Different Sensitivity of Flower-Visiting Diptera to a Neonicotinoid Insecticide: Expanding the Base for a Multiple-Species Risk Assessment Approach.

Insects play an essential role as pollinators of wild flowers and crops. At the same time, pollinators in agricultural environments are commonly exposed to pesticides, compromising their survival and the provision of pollination services. Although pollinators include a wide range of species from several insect orders, information on pesticide sensitivity is mostly restricted to bees. In addition, the disparity of methodological procedures used for different insect groups hinders the comparison of toxicity data between bees and other pollinators. Dipterans are a highly diverse insect order that includes some important pollinators. Therefore, in this study, we assessed the sensitivity of two hoverflies (Sphaerophoria rueppellii, Eristalinus aeneus) and one tachinid fly (Exorista larvarum) to a neonicotinoid insecticide (Confidor®, imidacloprid) following a comparative approach. We adapted the standardized methodology of acute contact exposure in honey bees to build dose-response curves and calculate median lethal doses (LD50) for the three species. The methodology consisted in applying 1 µL of the test solution on the thorax of each insect. Sphaerophoria rueppelli was the most sensitive species (LD50 = 10.23 ng/insect), and E. aeneus (LD50 = 18,176 ng/insect) the least. We then compared our results with those available in the literature for other pollinator species using species sensitivity distribution (SSD). Based on the SSD curve, the 95th percentile of pollinator species would be protected by a safety factor of 100 times the Apis mellifera endpoint. Overall, dipterans were less sensitive to imidacloprid than most bee species. As opposed to most bee species, oviposition and fecundity of many dipteran species can be reliably assessed in the laboratory. We measured the number of eggs laid following exposure to different insecticide doses and assessed the potential trade-off between oviposition and survival through the sublethal sensitivity index (SSI). Exposure to imidacloprid had a significant effect on fecundity, and SSI values indicated that oviposition is a sensitive endpoint for the three dipteran species tested. Future studies should integrate this information related to population dynamics in simulation models for environmental risk assessment.

Correction: Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

[This corrects the article DOI: 10.1371/journal.pone.0292043.].

Immunophenotyping of hemocytes from infected Galleria mellonella larvae as an innovative tool for immune profiling, infection studies and drug screening.

In recent years, there has been a considerable increasing interest in the use of the greater wax moth Galleria mellonella as an animal model. In vivo pharmacological tests, concerning the efficacy and the toxicity of novel compounds are typically performed in mammalian models. However, the use of the latter is costly, laborious and requires ethical approval. In this context, G. mellonella larvae can be considered a valid option due to their greater ease of use and the absence of ethical rules. Furthermore, it has been demonstrated that the immune system of these invertebrates has similarity with the one of mammals, thus guaranteeing the reliability of this in vivo model, mainly in the microbiological field. To better develop the full potential of this model, we present a novel approach to characterize the hemocyte population from G. mellonella larvae and to highlight the immuno modulation upon infection and treatments. Our approach is based on the detection in isolated hemocytes from G. mellonella hemolymph of cell membrane markers typically expressed by human immune cells upon inflammation and infection, for instance CD14, CD44, CD80, CD163 and CD200. This method highlights the analogies between G. mellonella larvae and humans. Furthermore, we provide an innovative tool to perform pre-clinical evaluations of the efficacy of antimicrobial compounds in vivo to further proceed with clinical trials and support drug discovery campaigns.

Effect of 2 sex-sorting time schedules on SIT facility management.

Improvements are needed in mosquito mass-rearing to effectively implement the sterile insect technique (SIT). However, managing this technique is challenging and resource intensive. SIT relies on mass rearing, sterilization, and release of adult males to reduce field populations. Maintaining an acceptable level of female presence, who can transmit viruses through biting, is crucial. Females are also essential for facility sustainability. Sex sorting plays a vital role in the production process, and our current mechanical sorting approach aims to obtain a high number of adult males with minimal female contamination within 24 h of pupation. Utilizing protandry helps control female contamination. While the 24-h sorting period achieves desired contamination levels, it may not yield enough females to sustain breeding lines, leading to increased labor costs that impact project sustainability. By delaying the sorting procedure to 48 h, we obtained sufficient females to sustain breeding lines, achieving a balance between male production and female contamination using the automatic version of the Fay-Morlan device as the sorting tool.

