Transcriptome changes reveal the genetic mechanisms of the reproductive plasticity of workers in lower termites.

BACKGROUND: The reproductive plasticity of termite workers provides colonies with tremendous flexibility to respond to environmental changes, which is the basis for evolutionary and ecological success. Although it is known that all colony members share the same genetic background and that differences in castes are caused by differences in gene expression, the pattern of the specific expression of genes involved in the differentiation of workers into reproductives remains unclear. In this study, the isolated workers of Reticulitermes labralis developed into reproductives, and then comparative transcriptomes were used for the first time to reveal the molecular mechanisms underlying the reproductive plasticity of workers. RESULTS: We identified 38,070 differentially expressed genes and found a pattern of gene expression involved in the differentiation of the workers into reproductives. 12, 543 genes were specifically upregulated in the isolated workers. Twenty-five signal transduction pathways classified into environmental information processing were related to the differentiation of workers into reproductives. Ras functions as a signalling switch regulates the reproductive plasticity of workers. The catalase gene which is related to longevity was up-regulated in reproductives. CONCLUSION: We demonstrate that workers leaving the natal colony can induce the expression of stage-specific genes in the workers, which leads to the differentiation of workers into reproductives and suggests that the signal transduction along the Ras-MAPK pathway crucially controls the reproductive plasticity of the workers. This study also provides an important model for revealing the molecular mechanism of longevity changes.

Hemocyte Morphology and Cellular Immune Response in Termite (Reticulitermes speratus).

Because termites (Reticulitermes speratus) are very small, it is difficult to conduct experiments involving pathogen injection and hemocyte collection. Therefore, to observe hemocyte-mediated immune responses against foreign substances, in vitro hemocyte culture is essential. After collecting about 3 µl of hemolymph, hemocytes were cultured for 7 d, during which the cells maintained full function. Four types of hemocyte were identified, namely, granulocytes, plasmatocytes, oenocytoids, and prohemocytes, among which granulocytes are the main immune hemocytes that fight invasion by foreign substances. Most hemocytes were alive and/or functioning after 7 d of culture, but then either died or lost function.

The transition path from female workers to neotenic reproductives in the termite Reticulitermes labralis.

Termite workers are characterized by unique flexibility in that a worker can develop in one of three ways: remain a worker, become a soldier within two successive moults, or become a neotenic reproductive (NR) within a single moult. However, is it true that workers can transform into NRs within a single moult? Actually, the developmental pathways of workers turning into NRs remains unclear. In this study, we show for the first time that the female workers of Reticulitermes labralis develop into NRs after a pre-NRs stage. We found that a female worker became a NR after two successive moults, whereas the male workers copulated directly with queens without undergoing any moults. After the first moult led the female workers into the pre-NR stage, the length of their abdomens, seventh sternites and ovaries significantly increased. After the second moult from the pre-NRs stage into NRs, the follicle cells returned to normal, and a few oocytes and follicle cells underwent apoptosis. These results demonstrated that the female pre-NR type was a transitional type during the development of female workers to the NR caste, and the starting point for oogenesis resumption was the NR stage. We confirmed there were fundamental differences in the reproductive pathway of the male and female workers. Therefore, we determined that the transformation process of the female NRs from workers may be a very complex process, and the reproductive biology of the workers has great potential to provide important and spectacular insights into the evolution strategy of termites.

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.

The fat body of termites: functions and stored materials.

The functions of the fat body in the different castes of termites, and accumulation of energy reserves, proteins and urates within this organ, are reviewed. The termite fat body is involved in multiple metabolic activities, including recycling of nitrogen. Termite fat body showed three different types of cells: adipocytes, urocytes and mycetocytes, the latter restricted to the species Mastotermes darwiniensis. Adipocytes synthesize and store lipids, glycogen and several proteins. These cells also elaborate important peptides, including some that act in immune processes. Urocytes are responsible for the storage of spherocrystals of urates, which vary quantitatively among the termite castes. The different metabolic functions of the fat body in the several castes and stages of termites are associated with specific adipocyte morphologies. The synthesis and storage of different compounds modify the structure of the fat body; this differentiation is coordinated by hormones involved with molting and reproductive cycles.

