Silkworms have been farmed for their silk since ancient times. After silk reeling, their chrysalides are consumed as food in several Asian countries. Despite the long rearing tradition of this insect, few studies have investigated the silkworm\'s microbiological safety all along the life cycle, focusing on detecting silkworm pathogens or on the safety of the dried chrysalis for food consumption. However, the in-farm rearing process, which takes around forty days, may affect the microbial load of the silkworm and of the rearing environment, as well as the quality of fresh cocoon and other performance parameters. No data is available on how microbial contamination changes during the rearing period and between different farmers. Furthermore, in light of the possible use of the chrysalis as food, it is crucial to understand how its microbial load varies according to the water content. To address these specific questions, we conducted an investigation involving the analysis of specific microbial indicators commonly used in the food chain. We collected environmental and silkworm samples from several farms. The examination covered the entire life cycle of silkworms, beginning with the first instar larvae and concluding with the scrutiny of both freshly harvested and dried pupae. Silkworm farms in Northeast Italy proved to be an appropriate model system for carrying out the experimentation. Additionally, an evaluation of rearing performance was conducted, with a focus on the quality of fresh cocoons and the survival rate of the insects.

The Oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive pest of quarantine importance affecting the global fruit trade. In managing B. dorsalis, methods like cultural, biological, chemical, sterile insect technique (SIT), and semiochemical-mediated attract-and-kill are in use with varying success. The SIT approach is the method of choice for a chemical-free, long-term suppression of B. dorsalis, followed in many countries across the globe. The nonspecific mutations caused by irradiation affect the overall fitness of flies, thus requiring a more precise method for a heritable, fitness-not-compromising approach. In this regard, CRISPR/Cas9-mediated genome editing enables the creation of mutations at the precise genomic location/s through RNA-guided dsDNA cleavage. Of late, DNA-free editing employing ribonucleoprotein complex (RNP) is preferred to validate the target genes at G0 stage embryos in insects. It requires characterizing genomic edits from adults after completing their life cycle, which may entail a few days to months, depending on longevity. Additionally, edit characterization is required from each individual, as edits are unique. Therefore, all RNP-microinjected individuals must be maintained until the end of their life cycle, irrespective of editing. To overcome this impediment, we predetermine the genomic edits from the shed tissues, such as pupal cases, to maintain only edited individuals. In this study, we have shown the utility of pupal cases from five males and females of B. dorsalis to predetermine the genomic edits, which corroborated the edits from the respective adults.

BACKGROUND: In 2017, several new housing districts were constructed on Bioko Island, Equatorial Guinea. This case study assessed the impact construction projects had on mosquito larval habitats and the effectiveness of larval source management in reducing malaria vector density within the surrounding area.
METHODS: Anopheline larval presence was assessed at 11 new construction sites by the proportion of larval habitats containing Anopheline pupae and late instar larval stages. Bacillus thuringiensis israelensis (Bti) larvicide was applied weekly to nine locations for 30 weeks, while two locations received no larvicide and acted as controls. Adult mosquito density was monitored via human landing collections in adjacent communities of six construction sites, including the two control sites.
RESULTS: The sites that received Bti had significantly lower observation rates of both pupae (3.2% vs. 18.0%; p < 0.001) and late instar Anopheles spp. mosquitoes (14.1 vs. 43.6%; p < 0.001) compared to the two untreated sites. Anopheles spp. accounted for 67% of mosquitoes collected with human landing collections and were captured at significantly lower levels in communities adjacent to treated construction sites compared to untreated sites (p < 0.001), with an estimated 38% reduction in human biting rate (IRR: 0.62, 95% CI IRR: 0.55, 0.69). Seven months after the start of the study, untreated sites were treated due to ethical concerns given results from treatment sties, necessitating immediate Bti application. The following week, the number of habitats, the proportion of larval sites with Anopheles spp. pupae, late instars, and adult biting rates in adjacent communities to these sites all decreased to comparable levels across all sites.
CONCLUSIONS: Findings suggest larval source management represents an effective intervention to suppress mosquito populations during infrastructure development. Incorporating larval source management into ongoing and planned construction initiatives represents an opportunity to fine tune vector control in response to anthropogenetic changes. Ideally, this should become standard practice in malaria-endemic regions in order to reduce viable mosquito habitats that are common by-products of construction.

