Imidacloprid-induced mortality, histopathology and biochemical impairments in the larvae of oriental latrine fly (Chrysomya megacephala).

The concentration-mortality response of third instar larvae of Chrysomya megacephala (Diptera: Calliphoridae) to a synthetic insecticide, imidacloprid, and its impact on histopathological, histochemical, and biochemical parameters were determined in laboratory assays. Larvae displayed a concentration and time-dependent mortality response for the insecticide. Histopathological studies exhibited quite noticeable modifications in the epithelial cells, peritrophic membrane, basement membrane and muscular layer of the larval midgut. The ultrastructural analysis demonstrated alterations in nuclei, lipid spheres, microvilli, mitochondria, rough endoplasmic reticulum and lysosomes. In addition, histochemical tests on the midgut were performed, which revealed a strong reaction for proteins and carbohydrates in the control group and a weak reaction in the group exposed to imidacloprid in a dose and time-dependent manner. Imidacloprid also caused a significant reduction in the total midgut content of carbohydrates, proteins, lipids and cholesterol. Larvae treated with imidacloprid also showed a reduction in the activities of acid and alkaline phosphatases at all concentrations compared to untreated larvae.

Comparative morphology of antennal ultrastructure in Tachinidae parasitoid flies (Diptera): The phylogenetic importance of antennal sensilla.

Tachinidae are one of the most diverse clades of Diptera. All tachinids are parasitoids of insects and other arthropods, and thus are considered an important source of biological pest control. Antennae are the most important olfactory organs of Tachinidae playing key roles in their lives, especially in locating hosts, and details of antennal ultrastructure could provide useful features for phylogenetic studies and understanding their adaptive evolution. Despite the ecological and evolutionary importance of antennae, the current knowledge of antennal ultrastructure is scarce for Tachinidae. Our study examined antennal sensilla of thirteen species belonging to thirteen genera within eleven tribes of all the four subfamilies (Phasiinae, Dexiinae, Tachininae, and Exoristinae): Beskia aelops Walker, Trichodura sp., Voria ruralis (Fallén), Zelia sp., Cylindromyia carinata Townsend, Phasia xenos Townsend, Neomintho sp., Genea australis (Townsend), Copecrypta sp., Hystricia sp., Belvosia sp., Leschenaultia sp., and Winthemia pinguis (Fabricius). Types, length and distribution of antennal sensilla were investigated via scanning electron microscopy (SEM). Our comparative analysis summarized 29 variable characters and we evaluated their phylogenetic signal for subfamilial, tribal and generic/specific levels, showing that antennal ultrastructure could be a reliable source of characters for phylogenetic analysis. Our findings demonstrate the remarkable diversity of the antennal ultrastructure of Tachinidae.

Hydrophobic-hydrophilic crown-like structure enables aquatic insects to reside effectively beneath the water surface.

Various insects utilise hydrophobic biological surfaces to live on the surface of water, while other organisms possess hydrophilic properties that enable them to live within a water column. Dixidae larvae reside, without being submerged, just below the water surface. However, little is known about how these larvae live in such an ecological niche. Herein, we use larvae of Dixa longistyla (Diptera: Dixidae) as experimental specimens and reveal their characteristics. A complex crown-like structure on the abdomen consists of hydrophobic and hydrophilic elements. The combination of these contrasting features enables the larvae to maintain their position as well as to move unidirectionally. Their hydrophobic region leverages water surface tension to function as an adhesive disc. By using the resistance of water, the hydrophilic region serves as a rudder during locomotion.

A Study of the Pupal Development of Five Forensically Important Flies (Diptera: Brachycera).