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.

MRT letter: A novel tegumental gland in female imagoes of the neotropical termite Cornitermes cumulans (Isoptera, termitidae, syntermitinae).

In general, the exocrine glands of social insects are structures involved in the chemical communication associated with social life. Here, we report the discovery of an unknown tegumental gland that is present in the female imagoes of Cornitermes cumulans and occurs next to the well-developed tergal glands that have previously been described. The tegumental glands release their secretion in the intersegmental membrane and are composed of bicellular units, a secretory cell and a canal cell, that are closely located to the epidermal cells in the inferior part of the eighth and ninth tergites. The ultrastructure of the glandular cells showed abundant smooth endoplasmic reticulum, suggesting that the secretion may be pheromonal, although its function is still unknown. These exocrine structures are facing the tergal glands, and we hypothesized that they act synergistically with the tergal glands to generate short-range attraction during tandem behavior.

The frontal gland in workers of Neotropical soldierless termites.

The presence of the frontal gland is well established in termite soldiers of Rhinotermitidae, Serritermitidae, and Termitidae. It is one of their main defensive adaptations or even an exclusive weapon. The gland was also occasionally reported in alate imagoes, but never in the worker caste. Here, we report the first observation of a frontal gland in workers of several Neotropical and one African species of Apicotermitinae. The ultrastructure of Aparatermes cingulatus and Anoplotermes nr. subterraneus is described in detail. In these two species, the gland is well-developed, functional and consists of class 1 secretory cells. The presence of envelope cells, wrapping the gland, is an unusual feature, as well as the presence of several zonulae adherens, connecting neighbouring glandular cells. The frontal gland of workers is homologous to this organ in soldiers and imagoes, as evidenced by the same position in the head and its connection to the same muscle. However, the defensive role of the frontal gland in workers remains to be confirmed.

Soldier-specific modification of the mandibular motor neurons in termites.

Social insects exhibit a variety of caste-specific behavioral tendencies that constitute the basis of division of labor within the colony. In termites, the soldier caste display distinctive defense behaviors, such as aggressively attacking enemies with well-developed mandibles, while the other castes retreat into the colony without exhibiting any aggressive response. It is thus likely that some form of soldier-specific neuronal modification exists in termites. In this study, the authors compared the brain (cerebral ganglion) and the suboesophageal ganglion (SOG) of soldiers and pseudergates (workers) in the damp-wood termite, Hodotermopsis sjostedti. The size of the SOG was significantly larger in soldiers than in pseudergates, but no difference in brain size was apparent between castes. Furthermore, mandibular nerves were thicker in soldiers than in pseudergates. Retrograde staining revealed that the somata sizes of the mandibular motor neurons (MdMNs) in soldiers were more than twice as large as those of pseudergates. The enlargement of MdMNs was also observed in individuals treated with a juvenile hormone analogue (JHA), indicating that MdMNs become enlarged in response to juvenile hormone (JH) action during soldier differentiation. This enlargement is likely to have two functions: a behavioral function in which soldier termites will be able to defend more effectively through relatively faster and stronger mandibular movements, and a developmental function that associates with the development of soldier-specific mandibular muscle morphogenesis in termite head. The soldier-specific enlargement of mandibular motor neurons was observed in all examined species in five termite families that have different mechanisms of defense, suggesting that such neuronal modification was already present in the common ancestor of termites and is significant for soldier function.

The fine structure of colleterial glands in two cockroaches and three termites, including a detailed study of Cryptocercus punctulatus (Blattaria, Cryptocercidae) and Mastotermes darwiniensis (Isoptera, Mastotermitidae).

