Biology of Callistethus plagiicollis (Coleoptera: Scarabaeoidea: Rutelinae) and comparative morphology of its larvae.

Larvae of the beetle subfamily Rutelinae are poorly described in the literature. Notably, the morphology of the larvae of Callistethus plagiicollis Fairmaire has not previously been analyzed. Here, we report for the first time that these larvae feed on the tubers and roots of Gastrodia elata Blume, an important traditional Chinese herbal medicine, which causes a reduction in the yield and economic value of G. elata. We employed scanning electron microscopy and light microscopy to investigate the morphology and occurrence regularity of egg, larvae, pupae, and adult specimens of C. plagiicollis collected from the G. elata planting base in Guizhou Province, China, with a focus on the ultrastructure of mature larvae. The results revealed one generation of C. plagiicollis per year in the study area and three instar stages of larvae. Mature larvae were identified by the following characteristics: raster without palidia with a large number of hamate setae, antennal apex containing seven sensilla basiconica, larval haptomerum containing eight sensilla styloconica and four enlarged heli, and seven longitudinally arranged stridulatory teeth on the stipes of the maxilla. The combination of scanning electron and light microscopy effectively revealed the difference between membranous and sclerotized structures, ensuring accurate identification of C. plagiicollis larvae. By determining the feeding characteristics and occurrence regularity of C. plagiicollis, this study has implications for improved pest management in G. elata crops. RESEARCH HIGHLIGHTS: We identified C. plagiicollis as a new pest of G. elata, a traditional Chinese medicine Scanning electron and light microscopy were combined to analyze the morphology of the mature larvae of C. plagiicollis for the first time We determined the feeding characteristics and occurrence regularity of C. plagiicollis, which can be used to develop effective pest management strategies.

Robustness of epithelial sealing is an emerging property of local ERK feedback driven by cell elimination.

What regulates the spatiotemporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination where simultaneous death of neighboring cells could impair epithelial sealing. Here, using the Drosophila pupal notum (a single-layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first showed that death of clusters of at least three cells impaired epithelial sealing; yet, such clusters were almost never observed in vivo. Accordingly, statistical analysis and simulations of cell death distribution highlighted a transient and local protective phase occurring near every cell death. This protection is driven by a transient activation of ERK in cells neighboring extruding cells, which inhibits caspase activation and prevents elimination of cells in clusters. This suggests that the robustness of epithelia with high rates of cell elimination is an emerging property of local ERK feedback.

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.

Crucial role of framework with cytoskeletal actin filaments for shaping microstructure of footpad setae in the ladybird beetle, Harmonia axyridis.

Insects that can walk on smooth surfaces have specialized structures, footpads, on their legs. Footpads play an important role in adhesion to the substrate surface. Although the morphology and function of footpads have been studied, the mechanism of their formation is still elusive. In the ladybird beetle (Harmonia axyridis), hairy footpads are present on the first and second tarsal segments of the legs. The footpads are covered with hundreds of hairs, i.e. setae, whose tips consist of four types: pointed, lanceolate, spatular, and discoidal. We examined the formation of the footpad during the pupal stage using immuno-staining and fluorescent-conjugated phalloidin staining. We found that a seta was composed of a shaft and a socket and some setae were accompanied by a neuron. By the mid-pupal stages, the shaft cells elongated to form a setal structure. Cytoskeletal actin bundles ramified to create a framework for the setal tip structure of the cells. We examined the effects of the application of cytochalasin D, which inhibits actin polymerization, on the formation of footpad setal structures. The results showed that the setal tips were deformed by the inhibition of actin polymerization. Our observations reveal that cytoskeletal actin filaments are involved in shaping the setae.

Microtubules provide guidance cues for myofibril and sarcomere assembly and growth.

BACKGROUND: Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. We utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. RESULTS: A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. CONCLUSIONS: Our observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units.

Morphological and histological examination of short-wing formation in the winter moth Protalcis concinnata (Insecta: Lepidoptera, Geometridae).

