caught around the nest showed a tentative correlation with the probability of Leucocyozoon infection of the nestlings. Among the subsample of 218 blackfly individuals that survived day one after capture, survival averaged 6.2 days. Our results suggest that Leucocytozoon transmission is complex and requires consideration of many factors, related to habitat and vector prevalence, especially given their temporal variation.

Secondary compounds from Cyrtocarpa procera bark inhibits the biological cycle of Haemonchus contortus: in vitro ovicidal and larvicidal studies

Cyrtocarpa procera is a tree from the Anacardiaceae family that has various biological properties such as antioxidant, antibacterial and anthelmintic. In this research, the in vitro ovicidal and larvicidal effect of a hydroalcoholic extract (HA-E) and two of its fractions (organic: EtAcO-F and aqueous: Aq-F) from dehydrated bark of C. procera on Haemonchus contortus, were evaluated. Additionaly, the extract and fractions were analyzed phytochemically by high-performance liquid chromatography (HPLC) for identification of major compounds. The HA extract showed a 100 % ovicidal effect with the maximum concentration evaluated (100 mg/mL), while the EtAcO fraction exhibited an ovicidal effect close to 100 % starting at 2.50 mg/mL, with the Aq fraction a lower ovicidal effect was obtained. In the larval stage, a greater larvicidal effect was observed in the EtAcO fraction compared to that observed in HA-E and Aq-F did not affect larval viability. The major compounds were gallic acid and two galloyl derivatives. It is concluded that the bark of the C. procera tree contains secondary compounds that affect the eggs and larvae of H. contortus; however, it is also concluded that to enhance the effect, the fractionation of HA-E is necessary to obtain a more phytochemically concentrated organic fraction, and with it develop in vivo research.

Native entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) provide effective biocontrol against Oriental leafworm moth, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) in cabbage

The Oriental leafworm, Spodoptera litura, is a polyphagous pest that attacks several crops. In this study, we studied the susceptibility of the larval and pupal stages of S. litura to native entomopathogenic nematodes Heterorhabditis indica NBAIRH80, and S. surkhetense NBAIRS81. Bioassay results revealed that upon inoculating 400 infective juveniles (IJ) larva−1 to fourth-instar S. litura larvae with H. indica and S. surkhetense, H. indica caused 100% mortality whereas S. surkhetense caused 54% mortality. When S. litura pupae were inoculated with 50 IJ of H. indica, the pupal mortality was only 10% when the IJ concentration was increased to 600 IJ pupa−1, the pupal mortality also increased to 80%. The percentage mortality in larvae and pupae of S. litura increased significantly with increase in the exposure time. Both nematode species were able to penetrate into the S. litura larvae and also completed their life cycle by producing large numbers of IJ. Pot and field experiments showed that cabbage plants sprayed with H. indica and flubendiamide significantly reduced the number of larvae, leaf injury (H. indica: 0.7: 0-4 scale flubendiamide: 0.4: 0-4 scale), head injury (H. indica: 0.5: 0-4 scale flubendiamide: 0.6: 0-4 scale) and increasing yield (H. indica: 23.57 tonnes ha−1 flubendiamide: 25.53 tonnes ha−1). These results showed that, H. indica performed equally well that of flubendiamide. Overall results showed that H. indica NBAIRH80 can be incorporated in integrated pest management programme for S. litura.

Environmental Modulators on the Development of the Raccoon Roundworm (Baylisascaris procyonis): Effects of Temperature on the Embryogenesis

Members of the Ascarididae family are common zoonotic pathogens in humans and play an economic role in domestic and livestock animal husbandry. This family includes the obligatorily parasitic nematodes of the genus Baylisascaris, with the raccoon roundworm Baylisascaris procyonis being the most well-known representative. B. procyonis uses the raccoon (Procyon lotor) as its primary host and can utilize a broad range of mammals as paratenic hosts. Sexual reproduction of the adult nematodes occurs in the small intestine. Eggs are excreted into the environment through feces, where they develop into the infectious stage under suitable conditions within a few days to weeks. Infection of primary and paratenic hosts occurs through the oral ingestion of these infectious eggs. Raccoons can also become infected by ingesting infected paratenic hosts. Humans serve as accidental hosts and can suffer significant damage to organ tissues, the visual system, and the central nervous system after ingesting infectious eggs. The aim of the study was to investigate the effects of ambient temperature on embryonic development and to document the morphological changes during embryogenesis. Live specimens were collected from the raccoon intestine and incubated. Single-celled eggs were collected during this process. The eggs were decorticated and then preserved. To test the effects of ambient temperature, the eggs were incubated at 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, and 38°C and monitored at 24-hour intervals for their developmental stages. Detailed photographic documentation of the developmental stages was conducted. An increase in ambient temperature led to a reduction in development time. The temperature range within which embryogenesis proceeded to the L1 larval stage was between 10°C and 30°C. Incubation at 5°C did not produce L1 larvae even after 11 months. Incubation at 35°C and 38°C resulted in the complete degeneration of the eggs before reaching the L1 larval stage.