Holometabolous insects undergo complete metamorphosis, and hence, they have different phases of development (egg, larva, pupa, and adult), which occupy distinct ecological niches. The pupae of several fly species are surrounded by the puparium, which is a rigid structure, usually formed by the integument of the last larval instar. The puparium presents unique characteristics distinct from those of the larval and adult phases. During intrapuparial development, it is possible to distinguish at least four fundamental and continuous steps, namely: 1) larval-pupal apolysis, 2) cryptocephalic pupa, 3) phanerocephalic pupa, and 4) pharate adult. The objective of this work was to describe the external morphology of the distinct phase of development for five species that were collected, identified, and raised in the laboratory; intrapuparial development was studied by fixing immature specimens at regular intervals; the morphological analyses were performed with the aid of both light and scanning electron microscopy. Under the conditions established (27 ± 1.0 or 23 ± 1.0°C, 60 ± 10% relative humidity, 12 h of photoperiod), the minimum time for intrapuparial development was: 252 h for Megaselia scalaris (Loew 1966) (Phoridae), 192 h for Piophila casei (Linnaeus 1758) (Piophilidae), Fannia pusio (Wiedemann 1830) (Fanniidae), and Musca domestica (Linnaeus 1758) (Muscidae), and 96 h for Chrysomya megacephala (Fabricius 1794) (Calliphoridae). Intrapuparial development has defined steps, and distinct species responded differently to the same environmental conditions. In addition, it is possible to establish a sequential rule without ignoring the specific characteristics of each taxon.

Fine Structure of Maxillary Palps in Adults of Hermetia illucens (Diptera: Stratiomyidae).

A relevant species in waste management but also in forensic, medical, and veterinary sciences is the black soldier fly, Hermetia illucens (Linnaeus; Diptera: Stratiomyidae). An ultrastructural study by scanning electron microscopy (SEM) was conducted for the first time on maxillary palps of both sexes, describing in detail the morphology and distribution of sensilla and microtrichia. The maxillary palps, composed of two segments, show sexual dimorphism in length and shape. In both sexes, the first segment is covered only by microtrichia, but the second one is divided into two parts: the proximal one, covered only by microtrichia, and the distal one containing both microtrichia and sensory structures. These structures include two types of sensory pits and one of chaetic sensilla. Due to sexual dimorphism in palp size, females have a higher number of sensory pits. The sexual dimorphism of palps and the presence and role of sensilla in H. illucens was discussed in comparison to other species of the family Stratiomyidae and of other Diptera. This study may represent a base for further investigations on mouthpart structures of this species, involved in key physiological activities, such as feeding, mating and oviposition.

Morphology and ultrastructure of the spermatozoa of Lonchoptera lutea Panzer, 1809 (Diptera: Lonchopteridae).

Lonchoptera lutea males produce giant spermatozoa that are more than 2000 µm long and 1.4 µm wide. Unlike the typical brachyceran spermatozoon, they have a highly asymmetrical cross-section with only a single, albeit very large, mitochondrial derivative and a pair of massive accessory bodies, one of which extends throughout the entire length of the sperm tail. The accessory bodies consist of an electron-dense matrix in which numerous peculiar electron-lucid substructures are embedded. In the mated female, the giant spermatozoa are found inside two tubular spermathecae which are also extremely long, measuring 4000 µm or more.

Visual system characterization of the obligate bat ectoparasite Trichobius frequens (Diptera: Streblidae).

As an obligate ectoparasite of bats, the bat fly Trichobius frequens (Diptera: Streblidae) inhabits the same subterranean environment as their nocturnal bat hosts. In this study, we characterize the macromorphology, optical architecture, rhabdom anatomy, photoreceptor absorbance, and opsin expression of the significantly reduced visual system in T. frequens resulting from evolution in the dark. The eyes develop over a 21-22 day pupal developmental period, with pigmentation appearing on pupal day 11. After eclosion as an adult, T. frequens eyes consist of on average 8 facets, each overlying a fused rhabdom consisting of anywhere from 11 to 18 estimated retinula cells. The dimensions of the facets and fused rhabdoms are similar to those measured in other nocturnal insects. T. frequens eyes are functional as shown by expression of a Rh1 opsin forming a visual pigment with a peak sensitivity to 487 nm, similar to other dipteran Rh1 opsins. Future studies will evaluate how individuals with such reduced capabilities for spatial vision as well as sensitivity still capture enough visual information to use flight to maneuver through dark habitats.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.

