The ultrastructure of the rostral gland in soldiers of Verrucositermes tuberosus (Blattodea: Termitidae: Nasutitermitinae).

The soil-feeding habit is an evolutionary novelty found in some advanced groups of termites. The study of such groups is important to revealing interesting adaptations to this way-of-life. The genus Verrucositermes is one such example, characterized by peculiar outgrowths on the head capsule, antennae and maxillary palps, which are not found in any other termite. These structures have been hypothesized to be linked to the presence of a new exocrine organ, the rostral gland, whose structure has remained unexplored. We have thus studied the ultrastructure of the epidermal layer of the head capsule of Verrucositermes tuberosus soldiers. We describe the ultrastructure of the rostral gland, which consists of class 3 secretory cells only. The dominant secretory organelles comprise rough endoplasmic reticulum and Golgi apparatus, which provide secretions delivered to the surface of the head, likely made of peptide-derived components of unclear function. We discuss a possible role of the rostral gland of soldiers as an adaptation to the frequent encounter with soil pathogens during search for new food resources.

The ultrastructure of the intramandibular gland in soldiers of the termite Machadotermes rigidus (Blattodea: Termitidae: Apicotermitinae).

Machadotermes is one of the basal Apicotermitinae genera, living in tropical West Africa. Old observations suggested the presence of a new gland, the intramandibular gland, in Machadotermes soldiers. Here, by combining micro-computed tomography, optical and electron microscopy, we showed that the gland exists in Machadotermes soldiers only as an active exocrine organ, consisting of numerous class III cells (bicellular units made of secretory and canal cells), within which the secretion is produced in rough endoplasmic reticulum, and modified and stored in Golgi apparatus. The final secretion is released out from the body through epicuticular canals running through the mandible cuticle to the exterior. We also studied three other Apicotermitinae, Indotermes, Duplidentitermes, and Jugositermes, in which this gland is absent. We speculate that the secretion of this gland may be used as a general protectant or antimicrobial agent. In addition, we observed that the frontal gland, a specific defensive organ in termites, is absent in Machadotermes soldiers while it is tiny in Indotermes soldiers and small in Duplidentitermes and Jugositermes soldiers. At last, we could also observe in all these species the labral, mandibular and labial glands, other exocrine glands present in all termite species studied so far.

Biochemical, molecular, and morphological variations of flight muscles before and after dispersal flight in a eusocial termite, Reticulitermes chinensis.

Swarming behavior facilitates pair formation, and therefore mating, in many eusocial termites. However, the physiological adjustments and morphological transformations of the flight muscles involved in flying and flightless insect forms are still unclear. Here, we found that the dispersal flight of the eusocial termite Reticulitermes chinensis Snyder led to a gradual decrease in adenosine triphosphate supply from oxidative phosphorylation, as well as a reduction in the activities of critical mitochondrial respiratory enzymes from preflight to dealation. Correspondingly, using three-dimensional reconstruction and transmission electron microscopy (TEM), the flight muscles were found to be gradually deteriorated during this process. In particular, two tergo-pleural muscles (IItpm5 and III-tpm5) necessary to adjust the rotation of wings for wing shedding behavior were present only in flying alates. These findings suggest that flight muscle systems vary in function and morphology to facilitate the swarming flight procedure, which sheds light on the important role of swarming in successful extension and fecundity of eusocial termites.

Mineral analysis reveals extreme manganese concentrations in wild harvested and commercially available edible termites.

Termites are widely used as a food resource, particularly in Africa and Asia. Markets for insects as food are also expanding worldwide. To inform the development of insect-based foods, we analysed selected minerals (Fe-Mn-Zn-Cu-Mg) in wild-harvested and commercially available termites. Mineral values were compared to selected commercially available insects. Alate termites, of the genera Macrotermes and Odontotermes, showed remarkably high manganese (Mn) content (292-515 mg/100 gdw), roughly 50-100 times the concentrations detected in other insects. Other mineral elements occur at moderate concentrations in all insects examined. On further examination, the Mn is located primarily in the abdomens of the Macrotermes subhyalinus; with scanning electron microscopy revealing small spherical structures highly enriched for Mn. We identify the fungus comb, of Macrotermes subhyanus, as a potential biological source of the high Mn concentrations. Consuming even small quantities of termite alates could exceed current upper recommended intakes for Mn in both adults and children. Given the widespread use of termites as food, a better understanding the sources, distribution and bio-availability of these high Mn concentrations in termite alates is needed.

