A loop-mediated isothermal amplification (LAMP) assay for the detection of Cryptotermes brevis West Indian drywood termite (Blattodea: Kalotermitidae).

Cryptotermes brevis is one of the most destructive invasive termites in the subtropics and tropics and is a common biosecurity intercept at the Australian border. Drywood termite species are cryptic and difficult to identify morphologically in situations when soldiers or imagos are unavailable. We developed a novel DNA based loop-mediated isothermal amplification (LAMP) assay to detect C. brevis and differentiate it from other drywood termites. Validated voucher specimens of 30 different drywood termite species were obtained from several insect collections from which DNA was extracted and amplified. The amplicons containing partial mitochondrial 16S rRNA were sequenced and a DNA database was created from which C. brevis LAMP primers were developed, optimized, and tested. The assay was assessed against a range of target and non-target species and found to be specific, successfully amplifying the target specimens of C. brevis in under 30 min. Amplification success was variable against C. brevis faecal pellets due to minute, unmeasurable or degraded DNA. This LAMP test is a new tool for the rapid detection of C. brevis that will enable faster and less destructive management of drywood termite infestations.

Taxonomic and Ecological Notes on Termes propinquus Holmgren, 1914 Known from Sumatra (Blattodea: Termitoidae: Termitidae).

The genus Termes Linneus, 1758 consisting of a total of 24 valid named species known from the Old World, is a very heterogeneous group of termites and seems to involve many taxonomic obscurities and confusions. In the island of Sumatra, the sixth-largest island located in the Southeast Asian tropics, four species of Termes have been found, namely, T. comis, T. laticornis, T. rostratus, and T. propinquus. Termes propinquus is also known from Brunei, Indonesia (Kalimantan and Sumatra), Malaysia, and Thailand. However, previous authors have mentioned that T. propinquus has been poorly discriminated from the other congeners, especially T. rostratus. Therefore, the present study aimed at clarifying the discrimination of Termes propinquus from the morphologically similar congeners from Sumatra. A total of 14 nests were collected using a standardized sampling protocol and visual colony searching in Sumatra and its adjacent island. As a result of a careful morphological examination of the soldier caste, T. propinquus was discriminated from the three other congeners by a combination of the following characteristics: distinctly long frontal projection, larger head capsule, and 2nd antennal segment distinctly longer than the 3rd. The redescription of the soldier caste of T. propinquus and a key to Termes species known from Sundaland are provided. The nests of T. propinquus were attached to the bases of living trees, clinging to stumps or the bases of the dead tree, or were epigeal.

Revised systematic position of Nasutitermes brevipilus Emerson, 1925 (Isoptera: Termitidae: Nasutitermitinae) and the designation of Hyleotermes gen. nov.

A new monotypic nasute termite genus, Hyleotermes gen. nov., is proposed for Nasutitermes brevipilus Emerson, 1925. Hyleotermes brevipilus, comb. nov., is redescribed and illustrated based on the morphology of the imago, soldier, and worker castes. It is expanded into Amazonia. The soldier of Hyleotermes differs from that of Nasutitermes Dudley, 1890 in that the former has a long and cylindrical nasus and the head capsule lacks long setae and is covered with microscopic setae. Unlike the worker of Nasutitermes, the Hyleotermes worker has a short mixed segment and an enteric valve is adorned with narrow spines on conical bases. The phylogenetic position of H. brevipilus comb nov., is reconstructed based on a dataset with two mitochondrial markers (COI and 16SrRNA) for 36 terminals, under maximum likelihood and Bayesian inference. Results corroborate that this species is unrelated to Nasutitermes and should be excluded from the genus.

Caste development and sex ratio of the Ryukyu drywood termite Neotermes sugioi and its potential mechanisms.

Sex allocation is one of the most studied traits in evolutionary biology because its theoretical predictions match the empirical data. Here, using the Ryukyu dry-wood termite Neotermes sugioi, we investigated several factors that could bias the sex allocation in three populations (Okinawa, Ishigaki/Iriomote, and Yonaguni). Our survey showed that there were more queen-only colonies than king-only colonies in these populations, suggesting a longer lifespan of the queens than that of the kings. In this condition, sex-asymmetric reproductive value (SRV) theory predicts female bias, because even after the short-lived kings die, the long-lived queens can continue reproduction with their sons. However, sex allocation in this species seemed to be biased toward males. Furthermore, we examined the possibility of intrasexual competition among siblings (ICS). If ICS is the cause of the bias, the allocation is expected to change depending on the total investment in sexual offspring. However, the biomass of both male and female alates increased linearly with the increase in the total biomass of the alates in these populations. Thus, neither the SRV nor the ICS theory could explain the male-biased sex ratio of N. sugioi. On the basis of these results, we discuss the remaining possibilities in this species.

