Effect of CO2 Concentrations on Entomopathogen Fitness and Insect-Pathogen Interactions.

Numerous insect species and their associated microbial pathogens are exposed to elevated CO2 concentrations in both artificial and natural environments. However, the impacts of elevated CO2 on the fitness of these pathogens and the susceptibility of insects to pathogen infections are not well understood. The yellow mealworm, Tenebrio molitor, is commonly produced for food and feed purposes in mass-rearing systems, which increases risk of pathogen infections. Additionally, entomopathogens are used to control T. molitor, which is also a pest of stored grains. It is therefore important to understand how elevated CO2 may affect both the pathogen directly and impact on host-pathogen interactions. We demonstrate that elevated CO2 concentrations reduced the viability and persistence of the spores of the bacterial pathogen Bacillus thuringiensis. In contrast, conidia of the fungal pathogen Metarhizium brunneum germinated faster under elevated CO2. Pre-exposure of the two pathogens to elevated CO2 prior to host infection did not affect the survival probability of T. molitor larvae. However, larvae reared at elevated CO2 concentrations were less susceptible to both pathogens compared to larvae reared at ambient CO2 concentrations. Our findings indicate that whilst elevated CO2 concentrations may be beneficial in reducing host susceptibility in mass-rearing systems, they may potentially reduce the efficacy of the tested entomopathogens when used as biological control agents of T. molitor larvae. We conclude that CO2 concentrations should be carefully selected and monitored as an additional environmental factor in laboratory experiments investigating insect-pathogen interactions.

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach.

Bees are important pollinators that help to maintain the biodiversity of wild and cultivated plants. However, the increased and inappropriate use of agrochemicals has caused an imbalance in the populations of these insects visiting flowers for pollen and nectar collection. Therefore, new research methods for understanding the mechanisms of action of pesticides and their impacts on the brains of bees, such as neurotoxicity and cellular changes, in response to different active characteristics and dosages of insecticides are necessary. Thus, with the aim of developing tests with greater specificity at the level of cells or tissues, this study sought to standardize a method for the in vitro culture of the nervous tissue of Apis mellifera. For this purpose, the brains of six foragers bees were transferred to three different insect cell culture media and it supplementation with 10% foetal bovine serum (FBS): Grace, Schneider, Leibovitz, Grace + FBS, Schneider + FBS and Leibovitz + FBS media for each collection time. Nervous tissue was collected after 1, 6, 12 and 24 h of incubation in a humidified CO2 incubator at 32 °C, and histological sections of the organs were analysed. The results showed that Leibovitz medium and Leibovitz medium + serum are potential culture media for the cultivation of nervous tissue, since they resulted in less tissue spacing and tissue disarrangement. Therefore, additional supplements are necessary to obtain an ideal medium for the cultivation of A.mellifera nervous tissue.

Molecular Identification of a Densovirus in Healthy and Diseased Zophobas morio (Coleoptera, Tenebrionidae).

Zophobas morio is a tropical darkling beetle which is widely exploited for commercial large-scale insect growing. Outbreaks of a disease may occur causing total devastation of cultures. In the present paper, samples of diseased Z. morio were obtained and used for establishment of a laboratory model as they were found infective to the larvae of the same insect species from another source. It took about 1 month to develop symptoms of acute disease in mid-age larvae and about twice as much when younger larvae were used for infection. Affected larvae perished quickly, and within several days up to 90-100% of the colony could perish. Both in healthy and diseased larvae a virus was detected using PCR with degenerate primers specific for a gene coding for a non-structural protein (ORF3). The sequenced gene fragment (Genbank accession #MN732869) confirmed allocation of the virus to Densoviridae, with maximal similarity of 97.2% to Blatella germanica densovirus-like virus (#JQ320376) and 66.2% to B. germanica densovirus (#AY189948). Genomic DNA samples of Z. morio larvae from an independent colony devoid of symptoms of a disease were also positive for this virus with a slightly different (99.7% sequence similarity to the former sequence of the Z. morio densovirus) genotype (#MN732870).

Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae).

BACKGROUND: Numerous important characteristics of adult arthropods are related to their size; this is influenced by conditions experienced as immatures. Arthropods cultured in the laboratory for research, or mass-reared for novel control methods, must therefore be of a standard size range and known quality so that results are reproducible. METHODS: A simple two-step technique to assess laboratory culture methods was demonstrated using the mosquito Anopheles gambiae s.s. as a model. First, the ranges of key development outcomes were determined using various diet levels. The observed outcomes described the physiologically constrained limits. Secondly, the same outcomes were measured when using a standard operating procedure (SOP) for comparison with the determined ranges. RESULTS: The standard method resulted in similar development rates to those of high and medium diets, wing length between those resulting from the high and medium diets, and larval survival exceeding all benchmark diet level values. The SOP used to produce experimental material was shown to produces high-quality material, relative to the biologically constrained limits. CONCLUSIONS: The comparison between all possible phenotypic outcomes, as determined by biological constraints, with those outcomes obtained using a given rearing protocol is termed "benchmarking". A method is here demonstrated which could be easily adapted to other arthropods, to objectively assess important characters obtained, and methods used, during routine culture that may affect outcomes of research.

A Culture Method for Darkling Beetles, Blapstinus spp. (Coleoptera:Tenebrionidae).