Host stage and temperature for the rearing of Aridelus rufotestaceus (Hymenoptera: Braconidae), with notes on acceptance and suitability of 2 stink bug species.

Nezara viridula (L.) (Hemiptera: Pentatomidae) is a harmful pest of many agricultural crops in different parts of the world. This stink bug is the preferred host species of Aridelus rufotestaceus Tobias (Hymenoptera: Braconidae), an endoparasitoid of nymphs and adults of pentatomids. With the aim to improve the rearing procedure of this beneficial insect, the acceptance and suitability of all mobile stages of N. viridula (from first instar nymph to adult) were evaluated. At 25 °C, all host stages were accepted and suitable for A. rufotestaceus development, but the highest parasitoid cocoon and adult yields were obtained from second instar nymphs. The possibility to reduce the development time of A. rufotestasceus by increasing the rearing temperature was also evaluated, but 28 °C proved to be detrimental for parasitoid development, as shown by the very low cocoon and adult numbers obtained. The acceptance and suitability of the invasive pentatomid species Halyomorpha halys (Stål) for A. rufotestaceus was also tested. Female wasps were observed piercing H. halys nymphs with the ovipositor, but no cocoons were obtained, nor were larvae or head capsules detected in the exposed stink bugs.

Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

The "Sterile Insect Technique" (SIT), a promising method to control Aedes albopictus, the Asian tiger mosquito, is gaining increasing interest. Recently, the role of microbiota in mosquito fitness received attention, but the link between microbiota and larval diet in mass rearing programs for SIT remains largely unexplored. We characterized the microbiota of four larval instars, pupae and eggs of non-wild (NW) lab-reared Ae. albopictus fed with a diet based on Black soldier fly (Hermetia illucens) larvae powder and fish food KOI pellets. We compared it with wild (W) field-collected individuals and the bacterial community occurring in rearing water-diet (DIET). A total of 18 bacterial classes with > 0.10% abundance were found overall in the samples, with seven classes being especially abundant. Overall, the microbiota profile significantly differed among NW, W and DIET. Verrucomicrobiae were significantly more abundant in W and DIET, Bacteroidia were more abundant in NW and DIET, and Gammaproteobacteria were only more abundant in W than in DIET. W-eggs microbiota differed from all the other groups. Large differences also appeared at the bacterial genus-level, with the abundance of 14 genera differing among groups. Three ASVs of Acinetobacter, known to have positive effects on tiger mosquitoes, were more abundant in NW than in W, while Serratia, known to have negative or neutral effects on another Aedes species, was less abundant in NW than in W. The bacterial community of W-eggs was the richest in species, while dominance and diversity did not differ among groups. Our data show that the diet based on Black soldier fly powder and fish food KOI influences the microbiota of NW tiger mosquito immature stages, but not in a way that may suggest a negative impact on their quality in SIT programs.

Edible Insects: A Historical and Cultural Perspective on Entomophagy with a Focus on Western Societies.

The relationship between insects and humans throughout history has always been complex and multifaceted. Insects are both a source of fascination and fear for humans and have played important roles in human culture, economy, and health. Nowadays, there is growing interest in using insects as a sustainable and environmentally friendly source of protein and other nutrients. Entomophagy can be seen as a new opportunity for the food industry and global food security. In fact, insects require far fewer resources than traditional livestock, and there are many references to insect consumption in human history. The ancient Romans are known to have eaten various insects, including beetles, caterpillars, and locusts. Insects such as crickets, grasshoppers, and ants have been eaten for centuries and are still considered a delicacy in many parts of the world, especially in Africa, Asia, Latin America, and Oceania. Entomophagy has, thus, been a part of human history for thousands of years and continues to be an important food habit for many people around the world. These topics are explored in this article from a historical and cultural perspective (e.g., ecological, nutritional, spiritual, and socio-psychological), with a focus on the progressive acceptance of edible insects in Western societies, since this novel food has also its roots in the Western world.