The transmission routes of African swine fever during an outbreak in Serbia July–August 2023: African swine fever virus detections in environmental samples and insects

African swine fever (ASF) is a highly contagious viral disease of domestic pigs and wild boar (Sus scrofa) caused by an arbovirus- African swine fever virus (ASFV), which is classified into the family Asfarviridae. Even though the main transmission route of ASFV is direct contact between animals and carcasses releasing ASFV into the environment, the role of other transmission routes such as via environmental contamination or insects remains in great part unclear. During an epidemic f ASF in Serbia in 2023, environmental samples (soil, feed, water and swabs from the pig barns), and insects [resulting in collection of adult and/or larval stages of non-biting flies (Diptera: Calliphoridae and Muscidae)] were collected in four locations in South Banat district of Serbia. To assess the possibility that insects carry the ASFV in infected courtyards, sticky fly traps and open Petri dishes containing meat mixed with humid cotton wads were offered in three locations during the five days of the experiment in the Belgrade area. Furthermore, to confirm the role of flies in ASF in mechanical transmission pathway in Serbia, L2 and L3 larvae of flies (Lucilia sericata Meigan, 1826; Stomoxys calcitrans Linnaeus, 1758) were collected from the pig carcasses from infected farms, bred to adults in the Laboratory and an ASFV spiked meat meal was placed into cages with three-day-old flies (n = 52) to estimate positivity of flies and duration of mechanical transmission of ASFV. The results from the environmental samples showed no positive ASFV DNA detection, the same was true for the samples from meat-based traps and sticky fly traps, while ASFV DNA was detected in three samples containing eggs, L1 and L3 fly larvae collected from carcasses and adult flies (L. sericata). In experimental conditions, only one S. calcitrans fly tested positive on day 1 post-infection. The results implicate the possible role of Lucilia sp. flies in the mechanical transmission of ASFV as well as S. calcitrans in Serbia during an outbreak, while ASFV DNA was not detected in environmental samples in this study.

Description of the immature stages and bionomics of Anthonomus (Anthonomus) brunnipennis Curtis, 1840 (Coleoptera, Curculionidae, Anthonomini)

The developmental stages (larva, pupa) of Anthonomus brunnipennis are fully described for the first time. Despite great similarities of the adults of A. brunnipennis and A. rubi the differences between immature stages of both species could be demonstrated. Two different habitats of A. brunnipennis in Germany are described, and host plant data are critically reviewed. The only well-documented host plant of A. brunnipennis is Potentilla erecta.

Host plants of the weevil genus Heilipus Germar, 1824 (Coleoptera, Curculionidae, Molytinae, Molytini, Hylobiina)

Heilipus Germar, 1824 (Coleoptera, Curculionidae, Molytinae, Molytini, Hylobiina) is a largely understudied tropical lineage of weevils with 90 described species distributed from the southern United States to northern Argentina. Several of its species cause damage to avocado (Persea americana Mill.; Lauraceae Juss.) crops in the Americas. Apart from the species of economic importance, there is a paucity of information regarding the host plants of species of Heilipus. This study presents a comprehensive overview of the current knowledge on the habits and host plants of this group of weevils, based on a literature review and the examination of label data associated with entomological material in museums and scientific collections. The larval stages of 24 Heilipus species are borers of either seeds or trunks of 38 plant species belonging to 13 genera: 33 species (12 genera) from the family Lauraceae and five species (one genus) from the family Annonaceae Juss. Four new host plant records and the first identification of habits and host plants for H. albovenosus (Champion), H. empiricus (Pascoe), H. fassli (Voss) and H. mirus (Pascoe) are provided. The biology and host plants of the seed and trunk borers are discussed, as well as the oligophagous feeding behaviour exhibited by these two feeding guilds. Seed-boring Heilipus species constitute one of the primary groups of pre-dispersal seed predators of the family Lauraceae in the Americas. Furthermore, the high diversity of Heilipus and its host plants in the Brazilian Atlantic Forest suggests that this area may be a possible centre of origin for these weevils.