Winter geometrid moths exhibit sexual dimorphism in wing length and female-specific flightlessness. Female-specific flightlessness in insects is an interesting phenomenon in terms of sexual dimorphism and reproductive biology. In the winter geometrid moth, Protalcis concinnata (Wileman), adult females have short wings and adult males have fully developed wings. Although the developmental process for wing reduction in Lepidoptera is well studied, little is known about the morphology and the developmental pattern of short-winged flightless morphs in Lepidoptera. To clarify the precise mechanisms and developmental processes that produce short-winged morphs, we performed morphological and histological investigations of adult and pupal wing development in the winter geometrid moth P. concinnata. Our findings showed that (a) wing development in both sexes is similar until larval-pupal metamorphosis, (b) the shape of the sexually dimorphic wings is determined by the position of the bordering lacuna (BL), (c) the BL is positioned farther inward in females than in males, and (d) after the short pupal diapause period, the female pupal wing epithelium degenerates to approximately two-thirds its original size due to cell death. We propose that this developmental pattern is a previously unrecognized process among flightless Lepidoptera.

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.

Micro-computed tomography permits enhanced visualization of mycangia across development and between sexes in Euwallacea ambrosia beetles.

Symbiosis can facilitate the development of specialized organs in the host body to maintain relationships with beneficial microorganisms. To understand the developmental and genetic mechanisms by which such organs develop, it is critical to first investigate the morphology and developmental timing of these structures during the onset of host development. We utilized micro-computed tomography (µCT) to describe the morphology and development of mycangia, a specialized organ, in the Asian ambrosia beetle species Euwallacea validus which maintains a mutualistic relationship with the Ascomycete fungus, Fusarium oligoseptatum. We scanned animals in larval, pupal and adult life stages and identified that mycangia develop during the late pupal stage. Here we reconcile preliminary evidence and provide additional morphological data for a second paired set of structures, including the superior, medial mycangia and an inferior, lateral pair of pouch-like structures, in both late-stage pupae and adult female beetles. Furthermore, we report the possible development of rudimentary, or partially developed pairs of medial mycangia in adult male beetles which has never been reported for any male Xyleborini. Our results illustrate the validity of µCT in observing soft tissues and the complex nature of mycangia morphology and development.

Ultrastructure of the Immature Stages of Musca domestica (Diptera: Muscidae: Muscinae).

Musca domestica (Linnaeus, 1758) is a muscoid species that is widespread throughout the world and acts as a mechanical vector of different enteropathogens primarily in underdeveloped countries. The adult and its immature forms are associated with decaying organic matter and can be seen visiting human corpses and animal carcass, and the larvae can also cause an infestation on human and animal wounds, feeding on the tissues. These characteristics make them have a forensic significance and, mainly, a great sanitary importance. This study aimed to analyze and describe morphological aspects of their immature stages of M. domestica, including the eggs, the first-, second-, and third-instar larvae, and the puparium using scanning electron microscopy (SEM). The eggs have standard format that all muscoid flies shared. The exochorion has some modified cells that were used for embryonic respiration. The first-instar larvae present two openings on the posterior spiracle as in the second-instar larvae, but with the last one, we can observe the anterior spiracle structure. The third-instar larvae, as in some other Muscidae species, have a posterior spiracular opening with a sinuous form that is located near the edge of the spiracular plate. The puparium morphology is equal to the third instar with a respiratory structure that helps the breathing during the metamorphosis process. This article supports the need for knowledge over the morphological characteristics of the immature forms of the muscoid Diptera (Linnaeus, 1758) at the same time helping with the correct identification of this insect phase.

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.

Benzoquinone synthesis-related genes of Tribolium castaneum confer the robust antifungal host defense to the adult beetles through the inhibition of conidial germination on the body surface.

Insects fight against invading microbial pathogens through various immune-related measures that comprise 'internal', 'external' as well as 'social' immunities. The defenses by external immunity associated with the cuticular integument are supposed to be of particular importance in repelling entomopathogenic fungi that infect host insects transcutaneously. Among such integument-related defenses, external secretions of benzoquinone derivatives typical of tenebrionid beetles have been suggested to play important roles in the antimicrobial defenses. In the present study, by utilizing the experimental infection system composed of the red flour beetle Tribolium castaneum and generalist ascomycete entomopathogens Beauveria bassiana and Metarhizium anisopliae, we performed the functional assays of the three T. castaneum genes whose involvement in benzoquinone synthesis in the adults has been reported, namely GT39, GT62 and GT63. Observations by scanning electron microcopy (SEM) revealed that the conidia of the two fungal species did not germinate on the wild-type adult body surface but did on the pupae. The expression analyses demonstrated that the levels of GT39 and GT62 mRNA increased from middle pupae and reached high in early adults while GT63 did not show a clear adult-biased expression pattern. The RNA interference-based knockdown of any of the three genes in pupae resulted in the adults compromised to the infection of the both fungal species. SEM observations revealed that the gene silencing allowed the conidial germination on the body surface of the knockdown beetles, thereby impairing the robust antifungal defense of adult beetles. Thus, we have provided direct experimental evidence for the functional importance in vivo of these benzoquinone synthesis-related genes that support the antifungal defense of tenebrionid beetles.