Aedes albopictus Sterile Male Production: Influence of Strains, Larval Diet and Mechanical Sexing Tools.

The sterile insect technique (SIT) is a biologically based method of pest control, which relies on the mass production, sterilization, and release of sterile males of the target species. Since females can transmit viruses, it is important to develop a mass rearing system to produce a large number of males with a low presence of females. We evaluated the effects of different strains, larval diets and sexing tools on male productivity and residual female presence for the application of SIT against Aedes albopictus. Strains coming from Italy, Germany, Greece, and Montenegro, with different levels of colonization, were reared with three larval diets: IAEA-BY, BLP-B and SLP-BY. Developed pupae were sexed using two different mechanical methods: sieve or Fay-Morlan separator. The results proved that adoption of the Fay-Morlan separator increased the productivity and limited the female presence. The IAEA-BY diet showed the lowest female contamination. Strains with a high number of breeding generations showed a decreased productivity and an increased female presence. Increased female presence was found only in extensively reared strains and only when the sorting operation was conducted with sieves. We hypothesize that extensive colonization may determine a size reduction which limits the sexing tool efficiency itself.

Monarda didyma Hydrolate Affects the Survival and the Behaviour of Drosophila suzukii.

Drosophila suzukii (Matsumara) is an herbivorous pest whose control in the field with conventional chemical is particularly difficult and has important drawbacks. Here, we investigated the insecticidal properties of hydrolate from Monarda didyma, scarlet beebalm, an aromatic herb in the Lamiaceae family. The identification of volatile organic compounds (VOCs) by CG-MS systems revealed that thymol (38%) and carvacrol (59%) were the most abundant VOCs in the hydrolate. M. didyma hydrolate did not show fumigant toxicity. Conversely, in contact assays, M. didyma hydrolate showed a LC50 of 5.03 µL mL-1, 48 h after the application on D. suzukii adults. Expression of detoxification genes increased in flies that survived the LC50 application. Furthermore, toxicity persisted for 7 days after the treatment in the survival evaluation. Artificial diet assays with 100 and 1000 µL mL-1 of M. didyma hydrolate resulted in a significant decrease in total food intake in both male and female D. suzukii adults. In addition, electropenetrography (EPG) showed that the D. suzukii females' feeding behaviour was altered in hydrolate-treated diets. The hydrolate also caused a significant reduction in the number of eggs laid in two different oviposition assays. Overall, our findings provide a new perspective for the improvement of D. suzukii control strategies through M. didyma hydrolate.

Implementing Mass Rearing of Trissolcus japonicus (Hymenoptera: Scelionidae) on Cold-Stored Host Eggs.

Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), a pest of Asian origin, has been causing severe damage to Italian agriculture. The application of classical biological control by the release of Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae), an exotic egg parasitoid, appears to be one promising solution. In Italy, releases of T. japonicus in the field were authorized in 2020. In this study, some parameters that could influence the rearing of T. japonicus in insectaries were investigated. A significantly higher production of progeny was observed on host eggs stored at 6 °C (86.5%) compared to -24 °C (48.8%) for up to two months prior to exposure to parasitism. There were no significant differences in progeny production from single females in a vial provided with only one egg mass (83.2%) or 10 females inside a cage with 6 egg masses (83.9%). The exposure of parasitoids to refrigerated (6 °C) egg masses of H. halys for 72 h led to a significantly higher production of progeny (62.1%) compared to shorter exposures for 48 (44.0%) or 24 h (37.1%). A decline in production of progeny by the same female was detected between the first (62.1%) and the second parasitization (41.3%). Adult parasitoids stored at 16 °C for up to 90 days had an 87.1% survival rate, but a significant decrease in progeny production was detected. These parameters could be adjusted when rearing T. japonicus for specific aims such as the production of individuals for field release or colony maintenance.