An in-depth description of head morphology and mouthparts in larvae of the black soldier fly Hermetia illucens.

The larvae of the black soldier fly (BSF) Hermetia illucens are increasingly being used for waste management purposes given their ability to grow on a wide range of organic decaying materials. Although significant efforts have been spent to improve the mass rearing of BSF larvae on specific substrates and their bioconversion capability, little is known about the biology of this insect, especially with regards to the digestive system. In this study, we analyzed the morphology of the head and buccal apparatus of H. illucens larvae by using optical and scanning electron microscopy, evaluating the different mouthparts and their modifications during larval development. Our analysis showed that the larval head of H. illucens presents similarities to those of campodeiform insect larvae, whereas the mandibular-maxillary complex represents a food intake solution typical of Stratiomyidae that enables BSF larvae to ingest semiliquid food. The mouthparts resemble a "tunnel boring machine", where the hypopharynx separates finer organic particles from coarser and inorganic ones.

Mechanisms of programmed cell death in the midgut and salivary glands from Bradysia hygida (Diptera: Sciaridae) during pupal-adult metamorphosis.

Programmed cell death is involved with the degeneration/remodeling of larval tissues and organs during holometabolous development. The midgut is a model to study the types of programmed cell death associated with metamorphosis because its structure while degenerating is a substrate for the formation of the adult organ. Another model is the salivary glands from dipteran because their elimination involves different cell death modes. This study aimed to investigate the models of programmed cell death operating during midgut replacement and salivary gland histolysis in Bradysia hygida. We carried out experiments of real-time observations, morphological analysis, glycogen detection, filamentous-actin localization, and nuclear acridine orange staining. Our findings allow us to establish that an intact actin cytoskeleton is required for midgut replacement in B. hygida and nuclear condensation and acridine orange staining precede the death of the larval cells. Salivary glands in histolysis present cytoplasmic blebbing, nuclear retraction, and acridine orange staining. This process can be partially reproduced in vitro. We propose that the larval midgut death involves autophagic and apoptotic features and apoptosis is a mechanism involved with salivary gland histolysis.

Evolutionary adaptations in four hippoboscid fly species belonging to three different subfamilies.

Lipoptena cervi (Linnaeus, 1758), Lipoptena fortisetosa Maa, 1965, Hippobosca equina Linnaeus, 1758, and Pseudolynchia canariensis (Macquart, 1840) (Diptera: Hippoboscidae) are haematophagous ectoparasites that infest different mammal and bird species and occasionally attack humans. They are known for the health implications they have as vectors of pathogens to humans and animals, and for the injuries they inflict on their host's skin. This study focused on the morphological structures evolved by parasites in terms of their biology and the different environment types that they inhabit. To this aim, we examined four hippoboscid species, as well as their hosts' fur (ungulate and horse), and feather (pigeon) through light and Scanning Electron Microscopy (SEM) observations in order to highlight the main morphological features that evolved differently in these flies and to explain the effect of hosts' fur/feather microhabitats on the morphological specializations observed in the investigated ectoparasites. The studied species showed main convergent characters in mouthparts while remarkable differences have been detected on the antennal sensillar pattern as well as on the leg acropod that displayed divergent characters evolved in relation to the host.

A world review of reported myiases caused by flower flies (Diptera: Syrphidae), including the first case of human myiasis from Palpada scutellaris (Fabricius, 1805).

Rat-tailed larvae of the syrphid species Palpada scutellaris (Fabricius, 1805) are documented causing an enteric human myiasis in Costa Rica. This is the first time that the genus Palpada is recorded as a human myiasis agent. We report a 68-year-old woman with intestinal pain and bloody diarrhea with several live Palpada larvae present in the stool. Using molecular techniques (DNA barcodes) and both electronic and optical microscopy to study the external morphology, the preimaginal stages of the fly were unambiguously identified. An identification key to all syrphid genera actually known as agents of human and animal myiases is provided for larvae, puparia, and adults. Moreover, a critical world review of more than 100 references of Syrphidae as myiasis agents is also given, with emphasis on the species with rat-tailed larvae.