Delineating early life stages of Belone belone: analysis of external morphology and postcranial skeletal development of the garfish

During fish larvae development functional, morphological, and physiological adaptations are key in larval survival strategies and can determine mortality bottlenecks. For many fish groups such as needlefish, which play crucial roles in marine food webs, studies on early life stages are almost lacking. Herein, the development, with focus on the postcranial skeleton, of the garfish Belone belone (Linnaeus, 1761) is described and a staging system is introduced. Ten larval and juvenile life stages are proposed in three main phases: yolk sac, larval, and juvenile development. During the yolk sac phase the garfish deplete their yolk reservoirs and finish the development of their unpaired fins. During the larval period the lower jaw elongates and the needlenose stage is reached. In the juvenile phase, the upper jaw elongates until both jaws are almost equally long. Since B. belone larvae develop much of their postcranial skeleton in the late embryonic and yolk sac phases they might not experience a severe bottleneck in this early stage such as many other marine fish species. Therefore, young garfish could be more resilient to environmental changes.

Effect of vitamin C injections on exercise muscular performance and biochemical parameters in Trichinella spiralis-infected mice

Background Trichinella spiralis is a worldwide intestinal nematode that can parasitize the striated muscles of its hosts at the larval stage. This study aims to evaluate potential of vitamin C for treating trichinellosis-related pathological problems in the infected muscles of mice. Materials and Methods Thirty CD1 male Albino mice were divided into three groups (10 mice per group). Negative and positive control groups (0.9% NaCl) and the infected vitamin C group (10 mg/kg body weight). Two weeks post-infection, each group was intraperitoneally injected daily for two weeks with Vitamin C or saline. The performance of the muscles was assessed both before and after the treatment. After dissection, constant parts of striated muscles were removed for further assays. The scoring of the histological changes of infected muscles was carried out. In addition to muscle malondialdehyde levels, superoxide dismutase and catalase activities were measured for the oxidative and antioxidant states. Creatine kinase and aspartate aminotransferase were also measured in tissues to reflect the degree of muscular damage. Results Vitamin C enhances the weakness of the muscular performance resulting from the infection. Vitamin C was able to repair some of the histological lesions that resulted from the infection. Trichinellosis caused severe changes in the biochemical markers in positive control animals. Muscle damage biomarkers and, besides, oxidative and antioxidant conditions were greatly ameliorated in infected vitamin C animals. Summing up, vitamin C can be used as a complementary drug due to its efficiency in improving pathogenesis following a trichinellosis infection. The supplement also must be tested in the intestinal stage of infection after showing promising results in the muscular stage.

Temperature Adaptation in the Postembryonic Development of Acanthocephalus tenuirostris (Palaeacanthocephala: Echinorhynchidae)

Under experimental conditions, the larval development of a fish parasite, Acanthocephalus tenuirostris was studied. This species is common in the eastern regions of Russia from Primorye Territory to the upper reaches of the Kolyma River. The material for the experiments was collected in different areas of the Magadan region which is located mainly in the subarctic climate zone. In several series of experiments, the infection and maintenance of intermediate hosts, the isopod Asellus hilgendorfii, took place at a constant temperature 15°C. In all cases, no more than 45 days were required for the formation of the final cystacanth stage. At the same time, in the hemocoel of isopods a large number of other larval stages were present simultaneously with cystacants. An additional experiment was conducted for the first 45 days at 15°C and the next 207 days at 4–6°C, showed the possibility of further development of larvae at low temperatures. Based on the comparison with the data available in the literature on the development times of European Acanthocephalus species living in temperate climates, it is concluded that the A. tenuirostris has anadaptation to a cold climate, whichprovides a twofold acceleration of the rate ofits development relative to larval A. lucii in Europe.

