Molt Cycle Research Articles

Redox Signaling of NADPH Oxidases Regulates Oxidative Stress Responses, Immunity and Aging.

An accumulating body of evidence suggests that transient or physiological reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases act as a redox signal to re-establish homeostasis. The capacity to re-establish homeostasis progressively declines during aging but is maintained in long-lived animals to promote healthy aging. In the model organism Caenorhabditis elegans, ROS generated by dual oxidases (Duox) are important for extracellular matrix integrity, pathogen defense, oxidative stress resistance, and longevity. The Duox enzymatic activity is tightly regulated and under cellular control. Developmental molting cycles, pathogen infections, toxins, mitochondrial-derived ROS, drugs, and small GTPases (e.g., RHO-1) can activate Duox (BLI-3) to generate ROS, whereas NADPH oxidase inhibitors and negative regulators, such as MEMO-1, can inhibit Duox from generating ROS. Three mechanisms-of-action have been discovered for the Duox/BLI-3-generated ROS: (1) enzymatic activity to catalyze crosslinking of free tyrosine ethyl ester in collagen bundles to stabilize extracellular matrices, (2) high ROS bursts/levels to kill pathogens, and (3) redox signaling activating downstream kinase cascades to transcription factors orchestrating oxidative stress and immunity responses to re-establish homeostasis. Although Duox function at the cell surface is well established, recent genetic and biochemical data also suggests a novel role for Duoxs at the endoplasmic reticulum membrane to control redox signaling. Evidence underlying these mechanisms initiated by ROS from NADPH oxidases, and their relevance for human aging, are discussed in this review. Appropriately controlling NADPH oxidase activity for local and physiological redox signaling to maintain cellular homeostasis might be a therapeutic strategy to promote healthy aging.

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas

BackgroundEcdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis.ResultsTranscriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult.In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis.ConclusionsThe data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.

The crustacean cuticle does not record chronological age: New evidence from the gastric mill ossicles

A proposed method to determine chronological age of crustaceans uses putative annual bands in the gastric mill ossicles of the foregut. The interpretation of cuticle bands as growth rings is based on the idea that ossicles are retained through the moult and could accumulate a continuous record of age. However, recent studies presented conflicting findings on the dynamics of gastric mill ossicles during ecdysis. We herein study cuticle bands in ossicles in four species of commercially important decapod crustaceans (Homarus gammarus, Nephrops norvegicus, Cancer pagurus and Necora puber) in different phases of the moult cycle using dissections, light microscopy, micro-computed tomography and cryo-scanning electron microscopy. Our results demonstrate that the gastric mill is moulted and ossicles are not retained but replaced during ecdysis. It is therefore not plausible to conclude that ossicles register a lifetime growth record as annual bands and thereby provide age information. Other mechanisms for the formation of cuticle bands and their correlation to size-based age estimates need to be considered and the effect of moulting on other cuticle structures where ‘annual growth bands’ have been reported should be investigated urgently. Based on our results, there is no evidence for a causative link between cuticle bands and chronological age, meaning it is unreliable for determining crustacean age.

Exogenous 20-hydroxyecdysone induces epidermal carbonic anhydrase but inhibits exoskeletal calcification in the post-ecdysial blue crab, Callinectes sapidus