The importance of insect sperm: Sperm ultrastructure of Hermetia illucens (black soldier fly).

Sperm structure and ultrastructure of Hermetia illucens was determined by light microscopy and transmission electron microscopy. The main sperm components were similar as for other Dipteran subspecies, while the ultrastructure revealed distinguishing features in the zone of overlap and anterior flagellar region. Sperm varied in size indicating sperm polymorphism. The head region is lacking an acrosome. The zone of overlap consisted of uniquely organized centriolar adjunct material, partly forming electron dense areas to finally form an outer ring separating the mitochondrial derivatives from the 9 + 9 + 2 axoneme. Accessory bodies arising from the zone of overlap are flanked by smaller to large mitochondrial derivatives into the anterior flagellum. This study confirms sperm structure diversity between brachyceran subspecies and support its relationship with nematoceran subspecies.

Insecticide activity of Curcuma longa (leaves) essential oil and its major compound α-phellandrene against Lucilia cuprina larvae (Diptera: Calliphoridae): Histological and ultrastructural biomarkers assessment.

Lucilia cuprina, known as the Australian blowfly, is of high medico-sanitary and veterinary importance due to its ability to induce myiasis. Synthetic products are the most frequent form of fly control, but their indiscriminate use has selected for resistant populations and accounted for high levels of residues in animal products. This study aimed to assess the effect of essential oil from leaves of Curcuma longa (CLLEO), and its major compound α-phellandrene against L. cuprina L3. An additional goal was to determine the morphological alterations in target organs/tissues through ultrastructural assessment (SEM) and light microscopy, as well as macroscopic damage to cuticle induced by CLLEO. Groups of 20 L3 were placed on filter paper impregnated with increasing concentrations of CLLEO (0.15 to 2.86 µL/cm2) and α-phellandrene (0.29 to 1.47 µL/cm2). Efficacy was determined by quantifying L3 mortality 6, 24 and 48 h after contact with CLLEO and by measuring the structural damage to L3. CLLEO and α-phellandrene inhibited adult emergence by 96.22 and 100%, respectively. Macroscopic cuticle damage, appeared as diffuse pigment and darkening of larval body, was caused by both extracts. The SEM revealed dryness on the cuticle surface, distortion of the sensorial structures and general degeneration in treated L3. Furthermore, alterations in target organs (digestive tract, fat body and brain) were noticed and shall be used as biomarkers in future attempts to elucidate the mechanism of action of these compounds. The vacuolar degeneration and pyknotic profiles observed in the brain tissue of treated larvae with both extracts and the decreased motility within <6 h after treatment leads us to suggest a neurotoxic activity of the products. This work demonstrates the potential use of CLLEO and α-phellandrene as bioinsecticides to be used against L. cuprina, representing an ecofriendly alternative for myiasis control in humans and animals.

Characterization of antennal chemosensilla and associated odorant binding as well as chemosensory proteins in the Eupeodes corollae (Diptera: Syrphidae).