The Possible Role of Microorganisms in Mosquito Mass Rearing.

In Europe, one of the most significant mosquitoes of public health importance is Aedes albopictus (Skuse), an allochthonous species of Asian origin. One of the most promising control methods against Aedes albopictus is the sterile insect technique (SIT), which consists of mass rearing the target species, separation of males from females, and male exposure to sterilizing ionizing radiation. Once released in the environment, the sterile males are expected to search for wild females to mate with. If mating occurs, no offspring is produced. The quality of sterile males is a crucial aspect in SIT programs in order to optimize effectiveness and limit production costs. The integration of probiotic microorganisms in larval and adult mosquito diets could enhance the quality parameters of the released sterile males. In this review, we attempt to give the most representative picture of the present knowledge on the relationships between gut microbiota of mosquitoes and the natural or artificial larval diet. Furthermore, the possible use of probiotic microorganisms for mosquito larvae rearing is explored. Based on the limited amount of data found in the literature, we hypothesize that a better understanding of the interaction between mosquitoes and their microbiota may bring significant improvements in mosquito mass rearing for SIT purposes.

Halyomorpha halys (Hemiptera: Pentatomidae) on Kiwifruit in Northern Italy: Phenology, Infestation, and Natural Enemies Assessment.

The brown marmorated stink bug (BMSB), Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is an Asian invasive plant-feeding insect and an emerging kiwifruit pest. Knowledge about the BMSB dynamics and damage to kiwifruit outside the bug native range is scarce. This 2-yr study was aimed at describing phenology and infestation dynamics of BMSB in green-fleshed and yellow-fleshed kiwifruits. Natural enemies were investigated as well. Field surveys were performed weekly in two sites in Northern Italy by pheromone-baited traps and visual samplings, from early Spring to mid-Autumn. All BMSB life stages were collected and kept under observation to assess occurrence of parasitoids. A sample of fruit was dissected weekly to detect BMSB feeding injuries. In 2018-2019, BMSB was found throughout the growing season with two generations per year with a large overlapping of all life stages. Pheromone-baited traps and visual samplings gave consistent results and can be both considered effective sampling methods on kiwifruit. Fruit injuries were significantly related to kiwifruit development stage, weekly captures of BMSBs by traps, and cultivar. Several BMSB egg masses were found on kiwifruit leaves. Anastatus bifasciatus Geoffroy (Hymenoptera: Eupelmidae), a native egg parasitoid with Palearctic distribution, emerged from 20.14% of eggs in 2018, but the percent parasitism dropped to 0.47% in 2019. In both years, other natural enemies were found exerting negligible pressure on BMSB populations. The determination of reliable economic thresholds for BMSB on kiwifruit is urgently needed to develop a robust and sustainable integrated pest management (IPM) strategy, and this study provides data towards that direction.

Improvements in the Rearing of the Tachinid Parasitoid Exorista larvarum (Diptera: Tachinidae): Influence of Adult Food on Female Longevity and Reproduction Capacity.

Exorista larvarum (L.), a polyphagous gregarious larval parasitoid of lepidopterans, can be mass produced both in vivo, using the greater wax moth Galleria mellonella (L.) (Lepidoptera: Pyralidae) as a factitious host, and in vitro, on artificial media composed of crude components. The present study was focused on another aspect of E. larvarum rearing, namely the influence of adult food on parasitoid performance. The standard food, consisting of lump sucrose and cotton balls soaked in a honey and water solution (1), was compared with other foods or food combinations, namely lump sucrose alone (2), honey and water solution (3), sucrose and water solution either alone (4) or combined with bee-collected pollen (5), and, finally, pollen alone (6). All foods were provided together with distilled water supplied in drinking troughs. Based on the parameters considered (i.e., female longevity, number of eggs laid on host larvae, puparia obtained from eggs, and adults emerged from puparia), pollen alone was deemed to be the most suitable food for adult females of E. larvarum. In particular, the pollen showed a longevity-promoting effect, increasing the number of eggs laid on host larvae throughout the female lifespan. The use of this adult food may also result in a higher flexibility of the management of E. larvarum colonies because it can be replaced weekly, as no desiccation or mold infections were ever found to occur.