Amorphous calcium phosphate in the pupal cuticle of Bactrocera dorsalis Hendel (Diptera: Tephritidae): A new discovery for reconsidering the mineralization of the insect cuticle.

It is now widely accepted that Hexapoda emerged from Crustacea. Compared to the ubiquitous calcified exoskeleton in crustaceans, a mineralized cuticle in insects is extremely rare. Catecholamine-driven protein cross-links play a leading role in the sclerotization of insect cuticle. In this study, mineralization was discovered in the pupal cuticle of Bactrocera dorsalis (Diptera: Tephritidae), a common pest of fruit farms. We mainly profiled the features of mineralized pupal cuticles from B. dorsalis and its white mutant B. dorsaliswh and unmineralized cuticle from Musca domestica using high-resolution field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with structural analysis involving infrared (IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and synchrotron X-ray absorption near-edge structure (XANES) spectroscopy. We also compared the thermodynamic and mechanical properties of different pupal cuticles. The results showed that the pupal cuticles of B. dorsalis contain a phase of stable amorphous calcium phosphate (ACP) with a high level of magnesium, which is mainly distributed in the exocuticle and assists in the formation of a graded, stiffened cuticle structure. Unexpectedly, this ACP possesses a very low Ca/P ratio and has a composition similar to that of CaHPO4·2H2O. The degree of mineralization in the pupal cuticle of B. dorsaliswh (approximately 22 wt%) is significantly greater than that of wild-type B. dorsalis (approximately 12 wt%), which indicates that there may be a connection between the biomineralization and tyrosine-mediated tanning pathways. These findings provide new evidence for the mineralization of the insect cuticle, which may shed new light on the evolutionary mechanism underlying the divergence of cuticle sclerotization between insects and crustaceans.

Honeybee pupal length assessed by CT-scan technique: effects of Varroa infestation, developmental stage and spatial position within the brood comb.

Honeybee pupae morphology can be affected by a number of stressor, but in vivo investigation is difficult. A computed tomography (CT) technique was applied to visualize a comb's inner structure without damaging the brood. The CT scan was performed on a brood comb containing pupae developed from eggs laid by the queen during a time window of 48 hours. From the CT images, the position of each pupa was determined by recording coordinates to a common reference point. Afterwards, every brood cell was inspected in order to assess the developmental stage of the pupa, the presence of Varroa destructor, the number and progeny of foundress mites. Using data on 651 pupae, the relationships between varroa infestation status, developmental stage and spatial position of the pupa within the brood comb, and its length were investigated. Pupae at 8 post-capping days were shorter than pupae at 7 post-capping days. Pupae in infected cells were significantly shorter than those in varroa-free cells and this effect was linked both to mite number and stage and to the position in the comb. Overall, the results suggest that the CT-scan may represent a suitable non-invasive tool to investigate the morphology and developing status of honeybee brood.

Can exposure to neem oil affect the spermatogenesis of predator Ceraeochrysa claveri?

Novel biological control methods and integrated pest management strategies are basic requirements for the development of sustainable agriculture. As a result, there is a growing demand for research on the use of plant extracts and natural enemies such as the green lacewing, Ceraeochrysa claveri, as natural pest control methods. Studies have shown that although natural compounds such as neem oil (Azadirachta indica) are effective as pest control strategies, they also cause sublethal effects on nontarget insects, such as C. claveri. The aim of this study was to examine the effects of neem oil on C. claveri testes. C. claveri larvae were fed Diatraea saccharalis eggs, which were pretreated with 0.5%, 1%, and 2% neem oil. Testes were collected from larvae, pupae, and adults and analyzed using light and electron (transmission and scanning) microscopy. Changes in cellular stress and possible cell death were also determined by TUNEL assay and the marker HSP-70. The results showed that neem oil affects the organization and distribution of cysts in the testes and the normal sequence of cyst development, causing a delay in spermatogenesis in the testes of treated insects. Tests for cellular stress and DNA fragmentation indicated there was no cellular alteration in the treated groups. Although neem oil does not induce cell death or changes in HSP-70 expression, this biopesticide negatively impacts the process of spermatogenesis and could decrease the perpetuation of this species in the agroecosystem, indicating that the use of neem oil in association with green lacewings as a biological control should be carefully evaluated.