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.

Comparative anatomy of the gut of the South American species of Amitermes, with description of two new species and an identification key based on soldiers and workers.

In this contribution we present updates on the taxonomy and morphology of the South American species of Amitermes. Two new species are described: Amitermes bandeirai, sp. n., from Brazil, and Amitermes lilloi, sp. n., from Argentina. Amitermes nordestinus is a junior synonym of Amitermes aporema. The imago of A. aporema is described for the first time. Detailed comparative gut anatomy of the eight species is presented for the first time. The geographic distribution of Amitermes in South America is expanded and the distribution patterns of some species are discussed.

Anatomical specializations of the gizzard in soil-feeding termites (Termitidae, Apicotermitinae): Taxonomical and functional implications.

The anatomy of the workers' digestive tube is essential in taxonomical studies of soil-feeding Apicotermitinae termites, especially in soldierless lineages. Two structures, the mesenteric-proctodeal junction and the enteric valve, have long been important to distinguish genera and species. By contrast, the gizzard (proventriculus) has been almost ignored by taxonomists because of its generally regressed state in soil-feeding termites. In this study, we document in detail for the first time the sclerotized structures and ornamentations in the gizzard in the Apicotermitinae subfamily. We identified two main clusters of species: those without ornamentations and those exhibiting a sclerotized pulvillar armature, which may include spicules or spines of diverse sizes, numbers and dispositions. The latter group comprises the majority of African soldierless species, a widely diverse and dominant group in tropical forests and savannas. We outline the potential role of the anatomy of the gizzard in the taxonomy of Apicotermitinae based on the interspecific anatomical variation of the pulvillar armatures. We suggest that sclerotized ornamentations regulate the flow of food particles and break or lacerate aggregates to facilitate the access of enzymes in the midgut.

Development and comparative morphology of the reproductive system in different aged males of the drywood termite Cryptotermes brevis (Blattaria, Isoptera, Kalotermitidae).

Termites are eusocial cockroaches, which have received great attention due to their diversity of reproductive strategies. Although these novelties allow new interpretations concerning the mating biology of these insects, studies highlighting the structure of the reproductive system are limited to some termite lineages. Here we provide the first comparative analysis of the reproductive system of a drywood termite, using different aged males of Cryptotermes brevis as models. This species represents an important structural pest in tropical regions, and most aspects of its reproductive biology remain unknown, especially on males. The reproductive apparatus of C. brevis is equipped with paired testes, composed of seven testicular lobes, in which developing spermatozoa are located. The basal portion of the lobes connects to the vasa deferentia and transport spermatozoa to a pair of enlarged chambers, the seminal vesicles. These structures join in a median ejaculatory duct, which opens to the external region through a retractile penis. Spermatozoa were observed in all C. brevis males, exhibiting elongated morphology and measuring about 10 µm in length/4 µm in width. Compared with last-instar nymphs and alates, functional kings showed enlarged testes and seminal vesicles, as well as an intense secretory activity towards the lumen of the latter structures. Histochemical tests evidenced strongly PAS and xylidine Ponceau positive reactions of the secretion only in functional kings, indicating the occurrence of glycoproteins. Thus, we suggest that morphophysiological changes establish during the maturation of the reproductive system in C. brevis.

Comparative study with scanning electron microscopy on the antennal sensilla of two main castes of Coptotermes formosanus Shiraki (Blattaria:Rhinotermitidae).

Sensilla on antennae of the workers and soldiers of Coptotermes formosanus Shiraki were examined by scanning electron microscopy in this study. As the two castes were allocated totally different tasks in the termite colony, we wondered if there was a big difference between their antennae which were recognized as the main sensory appendages of insects. Therefore, detailed information about the morphology, distribution and abundance of various types of sensilla was described in this report. However, our results showed no obvious caste dimorphism was observed. The morphology of antennae and sensilla as well as the general sensilla distribution pattern did not differ between the workers and soldiers of C. formosanus. In total, seven types of sensilla including sensilla chaetica (I, II, III), Böhm bristles, sensilla campaniformia (I, II, III), sensilla trichodea, sensilla basiconica, sensilla trichodea curvata and sensilla capitula were found on the antennae. Additionally, small apertures were found scattered randomly in the antennal cuticle. Functions of these sensilla or structures were proposed to be mechanoreceptors, chemo-receptors, thermo-hygroreceptors, co2-receptors etc. which probably play crucial roles in their various social behaviors.