The European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), is a univoltine species that overwinters at pupal stage. Under optimum overwintering conditions pupae will develop into adults the next spring. Shorter or longer than optimum chilling periods induce prolonged pupae dormancy. Pupae that enter prolonged dormancy due to a short chilling period exhibit high emergence rates after a second cycle of cold/warm periods. Adults found to be larger and less fecund compared to their counterparts from pupae with annual diapause. On the other hand, extreme long chilling periods at pupal stage results in high mortality rates. However, for one Greek population, a substantial number of adults emerged following prolonged chilling of pupae (ca. 18 consecutive months). In this study, we used three R. cerasi populations in order to address possible geographical variation in fitness cost of adults from pupae with prolonged dormancy. In addition, the fitness traits of these adults emerging after prolonged pupae chilling were compared with that of their counterparts from pupae with annual diapause or prolonged dormancy. Our results reveal no population-specific variation in fitness cost of adults from pupae with prolonged dormancy. Within a population, lifetime fecundity did not differ between adults emerged from pupae with prolonged dormancy and those emerged after prolonged pupae chilling. Adults emerged from pupae exposed to prolonged chilling suffer an additional reduction in adult longevity compared to adults from pupae with prolonged dormancy. Hence, fitness of R. cerasi adults is regulated by diapause regimes of pupae.

BACKGROUND: Insect metamorphosis from larvae to pupae is one of the most important stages of insect life history. Relatively comprehensive information related to gene transcription profiles during lepidopteran metamorphosis is required to understand the molecular mechanism underlying this important stage. We conducted transcriptional profiling of the brain and fat body of the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) during its transition from last instar larva into pupa to explore the physiological processes associated with different phases of metamorphosis.
RESULTS: During metamorphosis, the differences in gene expression patterns and the number of differentially expressed genes in the fat body were found to be greater than those in the brain. Each stage had a specific gene expression pattern, which contributed to different physiological changes. A decrease in juvenile hormone levels at the feeding stage is associated with increased expression levels of two genes (juvenile hormone esterase, juvenile hormone epoxide hydrolase). The expression levels of neuropeptides were highly expressed at the feeding stage and the initiation of the wandering stage and less expressed at the prepupal stage and the initiation of the pupal stage. The transcription levels of many hormone (or neuropeptide) receptors were specifically increased at the initiation of the wandering stage in comparison with other stages. The expression levels of many autophagy-related genes in the fat body were found to be gradually upregulated during metamorphosis. The activation of apoptosis was probably related to enhanced expression of many key genes (Apaf1, IAP-binding motif 1 like, cathepsins, caspases). Active proliferation might be associated with enhanced expression levels in several factors (JNK pathway: jun-D; TGF-β pathway: decapentaplegic, glass bottom boat; insulin pathway: insulin-like peptides from the fat body; Wnt pathway: wntless, TCF/Pangolin).
CONCLUSIONS: This study revealed several vital physiological processes and molecular events of metamorphosis and provided valuable information for illustrating the process of insect metamorphosis from larvae to pupae.

The mosquito species Aedes (Ochlerotatus) coluzzii Rioux, Guilvard & Pasteur, 1998 was distinguished from its sibling species Aedes detritus (Haliday, 1833) using an isoenzymatic method that required the destruction of the entire specimen, therefore no holotype was designated by the species authors. We aimed to designate a neotype for Ae. coluzzii from specimens collected from the type-locality and individually reared up to adult stage. Genomic DNA was extracted from pupal exuvia and ITS2 was sequenced, enabling verification of the identity of each specimen as Ae. coluzzii or Ae. detritus. Among the series of Ae. coluzzii, a male was designated as neotype and deposited in a collection. To our knowledge, this is the first time the type of a mosquito species is deposited thanks to its molecular identification from its pupal exuvia. The set of identified specimens allowed additional phylogenetic and morphologic studies.
UNASSIGNED: Utilisation d’une exuvie nymphale pour désigner le néotype intact d’une espèce appartenant à un complexe d’espèces jumelles - le cas d’Aedes coluzzii (Diptera, Culicidae).
UNASSIGNED: L’espèce de moustique Aedes (Ochlerotatus) coluzzii Rioux, Guilvard & Pasteur, 1998 a été distinguée de son espèce jumelle Aedes detritus (Haliday, 1833) par une méthode isoenzymatique qui a nécessité la destruction de l’ensemble du spécimen, et donc aucun holotype n’a été désigné par les auteurs de l’espèce. Notre objectif était de désigner un néotype pour Ae. coluzzii à partir de spécimens collectés dans la localité-type et élevés individuellement jusqu’au stade adulte. L’ADN génomique a été extrait de l’exuvie nymphale et l’ITS2 a été séquencé, permettant la vérification de l’identité de chaque spécimen comme Ae. coluzzii ou Ae. détritus. Parmi la série d’Ae. coluzzii, un mâle a été désigné comme néotype et déposé dans une collection. À notre connaissance, c’est la première fois que le type d’une espèce de moustique est déposé grâce à l’identification moléculaire à partir de son exuvie nymphale. L’ensemble des spécimens identifiés a permis des études phylogénétiques et morphologiques complémentaires.

