Anatomical damage caused by Bacillus thuringiensis variety israelensis in yellow fever mosquito Aedes aegypti (L.) larvae revealed by micro‑computed tomography

With micro-computed tomography techniques, using the single-distance phase-retrieval algorithm phase contrast, we reconstructed enhanced rendered images of soft tissues of Aedes aeqypti fourth instar larvae after Bti treatment. In contrast to previous publications based on conventional microscopy, either optical or electron microscopy, which were limited to partial studies, mostly in the form of histological sections, here we show for the first time the effects of Bti on the complete internal anatomy of an insect. Using 3D rendered images it was possible to study the effect of the bacterium in tissues and organs, not only in sections but also as a whole. We compared the anatomy of healthy larvae with the changes undergone in larvae after being exposed to Bti (for 30 min, 1 h and 6 h) and observed the progressive damage that Bti produce. Damage to the midgut epithelia was confirmed, with progressive swelling of the enterocytes, thickening epithelia, increase of the vacuolar spaces and finally cell lysis, producing openings in the midgut walls. Simultaneously, the larvae altered their motility, making it difficult for them to rise to the surface and position the respiratory siphon properly to break surface tension and breathe. Internally, osmotic shock phenomena were observed, resulting in a deformation of the cross-section shape, producing the appearance of a wide internal space between the cuticle and the internal structures and a progressive collapse of the tracheal trunks. Taken together, these results indicate the death of the larvae, not by starvation as a consequence of the destruction of the epithelia of the digestive tract as previously stated, but due to a synergic catastrophic multifactor process in addition to asphyxia due to a lack of adequate gas exchange.Consejería de Universidad, Investigación e Innovación of Junta de Andalucia (Spain)FEDER Programe through the research projects: “Caracterización de variantes de toxinas Cry activas frente a la Mosca de la Fruta del Mediterráneo (Ceratitis capitata) obtenidas mediante la tecnología del despliegue de proteínas en fagos” (B-BIO-081-UGR18)Búsqueda de nuevas toxinas Cry con actividad frente al ectoparásito de la abeja Varroa destructor mediante la evolución in vitro de proteínas y la técnica del despliegue de proteínas en fago” (A-BIO-424-UGR20

Asian citrus psyllid stylet morphology and applicability to the model for inter-instar stylet replacement in the potato psyllid

In Hemiptera, presumptive stylets for each consecutive postembryonic instar are manufactured prior to ecdysis to replace the ecdysial stylets discarded with the exuviae. With the discovery that the bacterium “Candidatus” Liberibacter solanacearum accesses the tissues involved in the stylet replacement process of the potato psyllid, a hypothesis was formed that the bacterium could adhere to the stylets of freshly emerged instars and hence gain access to the host plant when feeding is resumed. Although unproven, it was imperative that a model for stylet replacement be built. Stylet morphology and the stylet replacement process of the Asian citrus psyllid (ACP), vector of “C.” L. asiaticus, causal pathogen of citrus greening disease, are comparable to the potato psyllid model system. Morphology consists of a basal terminus with its tab-shaped auricle, a base, shaft, and an apical terminus. Each of the four auricles act as a platform for the replacement apparatus, which is compacted into a tight aggregate of cells, the ‘endcap’. As modeled, on apolysis of larval instar hypodermis, the aggregate ‘deconstructs’ and expands into a snail shell-shaped tube, the ‘atrium’, that houses the presumptive stylet as it is synthesized. Completed stylets then despool from the atrium and are fitted into their functional positions as the next instar emerges from its exuviae.Funding was provided by a grant from USDA-NIFA Award 2014- 70016-23028, 2015-2020, “Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease”

Observing the devastating coffee berry borer (Hypothenemus hampei) inside the coffee berry using microcomputed tomography

The coffee berry borer is the most devastating insect pest of coffee throughout the world. The insect spends most of its life cycle inside the coffee berry, which makes it quite difficult to observe its behaviour. Micro-computed tomography (micro-CT) was used to observe all developmental stages of the coffee berry borer inside coffee berries (Coffea canephora). An interesting oviposition pattern involving a sequential placement of eggs starting in the periphery of the seed and moving inwards was observed. Micro-CT should be useful in elucidating unknown life history aspects of other seed-feeding bark beetles as well as of bark and ambrosia beetles in general

Micro-CT to Document the Coffee Bean Weevil, Araecerus fasciculatus (Coleoptera: Anthribidae), Inside Field-Collected Coffee Berries (Coffea canephora)