Aphidophagous syrphids are important for pest control and pollination in various agroecosystems. However, the mechanism underlying olfaction, which is critical for insect' behavioral processes and fitness, has not been well understood in the family Syrphidae. Hence, we performed a systematic identification and characterisation of the antennal sensilla and two groups of soluble proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), in the hoverfly Eupeodes corollae. (i) With scanning electron microscopy, four major types of sensilla (chaetic sensilla [two subtypes], trichoid sensilla, basiconic sensilla [two subtypes] and coeloconic sensilla), with numerous microtrichia, were first observed along the entire surface of aristate antennae of both sexes of E. corollae. Of these, only chaetic sensillum was found on the first two antennal segments, scape and pedicel, while the other types of sensilla were located on the flagellum. No marked difference was observed in the morphological structure or distributional pattern of any of the sensilla between the two sexes. (ii) By molecular cloning and bioinformatic analysis, 7 EcorCSPs and 28 EcorOBPs (20 classic OBPs, 5 minus-C OBPs, and 3 plus-C OBPs) were directly identified from the species, which all share the characteristic hallmarks of their family, including the presence of a signal peptide and conserved cysteine signature. (iii) RT-qPCR of these chemosensory genes showed predominately tissue-biased expression patterns; 32 of the 35 EcorOBPs/CSPs were uniquely or primarily expressed in the main olfactory organs, either the antennae or head. (iv) Among these, several genes (EcorCSP2 and EcorOBP1, 9, 12, 15-17, 20) appeared to be antenna-biased. In situ hybridization assays indicated that each antenna-biased chemosensory gene was expressed in a different number of cells, suggesting they might play a more vital role in odour recognition and perception and could be potential candidates to study their biological functions in vivo and in vitro. Together, our current findings provide a basis for future studies on how syrphids utilize chemical cues to regulate their behavior during interactions among the natural enemy, its prey, and host plant in agro-ecosystems.

Comparative morphology of the deer ked Lipoptena fortisetosa first recorded from Italy.

Hippoboscidae flies parasitize various animal species. Knowledge about these insects remains sparse, although they are known to cause stress and damage to their hosts, and can also accidentally infest humans, causing different sanitary risks. Research conducted in Tuscany assessing the biology and distribution of Lipoptena cervi (Linnaeus, 1758) (Diptera: Hippoboscidae), the most common ectoparasite of ungulates in Italy, revealed the presence of Lipoptena fortisetosa Maa, 1965 in Italy for the first time. This study includes a morphological comparative description of L. cervi and L. fortisetosa, emphasizing the peculiar differences between the two species to facilitate their accurate identification. The most pertinent morphological differences between the two species are highlighted, such as the external features of the antennae, distribution of bristles, and different features in the external genitalia. In both species, scanning electron microscopy of mouthparts revealed strong adaptive convergence in the feeding apparatus. Modified palps and a very thin proboscis are described in relation to feeding behaviour.

Molecular and morphological characterization of third instar Palaearctic horse stomach bot fly larvae (Oestridae: Gasterophilinae, Gasterophilus).

Species of Gasterophilus are obligate parasites of equids and may induce severe, even lethal myiasis. However, identification of the third instar Gasterophilus larva at the species level is still problematic predominantly due to a shortage of diagnostic morphological features and incomplete molecular libraries. Testing the suitability of three different molecular markers showed that the traditional 650 bp barcode region near the 5' terminus of the cytochrome c oxidase subunit I (COI) served as a better tool for species-level identification than a 663 bp region near the 3' terminus of COI and a 554 bp region near the 5' terminus of the large subunit ribosomal RNA. We found that barcoding discriminates G. intestinalis, G. nasalis, G. nigricornis and G. pecorum but not G. haemorrhoidalis and G. inermis. A comparative morphological study using scanning electron microscopy was conducted to promote the identification of the third instar larvae. Photographs of fresh mature third instar larvae are provided for all species, and the remarkable green body colour of third instar G. nigricornis is fully documented for the first time. Two morphological keys are provided, one is suitable for quick identification, and the other based on ultrastructural details is provided for further comparative morphological investigation. A new term 'oral plate' instead of 'mandible' was proposed for a pair of sclerites of uncertain homology emerging from the secondary mouth opening. Our data shows that DNA barcodes cannot replace morphology for identification of third instars of Gasterophilus species, and a scaffold is provided for an integrated taxonomic reference system, which will contribute to monitoring gasterophilosis for equid welfare and protection, and also facilitate further studies in functional anatomy, phylogenetic analyses and host-parasite co-evolutionary investigations of Gasterophilus.

Morphology of immature blow fly Hypopygiopsis infumata (Bigot) (Diptera: Calliphoridae), a potential species of forensic importance.