Drywood Pest Termite Cryptotermes brevis (Blattaria: Isoptera: Kalotermitidae): a Detailed Morphological Study of Pseudergates.

The Kalotermitidae Cryptotermes brevis (Walker) presents colonies that lack a true worker caste. They have totipotent worker-like individuals named pseudergates. Few studies have characterized the morphology of immature instars, including pseudergates. In order to identify these instars and characterize the pseudergates, we conducted a comparison between morphometric and morphological variations among immature individuals of C. brevis colonies. Juvenile hormone analog (JHA) was used in the first instar nymphs to induce regressive molts and compare morphological differences between nymphs and pseudergates. Results showed the existence of three larval instars and four nymphal instars. These immatures were morphologically characterized. Individuals classified as third instar larvae presented white body, 10 to 12 antennal articles, absent or small non-pigmented compound eyes, and absence of wing buds. Pseudergates presented pigmented abdomen and sclerotized cuticle, 10 to 12 antennal articles, and absent or small compound eyes, and few specimens had large pigmented compound eyes and absence of wing buds. First instar nymphs had pigmented abdomen and sclerotized cuticle, 10 to 12 antennal articles, both large non-pigmented and pigmented compound eyes, the presence of wing buds. Bioassays using JHA on first instar nymphs resulted in a large percentage of nymph-soldier intercastes. We concluded that abdomen pigmentation and sclerotized cuticle are good characters to differentiate pseudergates from larvae and the absence of wing buds is a good character to differentiate pseudergates from nymphs. Our findings not only contribute to the basic biological and morphological information of this species but also help to identify correctly pseudergates in further studies that involve applied bioassays.

A new termite species of the genus Dihoplotermes Araújo (Blattaria, Isoptera, Termitidae) from the Brazilian Amazonian rainforest / Uma nova espécie de térmita do gênero Dihoplotermes Araujo (Blattaria, Isoptera, Termitidae) da floresta Amazônica Brasileira

Dihoplotermes, a previously monotypic genus, is now composed of two species. Dihoplotermes taurus sp. nov., a new Termitinae termite species, is illustrated and described based on morphological characters from soldiers, workers, and imago. The new species is distinguished from Dihoplotermes inusitatus Araujo by having a spine-like protuberance with blunt apex on the postmentum, the presence of a spine on the gizzard and the absence of dimorphic soldiers. Furthermore, the internal morphological characteristics of a Dihoplotermes worker are described for the first time. Both internal and external morphological characters were used to improve the characterization of the species.(AU)

Dihoplotermes, que era um gênero monotípico, agora é composto por duas espécies. Dihoplotermes taurus sp. nov., uma nova espécie de cupim Termitinae, é descrita e ilustrada com base nos caracteres morfológicos dos soldados, operários e imago. A nova espécie se diferencia de Dihoplotermes inusitatus Araujo por apresentar uma protuberância em forma de espinho não pontiagudo no posmento, presença de um espinho na moela e ausência de soldados dimórficos. Além disso, são descritas pela primeira vez as características morfológicas internas de um operário de Dihoplotermes e utilizamos ambos caracteres morfológicos internos e externos para melhor caracterizar as espécies.(AU)

Unique Morphogenesis in the Damp-Wood Termite: Abscission of the Stylus during Female Reproductive Caste Differentiation.

Eusocial insects organize their colonies based on division of labor and caste differentiation, in which caste-specific morphologies are sculpted during postembryonic development. In the differentiation between reproductive and sterile castes, characteristics related to mating and reproduction are developed in reproductives, and degenerated in neuters, although little is known about the developmental regulations during the differentiation. In some species of termites, a sensory protrusion at the posterior end of the abdomen, the stylus, is known to disappear in female reproductives. In the present study, we performed anatomical and histological analyses in the damp-wood termite Hodotermopsis sjostedti to elucidate the developmental process underlying the disappearance of the stylus during neotenic and alate differentiation. Although it was first hypothesized that styli were hidden beneath the enlarged seventh sternite, our observation results found out that the styli were completely lost in reproductive females. Further histological observations revealed that the stylus disappearance was not accomplished by degeneration process; rather, styli were separated from the abdomen and discarded with the exoskeleton (exuviae) during the molt into the reproductive caste. This phenomenon in which live tissues are discarded through developmental processes is suggested to be a case of abscission, as seen in plant leaves.