Overexpression of Lifeact in the C. elegans body wall muscle causes sarcomere disorganization and embryonic or larval lethality

Lifeact is a short peptide that is widely utilized as a probe for actin filaments in live imaging. However, high concentrations of Lifeact can alter actin filament dynamics and cause artificial modifications to the actin cytoskeleton. Here, I evaluated Caenorhabditis elegans strains expressing Lifeact fused to fluorescent proteins in the body wall muscle. I found that, while low-level expression of Lifeact from a single-copy transgene was appropriate for labeling sarcomeric actin filaments, overexpression of Lifeact from an extrachromosomal array causes severe disorganization of muscle sarcomeres and lethality at an embryonic or larval stage. Therefore, for imaging studies in C. elegans, Lifeact needs to be kept at a low level by proper management of the expression system.

ANÁLISE QUANTITATIVA DOS CASOS DE DENGUE NOS MUNICÍPIOS DO LITORAL NORTE DE SÃO PAULO NO PERÍODO DE JANEIRO DE 2014 A DEZEMBRO 2023

Dengue is characterized as a disease caused by a virus of the Flavivirus genus, transmitted by mosquitoes of the Aedes genus, with Aedes aegypti being the main vector. Due to its wide distribution and adaptation to urban and peri-domestic environments, dengue is predominantly found in urban areas. The ideal habitat for the mosquito is a tropical climate, which makes Brazil ideal for its proliferation. In addition to climatic factors, disorderly urbanization also contributes to the multiplication of the mosquito, increasing the places where it can reproduce. The vector's life cycle comprises the egg, four larval stages, a pupal stage and the adult, with an average lifespan of 10 to 42 days. Dengue symptoms generally include malaise, fever and weakness, and the disease tends to be self-limiting in most cases, being diagnosed through laboratory tests and clinical case. The aim of this study is to analyze the number of dengue cases in the municipalities of the north coast of São Paulo, Brazil, from January 2014 to December 2023, with the highest rate being observed in the municipality of São Sebastião with 14.95% of dengue cases in relation to the total population and the lowest rate of cases in Ilhabela with a percentage of around 7.37%. The year with the highest number of cases in the region was 2015 in the municipalities of Caraguatatuba, Ubatuba and São Sebastião and Ilhabela in 2014.

Immature stages and natural history of Eruga unilabiana (Hymenoptera: Ichneumonidae), an ectoparasitoid of two linyphiid spiders

AbstractWasps from the Polysphincta genus group (Hymenoptera: Ichneumonidae: Pimplinae) are currently the only parasitoid wasps that are ectoparasitoids of spiders. The species of the group exhibit several morphological and behavioral adaptations, which allow exploiting their host spiders. Here, we describe the external morphology, behavior and natural history of the immature stages of a Darwin wasp Eruga unilabiana Pádua & Sobczak, 2018, a parasitoid of sheet‐weaving spiders, Sphecozone sp. and Eurymorion sp. (Linyphiidae). The egg of E. unilabiana is a white mass that is deposited in the anterior region of the spider's abdomen. First stage larvae partially emerge from the egg's chorion. Second stage larvae have a body divided into 13 segments and induce spiders to build a modified web. The third stage larvae have eight pairs of retractable dorsal tubercles with tiny little hooks that help the larvae hang and move on the web threads after the spider dies. These results indicate that the immature stages of E. unilabiana present the same basic morphological and behavioral characteristics already known for the species of the group. However, the last stage larva's strategy of moving first to hang on the web threads before killing the spider is the first record in the group and more studies are needed to assess whether this behavior is adaptive for the wasp.

Morphological and molecular characterization of Purpureocillium lilacinum along with its biopesticidal effect against fall armyworm (Spodoptera frugiperda) in Southern Taiwan