The oral gland, a new exocrine organ of termites.

Termites have a rich set of exocrine glands. These glands are located all over the body, appearing in the head, thorax, legs and abdomen. Here, we describe the oral gland, a new gland formed by no more than a few tens of Class I secretory cells. The gland is divided into two secretory regions located just behind the mouth, on the dorsal and ventral side of the pharynx, respectively. The dominant secretory organelle is a smooth endoplasmic reticulum. Secretion release is under direct control of axons located within basal invaginations of the secretory cells. The secretion is released through a modified porous cuticle located at the mouth opening. We confirmed the presence of the oral gland in workers and soldiers of several wood- and soil-feeding species of Rhinotermitidae and Termitidae, suggesting a broader distribution of the oral gland among termites. The oral gland is the smallest exocrine gland described in termites so far. We hypothesise that the oily secretion can either ease the passage of food or serve as a primer pheromone.

Morphophysiological study of digestive system litter-feeding termite Cornitermes cumulans (Kollar, 1832).

Termites are the major decomposers of lignocellulosic biomass on Earth and are commonly considered as biological reactor models for lignocellulose degradation. Despite their biotechnological potential, few studies have focused on the morphophysiological aspects of the termite digestive system. We therefore analyze the morphology, ultrastructure and gut luminal pH of the digestive system in workers of the litter-feeding termite Cornitermes cumulans (Blattodea: Termitidae). Their digestive system is composed of salivary glands and an alimentary canal with a pH ranging from neutral to alkaline. The salivary glands have an acinar structure and present cells with secretory characteristics. The alimentary canal is differentiated into the foregut, midgut, mixed segment and hindgut, which comprises the ileum (p1), enteric valve (p2), paunch (p3), colon (p4) and rectum (p5) segments. The foregut has a well-developed chewing system. The midgut possesses a tubular peritrophic membrane and two cell types: digestive cells with secretory and absorptive features and several regenerative cells in mitosis, both cell types being organized into regenerative crypts. The mixed segment exhibits cells rich in glycogen granules. Hindgut p1, p4 and p5 segments have flattened cells with a few apical invaginations related to mitochondria and a thick cuticular lining. Conversely, the hindgut p3 segment contains large cuboid cells with extensive apical invaginations associated with numerous mitochondria. These new insights into the morphophysiology of the digestive system of C. cumulans reveal that it mobilizes lignocellulose components as a nutritional source by means of a highly compartmentalized organization with specialized segments and complex microenvironments.

Coendutermes tucum Fontes (Isoptera, Termitidae, Nasutitermitinae): description of the imago caste and additional notes.

Coendutermes Fontes, 1985 is a monotypic South American termite genus. Coendutermes tucum Fontes, 1985, was described based on morphological characters from soldiers and workers collected in Mato Grosso, Brazil, and Jodensavanne, Suriname. Herein, I describe the imago caste of C. tucum for the first time with additional notes on soldiers, workers, and new distributional records. The studied material is deposited at the Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil (MZUSP). I use the terminology of Fontes (1987) to describe worker mandibles, and that of Noirot (2001) for the different parts of the digestive tube of workers. I measured the imagoes morphometric characters following Roonwal (1970): LH, length of head capsule (9); WH, width of head capsule without eyes (18); OF, occipito-fontanelle distance (23); DE, diameter of eye (48); LO, length of ocellus (55); WO, width of ocellus (56); EOD, eye-ocellus distance (57); LP, length of pronotum (65); WP, width of pronotum (68); LT, length of hind tibia (85). I took photographs of all castes with a stereomicroscope (Leica M205C) attached to a video camera (Leica DFC295) and images of gizzard and enteric valve under a microscope (Leica DM750B) attached to a video camera (Leica ICC50HD), then I combined the stacks of images with the software Leica LAS EZ 2.0 or Helicon Focus 5.2.11 X64. For the scanning electron micrographs (SEM), one soldier was dried to critical point while directly mounted on a stub with double face adhesive tape, then coated with gold and photographed with the SEM (Zeiss LEO 440 ®).