Blow flies of the genus Hypopygiopsis are forensically-important, as their larvae are commonly associated with human corpses. Within a forensic entomology context, species identification of specimens collected from human corpses is the initial mandatory step in the investigation. Without identification, complete interpretation of entomological evidence is challenged. In this study, the ultrastructures of eggs, all instars, and puparia of Hypopygiopsis infumata (Bigot) are presented based on assessment with scanning electron microscopy (SEM) and light microscopy (LM). Distinctive features used for species identification of all stages are highlighted. Eggs have a slightly widening median area extending almost the entire length. Larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures (i.e., antennal dome, maxillary palpus and ventral organ). In the first instar, two tufts of cirri are observed along the dorsal margin of the mouth opening. In the second and third instars, six minute tubercles are present along the peripheral rims of the last abdominal segment. The anterior spiracle of the second, third instar, and puparia is fan-shape of single row, comprising 9-11 papillae. The cuticular spines between the 1st and 2nd thoracic segments of the third instar possess many rows of posteriorly-projecting acuminate spines in clusters. In puparia, at the latero-dorsal edge of the 1st abdominal segment, a cluster of ∼92 bubble membranes is present in young puparia (20-24 h). The peristigmatic tufts adjacent to the posterior spiracle of the second instar, third instar, and puparia are heavily branches of long, fine hairs. Our results demonstrate the morphology of eggs, larvae, and puparia of H. infumata are similar to other species in Hypopygiopsis. This study highlighted the main features of cephaloskeleton of H. infumata larvae as observed under LM. Particular attention is given to oral sclerite and rough surface of dorsal cornua which can distinguish between H. infumata and H. tumrasvini.

Bioprospection of immature salivary glands of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae).

Larval therapy (LT) comprises the application of sterile Calliphoridae larvae for wound debridement, disinfection, and healing in humans and animals. Larval digestion plays a key role in LT, where the salivary glands and gut produce and secrete proteolytic and antimicrobial substances. The objective of this work was to bioprospect the salivary glands of Chrysomya megacephala (Fabricius, 1794) larvae, using ultrastructural, morphological, and histological observations, and the total protein electrophoretic profile. The salivary glands present a deferent duct, originating from the buccal cavity, which bifurcates into efferent ducts that insert through a slight dilatation to a pair of tubular-shaped tissues, united in the region of fat cells. Histologically, the secretion had protein characteristics. Cell cytoplasm presented numerous free ribosomes, autophagic vacuoles, spherical and elongated mitochondria, atypical Golgi complexes, and dilated rough endoplasmic reticulum. In the apical cytoplasm, secretory granules and microvilli secretions demonstrated intense protein synthesis, basal cytoplasm with trachea insertions, and numerous mitochondria. The present work described the ultrastructure and morphology of C. megacephala third instar salivary glands, confirming intense protein synthesis and the molecular weight of soluble proteins.

Diversity and common themes in the organization of ocelli in Hymenoptera, Odonata and Diptera.

We show in a comparative analysis that distinct retinal specializations in insect ocelli are much more common than previously realized and that the rhabdom organization of ocellar photoreceptors is extremely diverse. Hymenoptera, Odonata and Diptera show prominent equatorial fovea-like indentations of the ocellar retinae, where distal receptor endings are furthest removed from the lens surface and receptor densities are highest. In contrast, rhabdomere arrangements are very diverse across insect groups: in Hymenoptera, with some exceptions, pairs of ocellar retinular cells form sheet-like rhabdoms that form elongated rectangular shapes in cross-section, with highly aligned microvilli directions perpendicular to the long axis of cross-sections. This arrangement makes most ocellar retinular cells in Hymenoptera sensitive to the direction of polarized light. In dragonflies, triplets of retinular cells form a y-shaped fused rhabdom with microvilli directions oriented at 60° to each other. In Dipteran ocellar retinular cells microvilli directions are randomised, which destroys polarization sensitivity. We suggest that the differences in ocellar organization between insect groups may reflect the different head attitude control systems that have evolved in these insect groups, but possibly also differences in the mode of locomotion and in the need for celestial compass information.