Abstract Background Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), a key agricultural pest, impacts a broad spectrum of crop species, such as cotton, maize, rice, sorghum, and a range of vegetables. Chemical pesticides and transgenic maize are commonly used to protect crops against this major pest. However, biological control offers a safer and more sustainable solution for long-term FAW management. Entomopathogenic fungi (EPF), a substitute for synthetic insecticides, have been proven to be an effective biocontrol agent for managing pest populations. The present study objective is to isolate and identify the EPF, Purpureocillium lilacinum from soil using morphological and molecular techniques and evaluate its pathogenicity against FAW eggs and various larval stages under different spore concentrations. Results The micro-morphological characteristics fluctuated in growth behavior, appearance, and color under diverse growth media and temperatures. Molecular analysis, which involved constructing a phylogenetic tree from ITS and TEF gene regions, confirmed that PT-02 was P. lilacinum. The pathogenicity of the fungus was tested on FAW eggs, neonates, and larvae at multiple concentrations (1 × 108, 1 × 107, and 1 × 106 spores/ml), resulting in variation mortality. The egg mortality rate at 1 × 108, 1 × 107, and 1 × 106 spores/ml was 98, 98 and 96%, respectively. The neonate mortality rate was 100%, while first and second instars larvae mortality ranged from 10–36% across three concentrations, seven days after treatment (DAT). The median lethal concentration (LC50) values of P. lilacinum PT-02 against FAW eggs, first and second instars, were 3.02 × 107, 1.38 × 108, and 2.56 × 108 spore/ml, respectively. Conclusions The findings revealed that the native strain of P. lilacinum exhibited strong virulence against FAW, suggesting its potential use as an effective biocontrol agent for managing field pest populations.

Establishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi

BackgroundGenetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions.MethodsTo address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi, we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti, exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR’s applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex (AngDsx) splicing module in An. stephensi. Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope.ResultsOur results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species.ConclusionsSEPARATOR’s independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes.Graphical

Ray-Resorption Syndrome in European Seabass, Dicentrarchus labrax (Linnaeus, 1758).

Fin abnormalities are common in reared fish. They mainly consist of partial to complete lack of rays and severe abnormalities of fin-supporting skeletal elements, which develop during the larval stage, up to the completion of fin skeleton ontogeny. This study reports a new abnormal condition, ray-resorption syndrome (RSS), which developed after the completion of fin ontogeny in initially normal European seabass larvae, leading to extensive loss of fin rays. At 49 days post-fertilisation (dpf) (15 mm standard length, SL) all fish presented normal fins. However, nineteen days later (21 mm SL), significant fin damage, characterised by ray loss or fractures, was observed in all studied populations. The dorsal fin was most affected (55%-84%), followed by the pelvic (27%-53%) and anal fins (7%-17%). Microscopically, multiple non-mineralised areas resembling resorption lacunae were evident along all fin lepidotrichia. By 98 dpf (40 mm SL), the fin-ray loss had advanced, reaching its higher frequency in the dorsal (74%-83%) and caudal fins (71%-94%). Gene expression analysis revealed a significant upregulation of bglap (osteoblast maturation marker), acp5a (osteoclast maturation marker) and mmp13a (extracellular-matrix remodelling marker) in RSS specimens. The results are discussed in respect of the possible causative factors of RSS.

Bioecological Parameters of the Black Fig Fly, Silba adipata (Diptera: Lonchaeidae), Collected from Fig Crops in Mexico

This study assessed several bioecological aspects of the black fig fly, Silba adipata McAlpine (Diptera: Lonchaeidae), the main pest of fig (Ficus carica L.). Figs were collected from eight sites in the Mexican states of Michoacán (Plan de Ayala, Los Tejones, Antúnez, Tangancícuaro, Indaparapeo, El Carrizal, and Charapendo) and Morelos (Telixtac). No infestation was recorded in figs collected in Charapendo, but, in the remaining sites, it was variable (2–33%). Figs from Plan de Ayala and El Carrizal were longer and contained more larvae than those from Telixtac and Los Tejones. Some figs (≤12) collected in Telixtac and Los Tejones contained few pupae or adults. The survival of larval and pupal stages (≤86%) and the proportion of females (40–53%) were determined at the sites where the infestation of figs was >6% (Telixtac, El Carrizal, Plan de Ayala, and Los Tejones). In the second part of this study, the development of individuals collected in Los Tejones was analyzed under constant conditions. The estimated larval duration time was between 13 and 15 d, whereas pupae lasted 11 d. The survival rate and longevity of females and males were very similar. Our results could help design a program for integrated pest management against S. adipata.