Secondary queens in the parthenogenetic termite Cavitermes tuberosus develop through a transitional helper stage.

In termite species with asexual queen succession (AQS), parthenogenetically produced immatures mostly differentiate into secondary queens, called "neotenics." In order to elucidate the ontogenetic origin of neotenics in Cavitermes tuberosus (Termitidae: Termitinae), a neotropical termite with AQS, we investigated developmental pathways of offspring according to their sex and genetic origin using both morphometric and genetic tools. The caste system of C. tuberosus follows the classical pathway of Termitidae. After the first larval instar, there is a bifurcation between two developmental lines. The apterous line is composed of a second larval instar, several worker instars, presoldiers, and soldiers. Workers display a consistent male bias and soldiers are female-only. The nymphal line is composed of five nymphal instars and the imago stage. We highlight that neotenic queens derive from third and fourth instar nymphs displaying peculiar morphological traits, here termed "aspirants," most of which are produced by parthenogenesis. Aspirants are present in all nests and perform worker tasks while waiting for the queen's death to differentiate into neotenic queens. Aspirants can successfully be used to demonstrate the occurrence of parthenogenesis in termite species whose reproductive cores are difficult to access.

Hypopharynx in termites: Morphological and functional aspects.

The hypopharynx morphology in workers, pseudergates, and soldiers was described in six families of Isoptera using scanning electronic microscopy. This is the first comparative study to detail the hypopharynx among different species of termites, and included 4 one-piece type (OP) and 9 multiple-pieces type termites (MP). The results showed different arrangements of the hypopharynx trichomes, with fewer of these structures in the frontal region of the hypopharynx in pseudergates and soldiers of OP termites. These characteristics did not apply to Serritermes serrifer, since its pseudergates displayed a large quantity of trichomes on the frontal surface. This species may not resemble other OP termites because it does not live inside a piece of wood, but rather are inquilines inside Cornitermes spp. nests. Similar morphology of this mouthpart was observed in the different species of MP termites, with workers exhibiting many unidirectional trichomes on the frontal surface and soldiers presenting fewer of these trichomes. The morphology of the hypopharynx seems to reflect the lifestyle of these species, because MP termites are able to exploit external water sources. Since different arrangements of hypopharynx trichomes are used for water intake via capillary action, the present data reinforce the soldiers' dependence on workers to obtain water and food through the process of trophallaxis. Three different types of sensilla were observed on the lateral region of the hypopharynx: chaetica, basiconica and campaniformia. Chaetica act as chemoreceptors, while basiconica and campaniformia are mechanoreceptors, used in feeding and environmental perception. Better understanding of hypopharynx morphology permits the inference of ecological habits among OP and MP termites, and reflects the physiological differences between workers and soldiers.

Symbiogenesis: Beyond the endosymbiosis theory?

Symbiogenesis, literally 'becoming by living together', refers to the crucial role of symbiosis in major evolutionary innovations. The term usually is reserved for the major transition to eukaryotes and to photosynthesising eukaryotic algae and plants by endosymbiosis. However, in some eukaryote lineages endosymbionts have been lost secondarily, showing that symbiosis can trigger a major evolutionary innovation, even if symbionts were lost secondarily. This leads to the intriguing possibility that symbiosis has played a role in other major evolutionary innovations as well, even if not all extant representatives of such groups still have the symbiotic association. We evaluate this hypothesis for two innovations in termites (Termitoidae, also known informally as "Isoptera"): i) the role of flagellate gut protist symbionts in the transition to eusociality from cockroach-like ancestors, and ii) the role of non-gut associated symbionts in the transition to 'higher' termites, characterized by the absence of flagellate gut protists. In both cases we identify a crucial role for symbionts, even though in both cases, subsequently, symbionts were lost again in some lineages. We also briefly discuss additional possible examples of symbiogenesis. We conclude that symbiogenesis is more broadly applicable than just for the endosymbiotic origin of eukaryotes and photosynthetic eukaryotes, and may be a useful concept to acknowledge the important role of symbiosis for evolutionary innovation. However, we do not accept Lynn Margulis's view that symbiogenesis will lead to a paradigm shift from neoDarwinism, as the role of symbiosis in evolutionary change can be integrated with existing theory perfectly.

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.

A taxonomic revision of the neotropical termite genus Diversitermes (Isoptera: Termitidae: Nasutitermitinae).