The coffee bean weevil, Araecerus fasciculatus (De Geer) (Coleoptera: Anthribidae), is a cosmopolitan insect with >100 hosts, and has been reported as a pest of stored coffee. During a study involving the coffee berry borer, we observed coffee bean weevils emerging from field-collected coffee berries and used micro-computerized tomography (micro-CT) scans to observe the insect inside the berry. Two eggs had eclosed inside the berry, resulting in observations of a newly eclosed adult beetle and a 5th instar larva, each feeding on one of the two seeds. This is the first time since 1775, when the insect was first described, that the insect has been observed inside a coffee berry

Anatomical study of the coffee berry borer (Hypothenemus hampei) using micro-computed tomography

Traditionally, the study of anatomy in insects has been based on dissection techniques. Microcomputed tomography (micro-CT) is an X-ray based technique that allows visualization of the internal anatomy of insects in situ and does not require dissections. We report on the use of micro-CT scans to study, in detail, the internal structures and organs of the coffee berry borer (Hypothenemus hampei), the most damaging insect pest of coffee worldwide. Detailed images and videos allowed us to make the first description of the aedeagus and the first report of differences between the sexes based on internal anatomy (flight musculature, midgut shape, hindgut convolutions, brain shape and size) and external morphology (lateral outline of the pronotum and number of abdominal tergites). This study is the first complete micro-CT reconstruction of the anatomy of an insect and is also the smallest insect to have been evaluated in this way. High quality rendered images, and additional supplementary videos and 3D models are suitable for use with mobile devices and are useful tools for future research and as teaching aids.This paper benefitted from sub-award agreement S15192.01 between Kansas State University (KSU) and the University of Granada, as part of the USDA-NIFA Award 2014-70016-23028 to Susan J. Brown (KSU), “Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease” (2015–2020)

Evidence of Different Thermoregulatory Mechanisms between Two Sympatric Scarabaeus Species Using Infrared Thermography and Micro-Computer Tomography

In endotherms insects, the thermoregulatory mechanisms modulate heat transfer from the thorax to the abdomen to avoid overheating or cooling in order to obtain a prolonged flight performance. Scarabaeus sacer and S. cicatricosus, two sympatric species with the same habitat and food preferences, showed daily temporal segregation with S. cicatricosus being more active during warmer hours of the day in opposition to S. sacer who avoid it. In the case of S. sacer, their endothermy pattern suggested an adaptive capacity for thorax heat retention. In S. cicatricosus, an active ‘heat exchanger’ mechanism was suggested. However, no empirical evidence had been documented until now. Thermographic sequences recorded during flight performance showed evidence of the existence of both thermoregulatory mechanisms. In S. sacer, infrared sequences showed a possible heat insulator (passive thermal window), which prevents heat transfer from meso- and metathorax to the abdomen during flight. In S. cicatricosus, infrared sequences revealed clear and effective heat flow between the thorax and abdomen (abdominal heat transfer) that should be considered the main mechanism of thermoregulation. This was related to a subsequent increase in abdominal pumping (as a cooling mechanism) during flight. Computer microtomography scanning, anatomical dissections and internal air volume measurements showed two possible heat retention mechanisms for S. sacer; the abdominal air sacs and the development of the internal abdominal sternites that could explain the thermoregulation between thorax and abdomen. Our results suggest that interspecific interactions between sympatric species are regulated by very different mechanisms. These mechanisms create unique thermal niches for the different species, thereby preventing competition and modulating spatio-temporal distribution and the composition of dung beetle assemblages

Morphological and histological description of the midgut caeca in true crabs (Malacostraca: Decapoda: Brachyura): origin, development and potential role

Background The decapods are a major group of crustaceans that includes shrimps, prawns, crayfishes, lobsters, and crabs. Several studies focused on the study of the digestive system of the decapods, constituted by the oesophagus, stomach, midgut tract, midgut gland, and hindgut. Nevertheless, in the midgut tract there are associated a set of organs called “midgut caeca”, which are among the most controversial and less studied digestive organs of this group. This work used the common spider crab Maja brachydactyla Balss, 1922 as a model to resolve the origin, development, and potential role of the midgut caeca. Such organs were studied in the larvae (zoea I, zoea II, megalopa), first juveniles, and adult phases, being employed traditional and modern techniques: dissection, micro-computed tomography (Micro-CT), and light and electron microscopical analyses (TEM and SEM). Results The common spider crab has a pair of anterior midgut caeca and a single posterior caecum that originate from the endoderm germ layer: they develop from the midgut tract, and their epithelium is composed by secretory cells while lacking a cuticle lining. The midgut caeca are small buds in the newly hatched larvae, enlarge linearly during the larval development, and then continue growing until became elongated and coiled blind-tubules in adults. The adult midgut caeca are internally folded to increase their inner surface. The electron microscopy observations showed that the midgut caeca are highly active organs with important macroapocrine and microapocrine secretory activity. Our results suggest that the role of the caeca might be related to the digestive enzyme secretion. The secretory activity should increase as the animal grows in size. Conclusion The present study resolves the embryonic origin of the midgut caeca (endoderm derived organs), development (general lengthening starting from small buds), and role (active secretory organs). The secretory activity of the midgut caeca should be incorporated in the current models of the digestive physiology in different decapod taxa.info:eu-repo/semantics/publishedVersio