Dehiscent organs used for defensive behavior of kamikaze termites of the genus Ruptitermes (Termitidae, Apicotermitinae) are not glands.

During Isoptera evolution, the caste of soldiers disappeared in some Apicotermitinae termites as in the Neotropical Ruptitermes. Paired dorsolateral structures located between the metathorax and abdomen of foraging workers of Ruptitermes were previously denominated dehiscent glands, and are responsible for releasing an adhesive secretion that immobilizes enemies, causing their death. In this study, we investigated the morphology of dehiscent organs of workers of Ruptitermes reconditus, Ruptitermes xanthochiton, and Ruptitermes pitan and also second instar larvae of R. reconditus using light, laser scanning confocal, and transmission electron microscopy. Additionally, we performed a preliminary protein analysis using SDS-PAGE to further characterize the secretion of Ruptitermes dehiscent organs. Our results showed that the dehiscent organs do not exhibit the typical characteristics of the exocrine glandular cells class I, II or III of insects, suggesting that they constitute a new type of defensive organ. Thus, the denomination dehiscent gland was not used but dehiscent organ. Dehiscent organs in larvae are formed by fat body cells. In workers, dehiscent organs are composed by compact masses of cells that accumulate a defensive secretion and are poor in organelles related to the production of secretion. Since the dehiscent organs are not glands, we hypothesize that the dehiscent organs originate from larval fat body. The defensive secretion may have been produced at younger developmental stages of worker or the defensive compounds were absorbed from food and accumulated in the worker fat body. Histochemical techniques and SDS-PAGE revealed that the secretion of Ruptitermes dehiscent organs is constituted mainly by a protein of high molecular weight (200 kDa). In conclusion, the dehiscent organs are extremely different from the exocrine glands of termites and other insects described until now. In fact, they seem to be a specialized fat body that is peculiar and exclusive of Ruptitermes termites.

Tarsomere and distal tibial glands: structure and potential roles in termites (Isoptera: Rhinotermitidae, Termitidae).

Social insects have numerous exocrine glands, but these organs are understudied in termites compared to hymenopterans. The tarsomere and distal tibial glands of the termites Heterotermes tenuis, Coptotermes gestroi and Silvestritermes euamignathus were investigated by scanning and transmission electron microscopy. Pore plates are visible in scanning micrographs on the distal tibial surfaces and on the ventral surface of the first and second tarsomeres of workers of H. tenuis and C. gestroi. In contrast, workers of S. euamignathus have isolated pores spread throughout the ventral surfaces of the first, second, and third tarsomeres and the distal tibia. In all three species each pore corresponds to the opening of a class-3 secretory unit, composed of one secretory and one canal cell. Clusters of class-3 glandular cells are arranged side by side underneath the cuticle. The main characteristics of these exocrine glands include their presence on all the legs and the electron-lucent secretion in the secretory cells. Possible functions of these glands are discussed.

The nasus gland: a new gland in soldiers of Angularitermes (Termitidae, Nasutitermitinae).

Termites have developed many exocrine glands, generally dedicated to defence or communication. Although a few of these glands occur in all termite species, or represent synapomorphies of larger clades, others are morphological innovations of a single species, or a few related species. Here, we describe the nasus gland, a new gland occurring at the base of the nasus of Angularitermes soldiers. The nasus gland is composed of class 1, 2, and 3 secretory cells, a rare combination that is only shared by the sternal and tergal glands of some termites and cockroaches. The ultrastructural observations suggest that the secretion is produced by class 2 and 3 secretory cells, and released mostly by class 3 cells. The base of the nasus has a rough appearance due to numerous pits bearing openings of canals conducting the secretion from class 3 secretory cells to the exterior. We tentatively assign a defensive function to the nasus gland, although further research is needed to confirm this function. Although the gland is described only from species of Angularitermes, other genera of Nasutitermitinae also present a rough nasus base, suggesting the presence of a similar, possibly homologous, gland.