Oviposition Strategies in Beneficial Insects.

AIMS AND SCOPE: The aim of this special collection is to highlight the importance of reproduction (ie, oviposition) in the life history, population dynamics, survival, and fitness of beneficial insects, broadly defined. Specific areas of interest include the (1) identification of natural products to boost oviposition; (2) importance of chemical cues in oviposition site selection; (3) influence of host plant defenses on oviposition success; (4) reproductive physiology and the frequency of egg laying; (5) trade-offs between maternal size and egg size; (6) foraging behavior, host selection, and oviposition in parasitoids; and (7) oviposition decisions in the face of intraguild predators.

Oviposition Strategies of Tachinid Parasitoids: Two Exorista Species as Case Studies.

Oviposition strategies and mechanisms of host selection in parasitoids may be crucial for the success of parasitization and parasitoid production. These aspects are far less known in tachinid parasitoids than in hymenopteran parasitoids. Depending on the species, parasitoid flies may adopt direct or indirect oviposition strategies. The 'direct type' females lay eggs on or, in relatively a few species, inside the host body. This review describes cues involved in host selection by tachinid parasitoids and their oviposition strategies and presents 2 case studies in more detail, focusing on Exorista larvarum and Exorista japonica. These 2 polyphagous parasitoids of Lepidoptera lay macrotype eggs directly on the host cuticle. Both species have been used as biological control agents in inoculative release against the gypsy moth Lymantria dispar in the Northern United States. Improved knowledge of the mechanisms involved in host selection and oviposition strategies may increase the possibility of eliciting oviposition by these tachinids on target lepidopterous hosts (and even artificial substrates), thus facilitating their rearing and ultimately making their exploitation as regulators of target insect pests more feasible and efficient.

Interactions between the Multicolored Asian Lady Beetle Harmonia axyridis and the Parasitoid Dinocampus coccinellae.

Harmonia axyridis (Pallas) has been introduced either intentionally or accidentally in different areas outside its native range, where it is often regarded as invasive. Dinocampus coccinellae (Schrank) has been recorded to parasitize H. axyridis in the field, both in the native and introduced areas, Italy included. The percent of parasitism found in our field investigation was low (four percent). The effect of exposure time of H. axyridis to D. coccinellae and the impact of parasitization on host longevity, oviposition capacity and egg fertility were evaluated in the laboratory. The acceptance and suitability of H. axyridis as host for D. coccinellae were then studied, in comparison with the native coccinellid Adalia bipunctata (L.), which shares the same ecological niche. The effects of parasitization on female longevity and reproduction capacity in the exotic vs. the indigenous lady beetle were also investigated. The overall results showed that D. coccinellae negatively affected the fitness of H. axyridis, more than that of A. bipunctata. The parasitoid may thus play a marginal role in controlling the populations of the Asian lady beetle, without representing a threat to A. bipunctata.

Supplementation of an Artificial Medium for the Parasitoid Exorista larvarum (Diptera: Tachnidae) With Hemolymph of Hermetia illucens (Diptera: Stratiomyidae) or Antheraea pernyi (Lepidoptera: Saturniidae).