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.

Properties of a novel carboxymethyl chitosan derived from silkworm pupa.

In this study, a carboxymethyl chitosan derived from silkworm pupa (SP-carboxymethyl chitosan) was prepared. The physical characteristics of the SP chitin, chitosan, and carboxymethyl chitosan were analyzed. The scanning electron microscopy results showed that the surfaces of the samples from SP were more uneven, with more surface fractures compared with those of the reference substance (RS). Thermal analysis, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy analysis showed that the main molecular chain structures of SP samples and RSs had no substantial differences. However, the crystallinity and thermal decomposition temperature of the SP samples were lower compared with those of the RSs. All of these results provide a theoretical basis for the development of applications for the SP-carboxymethyl chitosan.

The "transition zone" of the cilium-like regions in the Drosophila spermatocytes and the role of the C-tubule in axoneme assembly.

The fruit-fly Drosophila melanogaster harbours different types of ciliary structures: ciliary projections associated with neurons of type I and cilium-like regions (CLRs) found during male gametogenesis. The latter deserve particular attention since they are morphologically similar to vertebrate primary cilia and transform into the sperm axonemes during spermiogenesis. Although, all the centrioles are able to organize the CLRs, we found that the mother centriole docks first to the plasma membrane suggesting a new intrinsic functional asymmetry between the parent centrioles. We also show that the CLRs lack the Y-links that connect the axoneme doublets with the plasma membrane in conventional primary cilia. Moreover, the C-tubules, that are lacking in the axoneme of the primary cilia, persisted along the CLRs albeit modified into longitudinal blades. Remarkably, mutant flies in which the CLRs are devoid of the C-tubules or their number is reduced lack sperm axonemes or have incomplete axonemes. Therefore, the C-tubules are dispensable for the assembly of the CLRs but are essential for sperm axoneme elongation and maintenance in Drosophila.

Pronymphs, hatching, and proboscis assembly in leafhoppers and froghoppers (Hemiptera, Cicadellidae and Aphrophoridae).

Pharate 1st instar nymphs enclosed in the embryonic cuticle, referred to as pronymphs, were studied in a froghopper Aphrophora pectoralis Mats. (Aphrophoridae) and the leafhoppers Oncopsis flavicollis (L.), Populicerus populi (L.), Alebra wahlbergi (Boh.), Igutettix oculatus (Lindb.), and Scenergates viridis (Vilb.) (Cicadellidae). The species vary in the relative length of the pronymphal antennae and details of sculpturing of the cephalic region. No egg bursting structures were observed, except small denticles on the crown region of S. viridis pronymphs. Rudimentary mandibular and maxillary stylets of a pronymph are external, short, tubular appendages containing tips of the corresponding nymphal stylets, whose more basal parts develop inside of the head. Casting off of the embryonic cuticle results in the nymphal stylets being passively pulled out and assuming a close-set parallel orientation. Once the sheaths of unsclerotized cuticle secreted by the peripodial epithelium and enveloping each developing stylet have been cast off with the exuviae, the bare stylets become squeezed and interlocked into a functional bundle. The roles of the maxillary plates, clypeus, labrum, and labium in the stylet bundle assembly are discussed. The process repeats after each molt.

Polarization-resolved microscopy reveals a muscle myosin motor-independent mechanism of molecular actin ordering during sarcomere maturation.

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.

The Immatures of Culicoides trilineatus (Diptera: Ceratopogonidae) Potential Vector of the Bluetongue Virus.

The fourth instar larva and pupa of Culicoides trilineatus Fox (Diptera, Ceratopogonidae), a species considered as potential vector of the bluetongue virus in Central and South America, are described, illustrated, and photomicrographed for the first time by using binocular, phase-contrast, and scanning electron microscopy. The immatures were collected by using a siphon bottle in tree holes in Salta Province, Argentina, transported to the laboratory, and there reared to the adult's emergence. They are compared with the immatures of Culicoides debilipalpis Lutz (Diptera, Ceratopogonidae), another Neotropical species that develops in tree holes. Details on larval biology and habitat are given.