The rapid and economical monitoring of mosquitos is imperative to understanding the dynamics of both disease vectors and nuisance species. In light of technological advances in mosquito sampling and DNA sequencing, health agencies can now utilize the full potential of metabarcoding pipelines for rapid and standardizable surveillance. Here, we describe mosquito spatial and temporal variation, with particular focus on Mansonia Blanchard species, in the Madeira (Rondônia State) and the Ribeira (São Paulo) watersheds, Brazil using metabarcoding of the D2 rDNA marker. Sampling and molecular pipelines were used to evaluate the taxonomic contribution of mosquitos in pools of culicids collected en masse from macrophyte-roots (immatures) and from Mosquito Magnet traps and protected human landings (adults). Results for adult captures are comparable to morphological diagnoses and clarify previously unknown temporal and spatial species turnover. Metabarcoding of immature stages also confirmed the extent of the geographical distribution of some species and each taxon\'s association with macrophyte species. Given the benefits of metabarcoding, such as taxonomic acuity, high throughput processing, and objectivity, we suggest such techniques should be more fully incorporated into culicid monitoring schemes. The metabarcoding protocol described herein paired with standardized field sampling schemes, when used by mosquito monitoring professionals, offers substantial improvements in terms of practicality, speed and cost.

Predicting species responses to climate change requires tracking the variation in individual performance following exposure to warming conditions. One ecologically relevant approach consists of examining the thermal responses of a large number of traits, both related with population dynamics and trophic interactions (i.e. a multi-trait approach). Based on in situ climatic data and projections from climate models, we here designed two daily fluctuating thermal regimes realistically reflecting current and future conditions in Eastern France. These models detected an increase in mean temperature and in the range of daily thermal fluctuations as two local facets of global warming likely to occur in our study area by the end of this century. We then examined the responses of several fitness-related traits in caterpillars of the moth Lobesia botrana - including development, pupal mass, survival rates, energetic reserves, behavioral and immune traits expressed against parasitoids - to this experimental imitation of global warming. Increasing temperatures positively affected development (leading to a 31% reduction in the time needed to complete larval stage), survival rates (+19%), and movement speed as a surrogate for larval escape ability to natural enemies (+60%). Conversely, warming elicited detrimental effects on lipid reserves (-26%) and immunity (total phenoloxidase activity: -34%). These findings confirm that traits should differ in their sensitivity to global warming, underlying complex consequences for population dynamics and trophic interactions. Our study strengthens the importance of combining a multi-trait approach with the use of realistic fluctuating regimes to forecast the consequences of global warming for individuals, species and species assemblages.

Adult forms emerge from the relative growth of the body and its parts. Each appendage and organ has a unique pattern of growth that influences the size and shape it attains. This produces adult size relationships referred to as static allometries, which have received a great amount of attention in evolutionary and developmental biology. However, many questions remain unanswered, for example: What sorts of developmental processes coordinate growth? And how do these processes change given variation in body size? It has become increasingly clear that nutrition is one of the strongest influences on size relationships. In insects, nutrition acts via insulin/TOR signaling to facilitate inter- and intra-specific variation in body size and appendage size. Yet, the mechanism by which insulin signaling influences the scaling of growth remains unclear. Here we will discuss the potential roles of insulin signaling in wing-body scaling in Lepidoptera. We analyzed the growth of wings in animals reared on different diet qualities that induce a range of body sizes not normally present in our laboratory populations. By growing wings in tissue culture, we survey how perturbation and stimulation of insulin/TOR signaling influences wing growth. To conclude, we will discuss the implications of our findings for the development and evolution of organismal form.

Three new sulfated isoguanine alkaloid glycosides, designated as saikachinoside A monosulfate (1), saikachinoside A disulfate (2), and locustoside B disulfate (3), have been isolated from the pupal case of the wild bruchid seed beetle Bruchidius dorsalis (Chrysomelidae, Bruchinae) infesting the seed of Gleditsia japonica Miq. (Fabaceae). Their structures were determined by spectroscopic methods and the inhibitory activity of 2 and 3 against acid phosphatase was evaluated.