The effect of supplementing hemolymph of the black soldier fly, Hermetia illucens (L.), or the Chinese oak silkworm, Antheraea pernyi (Guérin-Méneville), to a basic insect-free artificial medium for the tachinid Exorista larvarum (L.) was investigated. The supplementation (20% w/w) was based on the assumption that insect additives may optimize the media for this parasitoid. Egg hatch, pupal and adult yields, and sex ratio did not differ among the enriched and basic media. Preimaginal development was faster on both hemolymph-enriched media than on the basic medium. Despite the shorter development on the medium supplemented with H. illucens hemolymph than on the basic medium, on the two media puparium weights were comparable. The female flies reared on the medium enriched with H. illucens hemolymph did not lay more eggs, but the latter yielded significantly more puparia compared with the control females. Conversely, the medium enriched with A. pernyi hemolymph yielded lower female puparium weights than the basic medium and produced only one ovipositing female out of the five obtained female adults. These results indicate that the in vitro development of E. larvarum improved when the basic artificial medium was enriched with H. illucens hemolymph, whereas the supplementation with A. pernyi hemolymph negatively affected the quality of the in vitro-reared females.

Studies on Aedes albopictus larval mass-rearing optimization.

To set up a sterile male technique program to control Aedes albopictus (Skuse) in areas in northern Italy, a pilot mass-rearing facility is under development. For this purpose, experiments were carried out to find the optimal larval density for the optimization of the rearing parameters, i.e., to obtain the fastest larval development, the highest larval and pupal survival rate, and large-sized pupae. Several different larval densities, from 40 to 2874 larvae per liter, were tested. For densities from 40 to 600 larvae per liter significant size differences were found among pupae obtained under different larval densities. The larvae raised at the lowest density tended to be smaller and to develop most slowly, i.e., longer pupation time. Also, increasing water volume and depth seemed to negatively affect the pupation success. Compared with the other larval densities tested, the larvae reared at a density of 2874 larvae per liter developed slightly faster and showed higher survival rates, indicating this density as appropriate for the development of a mass rearing, at least using the current larval diet.

Larval morphology and anatomy of the parasitoid Exorista larvarum (Diptera: Tachinidae), with an emphasis on cephalopharyngeal skeleton and digestive tract.

The endogenous development of the tachinid gregarious larval parasitoid Exorista larvarum L. (Diptera: Tachinidae) has been analyzed in the last larval instar of a factitious host, the wax moth Galleria mellonella L. (Lepidoptera: Pyralidae), with the use of histological techniques and scanning electron microscopy. This study has focused on the parasitoid internal body structures and their changes during the larval development. The first and second instars are enveloped by a host-derived hemocyte capsule attached to the respiratory funnel via a prominent anal hook located between 2 anal lobes. The third instar abandons the respiratory funnel and migrates free in the body cavity of the already dead host. Emphasis is given to the prominent cephalopharyngeal skeleton, highlighting the morphological aspects of its sclerotized as well as non-sclerotized components. In addition to the cephalopharyngeal skeleton, the anterior third of the larval parasitoid body is occupied by large salivary glands, massive proventriculus, and cerebral ganglia. The extensive digestive tract, which occupies the major part of the body, is differentiated into well-marked individual parts. The abdomen is predominantly filled with the extremely long mesenteron that increases in size during the larval development. The whole body is covered by an apparently thin integument, with strong spines that are especially numerous in the anterior and posterior body parts.

In vitro rearing of the parasitoid Exorista larvarum (Diptera: Tachinidae) from eggs laid out of host.

We evaluated the possibility of using the eggs laid out of host to rear Exorista larvarum (L.) (Diptera: Tachinidae), a larval parasitoid of Lepidoptera, on artificial media. In a first experiment, eggs oviposited on a plastic sheet (either by inexperienced or experienced females) showed the same in vitro hatching capability as those removed from the larvae of the factitious host Galleria mellonella L. Subsequently, eggs laid on the host integument or out of host, either by inexperienced or experienced females, were removed from the oviposition substrate and placed on a skimmed milkbased artificial medium. The percentages of hatched eggs, of puparia and adults, as well as the puparial weights did not differ significantly among the three treatments. These findings suggested that E. larvarum may be successfully reared in vitro with a total exclusion of the host insect. In a further test, no difference for the in vitro hatching time was found between the eggs oviposited either on a plastic sheet or on G. mellonella larvae in the same length of time (60 min). This result suggested that at oviposition the out-of-host eggs were unincubated, similarly to those that had been laid on the host larvae.