Compound eye formation in the termite Incisitermes minor (Isoptera: Kalotermitidae).

The postembryonic development and caste differentiation patterns of lower termites have been described multiple times in a variety of different species. However, most of these studies focused on gross ontogeny, without carefully describing the maturation of any particular organ or organ system. The few studies that have attempted to correlate caste development and organ differentiation have produced somewhat inconsistent results, especially in the area of eye formation. Therefore, in order to help further elucidate the relationship between eye formation and postembryonic differentiation in lower termites, we studied eye development in the termite, Incisitermes minor (Hagen). Eye formation in I. minor began in the earliest larvae, with only an eye primordium. However, in all later larval stages, characteristic eye structures were observed and were shown to progressively differentiate through larval and nymphal stages. Curiously, pigmentation began with three to eight groups of cells in early larvae and the number of these pigmented groups increased along the developmental time course. Ultimately, a uniformly pigmented eye area was formed by the early nymphal stage. The overall eye area also gradually increased along with normal caste development, but the characteristic lenses seen in a prototypical insect compound eye did not completely form until after the final nymphal stage. Electrophysiological measurements provided clear evidence that eyes were indeed functional at all stages of development where pigment was present. Based upon this data, the eye development pattern in I. minor appeared to follow a divergent pathway from holometabolous insects and an intermediate pathway between typical hemimetabolous eye development and the heterochronic shift observed in other termite species.

Location, morphology and function of nephrocytes in termites.

Insect nephrocytes are cells bathed in hemolymph and considered to have an excretory function. These cells have ambiguous nomenclature and are understudied in termites. This study is the first report on the occurrence, morphology and function of nephrocytes in different termite castes. Cytological characteristics in specific developmental stages and castes enable physiological functions to be inferred. Perforate diaphragms indicate a role in filtration, while the extensive peripheral invaginations of the cell membrane suggest active endocytosis. A sequence of morphologies in putative digestive vacuoles infers a lysosomal system and the occurrence of phosphatases suggests a function involving detoxification of substances sequestered from hemolymph. Pericardical nephrocytes took up the dye trypan blue injected in live termites, suggesting their activity connected to the filtration of the hemolymph. Additionally, histochemical tests showed the existence of stored proteins in their cytoplasm. These cells present a well-developed Golgi apparatus and abundant rough endoplasmic reticulum, consistent with protein synthesis. This study highlights the importance of nephrocytes in Isoptera and opens perspectives for further research of these cells.

The clypeal gland: a new exocrine gland in termite imagoes (Isoptera: Serritermitidae, Rhinotermitidae, Termitidae).

Social insects possess a rich set of exocrine organs producing diverse pheromones and defensive compounds. This is especially true for termite imagoes, which are equipped with several glands producing, among others, sex pheromones and defensive compounds protecting imagoes during the dispersal flight and colony foundation. Here, we describe the clypeal gland, a new termite exocrine organ occurring in the labro-clypeal region of imagoes of most Rhinotermitidae, Serritermitidae and Termitidae species. The clypeal gland of Coptotermes testaceus consists of class 1 (modified epidermal cell) and class 3 (bicellular gland unit) secretory cells. Ultrastructural features suggest that the gland secretes volatile compounds and proteins, probably after starting the reproduction. One peculiar feature of the gland is the presence of multiple secretory canals in a single canal cell, a feature never observed before in other insect glands. Although the function of the gland remains unknown, we hypothesize that it could produce secretion signalling the presence of functional reproductives or their need to be fed.

Labral gland in soldiers of the neotropical termite Cornitermes cumulans (Isoptera: Termitidae: Syntermitinae).

Cornitermes cumulans is a termite species of the subfamily Syntermitinae with a nasute mandibulate soldier caste, members of which are morphologically equipped with structures and organs specialized for the defence of the colony. We investigated the labrum of soldiers of C. cumulans and described the labral gland, an exocrine structure present in this appendage. The labrum of C. cumulans soldiers presented two distinct regions, the hyaline tip and the proximal region connected to the head. The hyaline tip exhibited a thick cuticle composed of a loose endocuticle involving an epithelium of class 1 cells, which synthesize the glandular product into a subcuticular space that function as a reservoir prior to release the final secretion. The proximal region of the labrum had an epithelium composed of class 1 and class 3 cells, which released the secretion onto both ventral and dorsal surfaces. The ultrastructure showed abundant smooth endoplasmic reticulum and glycogen in the class 1 cells, whereas the class 3 cells had many electron-lucent vesicles that varied in size. We associated the labral gland with the production of toxic substances that may act inside enemy wounds made by the soldiers' mandibles. Other possible functions for the labral secretion are also discussed.