The crustacean exoskeleton is composed primarily of chitin, proteins and various inorganic compounds. It is the inorganic compounds, such as calcium and magnesium, that underlie the exoskeletal mineralization process following ecdysis. Little is known about the hormonal mechanism for this process in crustaceans. Carbonic anhydrase (CA) in the epidermis has been suggested to aid in deposition of calcium and magnesium carbonates to the exoskeleton in crustaceans by generating bicarbonate ions, resulting in mineralization. Given a similar pattern of fluctuation in prevalence between epidermal CA and ecdysteroids during the crab molting cycle, it has been proposed in a previous study that post-ecdysial epidermal CA and subsequent metal deposition to the exoskeleton are controlled by the ecdysteroid molting hormones in the blue crab, Callinectes sapidus. This study sought to acquire evidence to support such a proposition. Early postmolt and early intermolt blue crabs were used to quantify epidermal CA mRNA expression when exposed in vitro to three physiologically relevant concentrations of 10 nM, 100 nM and 1 μM 20-hydroxyecdysone (20-HE), using the epidermis-with-exoskeleton (EWE) tissue assay method. It was found that 100 nM 20-HE significantly induced the mRNA of CasCAg, a CA isoenzyme, in epidermal tissues of early intermolt crabs and that injection of 20-HE at a dose of 15 ng/g significantly elevated epidermal CA activity in vivo in early intermolt crabs. These two lines of evidence clearly show that post-ecdysial epidermal CA is influenced by the molting hormone. Interestingly, exoskeletal calcium content was significantly lower in the 20-HE treated crabs than in the control, whereas magnesium content was unchanged. The inhibition of calcification in the post-ecdysial exoskeleton by the exogenous molting hormone may implicate that sclerotization and mineralization of the new shell must be coordinated and that enhanced deposition of carbonate salts as a result of increased epidermal CA activity following the administration of exogenous molting hormone would be inhibited to avoid the formation of a structurally disrupted exoskeleton.

Identification and functional analysis of transforming growth factor-β type I receptor (TβR1) from Scylla paramamosain: The first evidence of TβR1 involved in development and innate immunity in crustaceans

The transforming growth factor-β (TGF-β) receptor-mediated TGF-β signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-β receptor (TβR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTβR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTβR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTβR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTβR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTβR1 was silenced. Additionally, the expression levels of SpTβR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TβR1 antagonist or agonist for 24 h. These results indicated that TβR1 not only contributed to the crabs’ development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-β receptors in crustacean species and even in invertebrates.

The search for proteins involved in the formation of crustacean cuticular structures

Crustacean cuticular structures are key features formed during a molt cycle. These structures are complex biomaterials comprising chitin and different mineral forms in distinct scaffold organizations. The formation of these complex biomaterials is controlled by the organic extracellular matrix including structural proteins. Since cuticular structures are formed de novo during each molt cycle, the spatial and temporal expression patterns of structural proteins are tightly linked to molt cycle events. As a model scenario, we demonstrate the molt-related pattern of expression of the gene encoding GAP65, a core structural protein involved in the formation of the cuticular structures of Cherax quadricarinatus. Based on this typical pattern of expression and using a binary-patterning approach, which is a specialized tool for the study of molt-related proteins, we revealed and characterized additional candidate proteins involved in the formation of crustacean cuticular structures. We propose that our approach be applied as a framework in the search for proteins involved in the formation of the crustacean cuticle. To stimulate research on this important aspect of structural biology, we put forward a schematic representation of the extracellular matrix and its proteins in three cuticular structures of C. quadricarinatus, the gastroliths, the mandibles, and the mineralized cuticle.

Recognizing incipient epizootic shell disease lesions in the carapace of the American lobster, Homarus americanus

Causal factors leading to epizootic shell disease (ESD) lesions in American lobster, Homarus americanus H. Milne-Edwards, 1837, are not well understood. We explore the structural and physiological bases for development of ESD from preclinical stages invisible to unaided eye to early visible stages. We present a lobster shell model, which develops structural functional vulnerability and suggests plausible routes to ESD. Medallions of carapace cuticle were obtained from carapace fixed with protocols to minimize movement of mineral and macromolecular components. Rapid processing of medallions was used to encourage large sample sizes compatible with environmental surveys. One- and two-dimensional analytic maps of polished sections of the cuticle, obtained with an electron microprobe, described the composite mineral and polymeric structures. Micro-Raman spectroscopy was used to identify bond properties of phosphates and carbonates, as well as signatures of organic structures. The frequency and properties of structures identified can be monitored through the lobster molting cycle using a high throughput application of micro-computed tomography (μCT). We observed density differences in the calcite layer, exocuticle, and endocuticle, and the frequency and structure of CaCO3 structures in the endocuticle and membranous layer of carapace cuticle during chosen stages of the molting cycle. The correlative microscopy and μCT of shell structures provides improved understanding of the lobster cuticle structure. Detailed structural differences quantified through development and under different environmental conditions can provide insight into causes and vulnerabilities associated with ESD.