Cenozoic origins of the genus Calliarcys (Insecta, Ephemeroptera) revealed by Micro‑CT, with DNA barcode gap analysis of Leptophlebiinae and Habrophlebiinae

We are grateful to Michel Sartori (Lausanne, Switzerland) and José Ángel Martín del Arco (Salamanca, Spain) for donating the specimens of C. humilis to SMNS, BC CAS, and UŁ collections. We are also grateful to Christel and Hans-Werner Hoffeins (Hamburg, Germany), Mike Reich (BSPG, Munich, Germany), and Evgeny Perkovsky (Schmalhausen Institute of Zoology, Kyiv, Ukraine) for access to their collections of fossil mayflies from the Eocene Rovno amber. We would like to thank Kateřina Bláhová (IE, BC CAS) and Milan Pallmann (SMNS) for technical assistance with line drawings and the preparation of a set of macro photographs. Tomasz Mamos (UniLodz, Poland) is acknowledged for his help in the Bayesian reconstruction of phylogeny, and Łukasz Trębicki (UniLodz, Poland) for help in molecular laboratory. Comments from reviewers helped to improve the manuscript. JA-T thank the staff of Bruker SkyScan in Kontich (Belgium) for their effectiveness and fast support, for their constant improvements to the software, and for implementing the new options we requested. In this respect, we are especially indebted to Alexander Sasov (now at NeoScan, https:// neosc an. com), Stephan Boons, Xuan Liu, Phil Salmon, and Vladimir Kharitonov. We would like to thank the reviewers for their thoughtful comments and efforts towards improving our manuscript. LSID urn:lsid:zoobank. org:pub:C58BEE82-0EC6-4C59-A02D-1E5F796179B6Additional information Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598- 022- 18234-4.Funding Open access funding enabled and organized by the University of Łódź (Poland). RJG acknowledges the financial support of the Grant Agency of the Czech Republic (No. 21-05216S) and institutional support of the Institute of Entomology (Biology Centre of the Czech Academy of Sciences) RVO: 60077344. Acquisition of research equipment used in this study has been carried out within equipment subsidy granted by Alexander von Humboldt Foundation [Georg Forster Research Fellowship for Experienced Researchers] for RJG.Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. Additionally, we present first molecular data on the extant species Calliarcys humilis Eaton, 1881 from the Iberian Peninsula and the barcode gap analysis for Leptophlebiinae and Habrophlebiinae.Funding Open access funding enabled and organized by the University of Łódź (Poland)Grant Agency of the Czech Republic (No. 21-05216S)Institute of Entomology (Biology Centre of the Czech Academy of Sciences) RVO: 60077344Alexander von Humboldt Foundation [Georg Forster Research Fellowship for Experienced Researchers

Morphology and ultrastructure of the midgut gland ("hepatopancreas") during ontogeny in the common spider crab Maja brachydactyla Balss, 1922 (Brachyura, Majidae)

We studied the anatomy and cytology of the midgut gland (MGl) of the common spider crab Maja brachydactyla Balss, 1922 at several life stages (zoea, megalopa, first juvenile, and adult) using dissection, histology, electron microscopy, computed tomography, and micro-computed tomography (micro-CT). In newly hatched larvae, 14 blind-end tubules form the MGl. The length of the tubules increases during the larval development. In the late megalopa, the number of tubules also increases. In adults, 35,000 to 60,000 blind-ending tubules comprise the MGl. In all life stages, a square-net network of muscle fibers surround the tubules. We describe five cell types in the MGl in all larval stages, which have a similar location, histology, and ultrastructure in larvae and adults: embryonary (E-) cells, resorptive (R-) cells, fibrillar (F-) cells, blister-like (B-) cells, and midget (M-) cells. Major difference between larval and adult cells is the larger size of the adult cells. Microapocrine secretion occurs from the microvilli of the B-cells. No ultrastructural changes were observed during larval development, which suggests that the function of each cell type might be similar in all life stages. The role of each epithelial cell type in larvae and adults is discussed.info:eu-repo/semantics/acceptedVersio