Three-dimensional visualization of termite (Apicotermitinae) enteric valve using confocal laser scanning microscopy.

Humivorous termites are dominant members of tropical rainforest soil communities. In the soil-feeding subfamily Apicotermitinae (Termitidae), the enteric valve connecting the first section of the hindgut to the paunch often displays a complex sclerotized armature everted towards the lumen of the paunch. This structure is central in termite taxonomy but its function remains hypothetical. Here, we evaluate the potential of confocal laser scanning microscopy to provide detailed imaging of the valve of Anoplotermes parvus, by comparison with bright-field microscopy and scanning electron microscopy. We detected a strong far-red emission of the enteric valve armature that sharply contrasted with the surrounding tissues, providing a convenient method to highlight minute structural elements of the valve and its three-dimensional structure. The method is easy to use and is applicable to standard archival material as demonstrated by images of enteric valves of four other Apicotermitinae species. It may represent a valuable asset for the study of termite enteric valves, for the purpose of taxonomy or functional morphology.

Age-dependent changes in ultrastructure of the defensive glands of Neocapritermes taracua workers (Isoptera, Termitidae).

Protection against predators and competitors is one of the main concerns of termite colonies, which developed a specialised defensive caste, the soldiers. However, soldiers are rare or even missing in several lineages of termites, while workers often develop new defence strategies especially in soil-feeding species. Here, we describe the morphology and ultrastructure of the autothysis-associated glands of Neocapritermes taracua workers and report their age-related changes in structure. The defensive glands of N. taracua workers consist of a pair of labial and a pair of crystal glands, whose secretions mix together through autothysis. Autothysis always occurs at the line of weakness connecting the anterior parts of the crystal-bearing pouches. The crystal glands consist of groups of bicellular secretory units (secretory and corresponding canal cells) which secrete the blue crystal material into external pouches. Their secretory activity is maximal in the middle of worker life, and is considerably lower in very young and old workers. The labial glands are composed of two types of secretory cells: the central and the parietal cells. While the central cells are developed similarly to other termites and secrete proteinaceous secretion into labial gland ducts, the parietal cells develop proteinaceous granules which may eventually bud off the cells. The secretory function of parietal cells is so far unique to N. taracua and differs from other termite species in which they are only responsible of water uptake by acini. The defensive device of N. taracua is truly exceptional as it involves a new gland and a previously undescribed function for parietal cells, being a remarkable example of evolution of morphological innovation.

Armed reproductives: evolution of the frontal gland in imagoes of Termitidae.

The frontal gland of termites is a structure without any equivalent among other animals. Although this gland is well known in soldiers, it received almost no attention in other castes. Recently, we described it in imagoes of Rhinotermitidae and Serritermitidae. In order to provide a complete picture of the evolution of this gland in termite imagoes, we studied it in additional 34 species of Termitidae, representing 7 of the 8 subfamilies. The frontal gland of these species is formed by class 1 secretory cells only, and occurs in two basic shapes: epithelial with reservoir in Foraminitermitinae and Macrotermitinae, and epithelial without reservoir in all other subfamilies. The size variability of the gland is high, not only among Termitidae subfamilies, but also within subfamilies. Our data suggest that the ancestral form of the frontal gland is epithelial with reservoir, as found in Rhinotermitidae, Serritermitidae, and basal Termitidae. The reduction of the reservoir occurred at least two times and the gland was lost two times independently: in Protermes sp. and in Microtermes toumodiensis (both Macrotermitinae).

Colonization of termite hindgut walls by oxymonad flagellates and prokaryotes in Incisitermes tabogae, I. marginipennis and Reticulitermes flavipes.