The taxonomy of the neotropical termite genus Diversitermes Holmgren 1912 is revised. Three species are recognized as valid: D. castaniceps (Holmgren 1910), D. diversimiles (Silvestri 1901) and D. tiapuan, sp. n. Identification keys based on soldiers and workers are presented. Diversitermes aporeticus Mathews 1977 is transferred to the genus Velocitermes Holmgren 1912. Diversitermes needhami Roonwal, Chhotani & Verma 1981 is a junior synonym of Velocitermes heteropterus (Silvestri 1901), and D. eidmanni Roonwal, Chhotani & Verma 1981 is a junior synonym of D. castaniceps (Holmgren 1910). Eutermes strelnicovi John 1920 (=Nasutitermes strelnicovi) is a junior synonym of D. diversimiles. All castes of the three valid species are described and illustrated. Distribution maps are presented for all species.

A taxonomic revision of the Neotropical termite genus Rhynchotermes (Isoptera, Termitidae, Syntermitinae).

We herein revise the taxonomy of the Neotropical syntermitine genus Rhynchotermes Holmgren. Seven species were recognized and diagnosis given for all available castes. This includes two new species: Rhynchotermes amazonensis Constantini & Cancello sp. nov. and Rhynchotermes matraga Constantini & Cancello sp. nov. Rhynchotermes guarany Cancello is recognized as syn. nov. of Rhynchotermes nasutissimus (Silvestri) and Rhynchotermes nyctobius Mathews syn. nov. of Rhynchotermes diphyes Mathews. The three other formerly described species include Rhynchotermes perarmatus (Snyder), Rhynchotermes piauy Cancello and Rhynchotermes bulbinasus Scheffrahn. We also provide a key to the soldiers of the genus, a distribution map of the species as well as illustrations and biological notes for all species.

The Termite Worker Phenotype Evolved as a Dispersal Strategy for Fertile Wingless Individuals before Eusociality.

Termites are eusocial insects that evolved from solitary cockroaches. It is not known precisely what factors drove the evolution of termite eusociality, that is, skewed reproduction with distinct winged reproductive and wingless worker phenotypes. In other eusocial insects (bees and wasps), reproductive skew evolved first and phenotype differences evolved second. We propose that the reverse pattern occurred in termites, that is, that the winged-wingless diphenism evolved before eusociality. We discuss existing phylogenetic and pheromonal evidence supporting our hypothesis. We provide new experimental evidence from the most basal termite species (Mastotermes darwiniensis), suggesting that the ancestral state was indeed diphenic but presocial. We propose that the mechanism promoting a winged-wingless diphenism-in the absence of eusociality-was greater predation of aerial than terrestrial dispersers, and we support this with a game theoretic model. We augment our hypothesis with a novel explanation for the evolution of the developmental pathways leading to winged and wingless phenotypes in termites. An added benefit of our hypothesis is that it neatly explains the origin of termite eusociality itself: in the pre-eusocial ancestral species, the poor dispersal ability of the wingless phenotype would have led to clustering of relatives around shared resources-a prerequisite for nonparental care of close relatives.

Morphologically Specialized Termite Castes and Advanced Sociality in the Early Cretaceous.

A hallmark of animals that are eusocial, or those with advanced sociality, is reproductive specialization into worker and queen castes. In the most derived societies, these divisions are essentially fixed and in some arthropods, include further specialization--a tripartite system with a soldier caste that defends the colony. Eusociality has originated numerous times among insects but is believed to have appeared first in the termites (Isoptera), in the Early Cretaceous. However, all termites known from the Cretaceous have, until now, only been winged reproductives (alates and dealates); the earliest soldiers and definitive workers were known from just the Miocene (ca. 17-20 million years ago [mya]). Here, we report six termite species preserved in Early Cretaceous (ca. 100 mya) amber from Myanmar, one described as Krishnatermes yoddha gen. et sp. nov., comprising the worker/pseudergate, winged reproductive, and soldier, and a second species, Gigantotermes rex gen. et sp. nov., based on one of the largest soldier termites yet known. Phylogenetic analysis indicates that Krishnatermes are in the basal "Meiatermes-grade" of Cretaceous termites. Workers/pseudergates of another four species are briefly described, but not named. One of these workers/pseudergates reveals that ants--the most serious enemies of modern termites--lived in close proximity to termites in the Burmese paleofauna. These discoveries demonstrate the Mesozoic antiquity of specialized termite caste systems and corroborate that among all social species, termites probably had the original societies.