The colleterial glands of insects are organs associated with the female genital apparatus. In cockroaches, these glands produce secretions that cover two parallel rows of eggs during oviposition, and in oviparous species, these secretions become the tanned, sculpted, rigid outer casing of the ootheca. The goal of this study was to compare the gross anatomy of the colleterial glands and the ultrastructure of their component tubules in the phylogenetically significant genera Cryptocercus (Blattaria) and Mastotermes (Isoptera). Recent studies indicate that cockroaches in the genus Cryptocercus are the sister group of termites, and Mastotermes is the only termite known to produce a cockroach-like ootheca. One additional oviparous cockroach, Therea, and two additional termites, Zootermopsis and Pseudacanthotermes, were also examined. As in other cockroaches, the colleterial glands of Cryptocercus and Therea are asymmetrical, with a well developed bipartite left gland and a smaller right gland. In the termites Mastotermes, Zootermopsis, and Pseudacanthotermes, the colleterial glands are composed of a well-developed, paired, anterior gland and a small posterior gland; histological staining and cytological evidence suggest that these are homologues of the left and the right colleterial glands of cockroaches, respectively. At the ultrastructural level, colleterial gland tubules are made of cells belonging to a modified class 1 type cell in the cockroaches, in Mastotermes, and in Zootermopsis; the latter lays its eggs singly, without a surrounding ootheca-like structure. In the advanced termite Pseudacanthotermes, the tubules are made of secretory units belonging to the class 3 cell type. This study demonstrates that the cytological characteristics of colleterial glands in basal termites are similar to those of cockroaches, whether the termite secretes an oothecal casing that covers two parallel rows of eggs, as in Mastotermes, or lays its eggs singly, as in Zootermopsis. The function of colleterial glands in non-mastotermitid termites is unknown.

[Immunocytochemical localization of estrogen receptor in the spermatogenesis of termites].

The available information indicates that estrogen receptor(ER) play a physiological role in the regulation of spermatogenesis in vertebrates. However, the cellular distribution of ER in the testis is poorly understood in invertebrates. The aim of this study was to determine the presence and cellular distribution of ER in the spermatogenesis of termite (Reticulitermes aculabialis). Immunocytochemical analysis showed ER was present in the nucleus of the primary spermatocytes, and the expression of ER was relatively stronger in the primary spermatocytes of the swarming termites. Previous studies have demonstrated the procerebrum of the swarming male termites could strongly secrete FSH (Follicle Stimulating Hormone) and LH (Luteinizing Hormone) which stimulated estrogen secreting. In conclusion, we demonstrated here for the first time that ER might be an important factor in the regulation of the spermatogenesis of termites, and play an important role for starting and maintaining the meiosis cell division of spermatocytes.

Fat body of Prorhinotermes simplex (Isoptera: Rhinotermitidae): ultrastructure, inter-caste differences and lipid composition.

Ultrastructure of the fat body was studied in following castes and developmental stages of Prorhinotermes simplex: larvae of the first and the second instar, pseudergates, presoldiers, soldiers, nymphs, imagoes and mature ergatoid neotenic reproductives of both sexes. Fat body always consists of two principal cell types: adipocytes and urocytes. Adipocytes are characterized by a presence of large amounts of storage substances, namely lipid droplets, glycogen rosettes and proteins in the form of either biocrystals or vacuoles. Proportion of these components strongly varies during ontogeny. Adipocytes are equipped by a large central vacuole in which lipid droplets may resolve. Cytoplasm of urocytes contain glycogen rosettes and spherical or irregular concretions, other organelles are rare. Only adipocytes change their inner structure in the course of ontogeny: amount of glycogen decreases during the postembryonic development, it is the major kind of inclusion in the larvae but lacks in nymphs and imagoes; opposite trend is performed by lipids. The changes in protein content are less obvious but are explained and discussed. The total amount of triacylglycerols (TAGs) was found to be roughly 100microg in a pseudergate, 250microg in a nymph, and 30microg in a soldier. The most abundant fatty acids in TAGs are oleic (O), stearic (S), palmitic (P) and linoleic (L) acid. TAGs form a complex mixture with OOO, OPO, OLO and OOS being the most abundant isomers. Only negligible differences exist among the castes.

Intraspecific molecular phylogeny, genetic variation and phylogeography of Reticulitermes speratus (Isoptera: Rhinotermitidae).