We studied the colonization of the paunch wall of three lower termites, Reticulitermes flavipes, Incisitermes tabogae, and Incisitermes marginipennis, by light and electron microscopy. In addition to various prokaryotes, oxymonad flagellates were attached to the wall of the paunch in all three species. The prokaryotic layer found in R. flavipes is relatively thin, since most organisms are attached laterally. Large members of the flagellate genus Pyrsonympha protrude into the gut lumen. The prokaryotes are very abundant on the gut wall in I. tabogae and I. marginipennis, forming a thick carpet of mostly vertically attached rods and wavy spirochetes. The adhering oxymonads are relatively small and almost hidden in the thick bacterial biofilm. Three small morphotypes were seen in I. tabogae; two possessing a short rostellum and one amoeboid. The only oxymonad found in I. tabogae so far, Oxymonas clevelandi, is not identical to any of the present oxymonads. I. marginipennis contains a mid-sized oxymonad with ectobiotic spirochetes, probably identical to Oxymonas hubbardi, and a tiny unknown morphotype. The spatial organization of the pro- and eukaryotic microorganisms on the gut wall of the three termites is described and discussed concerning oxygen stress.

Tergal glands in termite soldiers of the subfamily Syntermitinae (Isoptera: Termitidae).

The subfamily Syntermitinae comprises 14 genera of termites that are exclusively neotropical. The present study reports morphological data about mandibulate nasute soldiers from termite species belonging to three different genera within this subfamily. We describe tergal glands that were present under all tergites of soldiers of the following species: Cornitermes cumulans, Procornitermes araujoi, Syntermes nanus, and Syntermes wheeleri. The tergal glands were composed of class 2 and class 3 cells. Class 2 cells never reached the cuticle and were located below a flat layer of epidermal cells. Class 3 cells, composed of secretory cells and canal cells, were sporadic, whereas class 2 secretory cells were abundant. Secretory cells of class 3 were narrow and their cytoplasms were filled with several clear, oval-shaped vesicles with limiting membranes. The ultrastructure of class 2 cells showed well-developed smooth endoplasmic reticulum, Golgi apparatus, elongated mitochondria, several electron-lucent vesicles, and electron-dense granules that contain paracrystalline structures in S. nanus. Scanning electron micrographs displayed pores, campaniform sensilla and hairs in the outer cuticle of the soldier tergites. We hypothesize that soldier tergal glands may be involved in the production of defensive compounds, which occur in similar glands of certain cockroaches, or of primer pheromones, that might act in the regulation of soldier differentiation in the termite colony. To date, tergal glands have only been described in termite imagoes, and their occurrence in these soldiers of basal Syntermitinae implies a specific role in this caste that is still speculative and needs to be clarified.

Contrasting micro/nano architecture on termite wings: two divergent strategies for optimising success of colonisation flights.

Many termite species typically fly during or shortly after rain periods. Local precipitation will ensure water will be present when establishing a new colony after the initial flight. Here we show how different species of termite utilise two distinct and contrasting strategies for optimising the success of the colonisation flight. Nasutitermes sp. and Microcerotermes sp. fly during rain periods and adopt hydrophobic structuring/'technologies' on their wings to contend with a moving canvas of droplets in daylight hours. Schedorhinotermes sp. fly after rain periods (typically at night) and thus do not come into contact with mobile droplets. These termites, in contrast, display hydrophilic structuring on their wings with a small scale roughness which is not dimensionally sufficient to introduce an increase in hydrophobicity. The lack of hydrophobicity allows the termite to be hydrophilicly captured at locations where water may be present in large quantities; sufficient for the initial colonization period. The high wettability of the termite cuticle (Schedorhinotermes sp.) indicates that the membrane has a high surface energy and thus will also have strong attractions with solid particles. To investigate this the termite wings were also interacted with both artificial and natural contaminants in the form of hydrophilic silicon beads of various sizes, 4 µm C(18) beads and three differently structured pollens. These were compared to the superhydrophobic surface of the planthopper (Desudaba psittacus) and a native Si wafer surface. The termite cuticle demonstrated higher adhesive interactions with all particles in comparison to those measured on the plant hopper.