Preference for host larval stages and density-dependent parasitism of Psyttalia parasitoid species in Bactrocera fruit flies

The study examined host preferences among three Psyttalia parasitoids (P. ponerophaga, P. concolor, and P. fletcheri) regarding the larval stages of two Bactrocera fruit flies (B. cucurbitae and B. oleae). All parasitoids preferred the third instar larvae, followed by the second and first instars. Psyttalia fletcheri had the highest parasitism rate on B. cucurbitae, while P. ponerophaga was most effective on B. oleae. The highest parasitism rates were observed at the host-parasitoid ratio (400:20) for both P. fletcheri on B. cucurbitae and P. ponerophaga on B. oleae. These results highlight the importance of understanding host-parasitoid dynamics for effective fruit fly management.

Pleiotropic effects of Ebony on pigmentation and development in the Asian multi-coloured ladybird beetle, Harmonia axyridis (Coleoptera: Coccinellidae).

Melanin plays a pivotal role in insect body pigmentation, significantly contributing to their adaptation to diverse biotic and abiotic environmental challenges. Several genes involved in insect melanin synthesis showed pleiotropic effects on insect development and reproduction. Among these, the N-β-alanyl dopamine synthetase gene (Ebony) is integral to the pigmentation process. However, the full spectrum of its pleiotropic impacts is not yet thoroughly understood. In this study, we identified and characterised the HaEbony gene in the Asian multi-coloured ladybird beetle (Harmonia axyridis) and found that HaEbony gene is a conserved gene within the Coleoptera order. We aimed to further explore the multiple roles of HaEbony in the physiology and behaviour in H. axyridis. The CRISPR/Cas9 system was applied to generate multiple HaEbony knockout allele (HaEbony+/-), showing nucleotide deletion in the G0 and G1 generations. Remarkably, the resultant HaEbony+/- mutants consistently displayed darker pigmentation than their wild-type counterparts across larval, pupal and adult stages. Furthermore, these HaEbony+/- individuals (G0) demonstrated an enhanced predatory efficiency, evidenced by a higher number of aphids consumed compared to the wild type. A significant finding was the reduced egg hatchability in both G0 and G1 generations of the HaEbony+/- group, highlighting a potential reproductive fitness cost associated with HaEbony deficiency. In conclusion, our study not only sheds light on the multifaceted roles of HaEbony in H. axyridis but also highlights the potential of employing CRISPR/Cas9-targeted modifications of the Ebony gene. Such genetic interventions could enhance the environmental adaptability and predatory efficacy of ladybirds, presenting a novel strategy in biological control application.

Carry-over effects of Bacillus thuringiensis on tolerant Aedes albopictus mosquitoes

BackgroundThe biological larvicide Bacillus thuringiensis subsp. israelensis (Bti) represents a safe and effective alternative to chemical insecticides for mosquito control. Efficient control of mosquitoes implicates continuous and extensive application of Bti. This massive use of Bti imposes strong selective pressure, but the complex mode of action of the numerous synergistic Bti endotoxins lower the risk of the emergence of resistance. Although resistance to Bti has not been identified at the population level in nature, some larvae can survive Bti exposure, suggesting tolerance mechanisms. Here we investigated whether Bti-tolerant Aedes albopictus larvae experience any fitness costs. We also studied how this tolerance affects different aspects of the phenotype of the emerging adults that could be relevant for arboviral transmission.MethodsWe exposed Ae. albopictus larvae to lethal concentration of Bti and studied the fitness and gut microbiota of tolerant larvae and their adult counterparts. We further compared the transcript abundance of nine key immunity genes in the gut of Bti-tolerant larvae and their emerging adults versus those not exposed to Bti.ResultsOur results showed that Bti exposure has multifaceted impacts on Ae. albopictus mosquitoes during both larval and adult stages. The carry-over effect of Bti exposure on tolerant larvae manifested in reduced adult emergence rate, shorter lifespan, and decreased fecundity. Bti also alters the gut microbiota of both larvae and adults. We observed higher microbial diversity in Bti-tolerant larvae and changes in the richness of core microbiota. Bti infection and the altered microbiota triggered immune responses in the larval and adult guts.ConclusionsThe observed reduction in mosquito fitness and changes in the composition of the microbiota of adults emerging from tolerant larvae could negatively influence mosquito vectorial capacity. Understanding these impacts is crucial for evaluating the broader implications of Bti-based insecticides in mosquito control programs.Graphical