Population structure was investigated in Reticulitermes speratus populations in the Korean Peninsula and the Japanese Archipelago. All trees derived from analyses of the combined sequence dataset of two mitochondrial genes, COII and COIII, showed that R. speratus populations cluster into two major clades comprising the Korean/southern Japanese populations and the north-ern Japanese populations. Analysis of population ge-netic structure showed strong genetic partitioning between populations of the two clades. To understand historical migration routes and current distributions, the phylogeographic history of R. speratus was inferred from intra-/interspecific phylogeny and diver-gence times estimated between the clades of the phylogenetic tree. The estimated migration route and divergence time of ancestral R. speratus are congruent with recent paleogeographic hypotheses involving land-bridge connections between the Asian continent and the Japanese Archipelago. We suggest that ancestral R. speratus separated into northern and southern Japanese populations after its migration into the Japanese main islands from East China during the early Pleistocene via the East China Sea basin, which may have been exposed during that period. The Korean populations seem to have diverged recently from southern Japanese populations; this may explain the current distribution of R. speratus in the Japanese Arachipelago, and account for why it is restricted to northern areas of the Tokara Strait.

[Ultrastructural basis of interactions between prokaryotes and eukaryotes in different symbiotic models]. / Basi ultrastrutturali delle interazioni tra procarioti ed eucarioti in differenti modelli simbiotici.

This paper reviews the Author's contribution to the knowledge of the ultrastructural basis of the prokaryote-eukaryote interactions in different models assessed by an ultrastructural approach. In agreement with the hypothesis of the origin of eukaryotic cells, which are chimeras of several prokaryotes with different morpho-functional specializations, symbiosis had major consequence for evolution of life. In Arthropods, one of the most successful lifestyles, the presence of endosymbiotic prokaryotes, plays an important role in their metabolism. In some cases, genome integration has occurred in the endosymbiotic relationships with the host, proving that intracellular symbiosis is not merely a nutritional supplement. Intracellular symbiotic bacteria are also described in nematodes. In particular, the presence of intracellular Wolbachia in filariae, even if its function is not yet completely known, influences positively the reproductive biology and the survival of the host, as proved by antibiotic treatment against this bacterium. The ultrastructural images reported in this review were obtained using different species of cockroaches, termites, ticks and filarial nematodes. The traditional methods of transmission (TEM), scansion (SEM) and immuno electron microscopy were used. In addition, also freeze-fracture and deep-etching techniques were employed. The cockroaches and the primitive termite Mastotermes darwiniensis host symbiotic bacteria in the ovary and in specialized cells (bacteriocytes) of the fat body. These bacteria have the typical cell boundary profile of gram-negative bacteria and are enveloped in a vacuolar membrane produced by the host cell. Molecular sequence data of 16S rDNA of endosymbionts of five species of cockroaches and M. darwiniensis indicate that they are members of the Flavobacteria-bacteroides group and that the infection occurred in an ancestor common to cockroaches and termites probably after the end of the Paleozoic (250 Ma BP). The symbiotic bacteria are transmitted transovarially and, during embryogenesis, they are integrated into the morphogenetic processes. In particular, we were able to demonstrate that the origin of the bacteriocyte should be looked for in the cells of the haemocyte line (embryonic plasmatocytes). The eggs are infected by the bacteria emerging from the bacteriocytes of the ovaric fat body and, at the end of the vitellogenesis, they are actively phagocytized by the egg membrane. In filarial nematodes, intracellular bacteria belonging to the genus Wolbachia have been described: they have evolved an obligatory mutualistic association with their host. In fact, antibiotic treatments lead to the clearance of bacteria and this loss produces a negative impact on reproduction and survival of the filarial host. We evidenced, by TEM, the degenerative events occurring during the embriogenesis of Brugia pahangi and Dirofilaria immitis after tetracycline treatment. The data suggest that the Wolbachia play a direct role in worm metabolism. Finally, a new additional model of the prokaryote-eukaryote interaction has been described: we have recently discovered a new intracellular alpha-proteobacterium, named Iric ES1, which resides in the ovarian tissues of the tick Ixodes ricinus. The intriguing characteristic of this bacterium is its ability to invade and consume the ovaric mitochondria. From an evolutionary perspective, it is interesting to note that Iric ES1 enters mitochondria in a similar way to that employed by the "predatory" bacterium Bdellovibrio bacteriovorus.