Darkling beetles, Blapstinus spp., have become a serious pest of Cucurbitaceae crops, especially in California. A culture method was sought to provide large numbers (>500) of adult beetles of known age and sex that could be used for laboratory testing when needed. A method previously developed for Alphitobius diaperinus (Panzer) using a diet of ground chick feed, with apple slices as a moisture source, was modified for use with Blapstinus spp. and then compared with the same method substituting apple slices with zucchini as the moisture source. Rearing boxes set up with apple slices produced significantly more pupae and adults than boxes containing zucchini slices. However, using either zucchini or apples as a moisture source yielded over the target of 500 adults per rearing box. A previous method designed to sex A. diaperinus based on the presence (♀) or absence (♂) of second valvifers in the pupal stage also proved to be effective for sexing the Blapstinus spp.

Life history traits and a method for continuous mass rearing of the planthopper Pentastiridius leporinus, a vector of the causal agent of syndrome 'basses richesses' in sugar beet.

BACKGROUND: The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of the γ-3 proteobacterium 'Candidatus Arsenophonus phytopathogenicus' which causes the syndrome 'basses richesses' (SBR) in sugar beet. SBR is a new and fast-spreading disease in Central Europe that leads to high yield losses. To date, the development of management strategies has been hampered by insufficient knowledge about general life history traits of the planthopper and, most importantly, the year-round availability of insects reared under controlled conditions. Rearing of P. leporinus has been considered challenging and to date no protocol exists. RESULTS: Here we describe a method for mass rearing P. leporinus on sugar beet from egg to adult that has produced five generations and over 20 000 individuals between June 2020 and March 2022. An alternative host such as wheat is not necessary for completing the life cycle. No-choice experiments showed that P. leporinus lays 139.1 ± 132.9 eggs on sugar beet, whereas no oviposition was observed on its nymphal host wheat. Head capsule width was identified as a trait that unequivocally distinguished the five nymphal instars. Developmental time from first instar to adult was 193.6 ± 35.8 days for males and 193.5 ± 59.2 days for females. Infection rates of adults were tested with a nested polymerase chain reaction. The results demonstrated that 70%-80% of reared planthoppers across all generations carried the SBR proteobacterium. CONCLUSION: The mass-rearing protocol and life history data will help overcome an important bottleneck in SBR research and enhance efforts in developing integrated pest management tools. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Establishment of White-Backed Planthopper Cell Lines.

The first continuous cell line of leafhopper was established over 50 years ago. Since then, leafhopper cell monolayers have been used extensively to assay the infectivity of plant viruses that multiply in their insect vectors and to elucidate the viral determinants for virus transmission via insects. We have established continuous insect cell lines of three rice planthoppers, which have been used to study the mechanisms for replication and spread of rice viruses. The notable advantage of the vector cell monolayer system is that it can reach a uniform infection rate of 100% of the cells in the culture inoculated with diluted viruses, and thus allows for synchronous virus multiplication. The self-propagative nature of leafhopper and planthopper cell lines under favorable conditions ensures the system both dynamic and stable for viral infection. The vector cell monolayer systems and molecular probes, along with reliable traditional methods, certainly facilitate studies on interactions between plant viruses and insect vectors at molecular and cellular levels.

Optimization of in vitro culture of honeybee nervous tissue for pesticide risk assessment.

The most used pesticides have neurotoxic action on the neurotransmitter system of target and non-targeted insects, such as honeybees. However, honeybees have foremost importance worldwide, which has encouraged the development of tools to evaluate the action of specific pesticide molecules on their nervous system, providing accurate data on damage to their brain. In this sense, our study aimed to optimize in vitro honeybee nervous tissue culture to assess pesticide risks. To this end, six forager honeybee brains were dissected and transferred to different combinations of Leibovitz-15 (L-15) culture medium supplemented with Fetal Bovine Serum (FBS), Hank's Balanced Salt Solution (HBSS), and Insect Medium Supplement (IMS). Nervous tissues were collected after different incubation times (1, 6, 12, and 24 h) for morphology and Kenyon cell analyses. Our results showed that L-15 medium supplemented with HBSS and with HBSS plus FBS were the best media for culturing honey nervous tissue for 24 h, as they resulted in less tissue spacing and cell disarrangement. Therefore, they may be assessed in future ecotoxicological tests.

Patterns of Genetic Diversity and Mating Systems in a Mass-Reared Black Soldier Fly Colony.

The black soldier fly (BSF), Hermetia illucens, is a promising candidate for the emerging insect farming industry with favourable characteristics for both bioremediation and production of animal delivered nutritive and industrial compounds. The genetic management of commercial colonies will become increasingly important for the sustainability of the industry. However, r-selected life history traits of insects pose challenges to conventional animal husbandry and breeding approaches. In this study, the long-term genetic effects of mass-rearing were evaluated as well as mating systems in the species to establish factors that might influence genetic diversity, and by implication fitness and productivity in commercial colonies. Population genetic parameters, based on microsatellite markers, were estimated and compared amongst two temporal wild sampling populations and four generations (F28, F48, F52, and F62) of a mass-reared colony. Furthermore, genetic relationships amongst mate pairs were evaluated and parentage analysis was performed to determine the oc-currence of preferential mate choice and multiple paternity. The mass-reared colony showed a reduction in genetic diversity and evidence for inbreeding with significant successive generational genetic differentiation from the wild progenitor population. Population-level analysis also gave the first tentative evidence of positive assortative mating and genetic polyandry in BSF. The homoge-neity of the mass-reared colony seems to result from a dual action caused by small effective popu-lation size and increased homozygosity due to positive assortative mating. However, the high ge-netic diversity in the wild and a polyandrous mating system might suggest the possible restoration of diversity in mass-reared colonies through augmentation with the wild population.