Transmission electron microscopic and immunohistochemical observations of resting follicles of feathers in chicken show massive cell degeneration

The molting cycle of feathers includes an anagen (growth) stage, a likely catagen stage where the feather follicles degenerate, and a resting stage where fully grown feathers remain in their follicles and are functional before molting. However, the cytological changes involved in the resting and molting stages are poorly known, so the results of an ultrastructural analysis of these processes in adult chick feathers are presented here. The study showed that the dermal papilla shrinks, and numerous cells present increased heterochromatin and free collagen fibrils in the extracellular matrix. Degeneration of the germinal epithelium of the follicle-the papillary collar-occurs with an initial substantial contraction of cells followed by an increase in heterochromatin, vesicle and lipid accumulation, and membrane and organelle degeneration. Desmosomes are still present between degenerating epithelial cells, but ribosomes and tonofilaments disappear. This suggests that cell necrosis initially proceeds as a major contraction resembling apoptosis-a process termed necroptosis, which was previously also shown to occur during the formation of barbs and barbules in mature down and pennaceous feathers. This study suggests that, aside from apoptosis, the collar epithelium degenerates due to external factors, in particular the retraction of blood vessels supplying the dermal papilla. In contrast, revascularization of the dermal papilla triggers a new phase of feather growth (anagen).

The consequences of daily cyclic hypoxia on a European grass shrimp: From short‐term responses to long‐term effects

Abstract Salt marshes are a key coastal environment for their important role as nursery habitats for marine and estuarine fish and crustaceans. Salt marshes are variable environments where species can experience daily cyclic hypoxic stress, characterised by profound variations in oxygen partial pressure (pO2) from supersaturated conditions (~42 kPa) to extremely hypoxic conditions (~3 kPa) in ~12‐hr. Here, under laboratory conditions, we assessed the physiological consequences of exposing the shrimp Palaemon varians, a species commonly found in the salt marshes of northern Europe, to the daily cyclic hypoxic regime currently experienced in its habitat in August (7.1 ± 1.8 hr/day below 4.0 kPa). In the laboratory, adults were kept at water pO2 < 4.5 kPa for 7 hr each night and in normoxic conditions for the rest of the time. We recorded an acceleration of P. varians’ moult cycle, which was 15% shorter in animals kept in cyclic hypoxia compared to animals in normoxia. Similarly, the pattern of expression of two cuticular proteins over an entire moult cycle indicated an effect of cyclic hypoxia on moult stage‐related genes. After 16 days, morphological changes to the gills were detected, with shrimps in cyclic hypoxia having a 13.6% larger lamellar surface area (measured in μm2/mg animal) than normoxic animals, which could improve gas exchange capacity. Overall, phenotypic and morphological data indicate that faster moulting is triggered in response to cyclic hypoxia, with the benefit that gill modifications can be prompted more rapidly in order to meet oxygen requirements of the body. On the first experimental day, in cyclic hypoxic‐exposed animals, we recorded a 50% decrease in feeding rates (during hypoxic conditions) in comparison with normoxic animals. Similarly, ammonium excretion was reduced by 66%–75% during the 1st and 21st experimental day. Body size was reduced by ~4% after 28 days. Females that reproduced in cyclic hypoxic conditions reduced the amount of yolk in each egg by ~24%. Overall, results underline how, in a decapod shrimp living in a key coastal environment, many physiological parameters are impaired by a cyclic hypoxic regime that is currently found in its natural habitat. A plain language summary is available for this article.

Characterization, expression patterns of molt-inhibiting hormone gene of Macrobrachium nipponense and its roles in molting and growth.

The oriental river prawn, Macrobrachium nipponense, is an important commercial aquaculture resource in China. In order to overwinter, M. nipponense displays decreased physiological activity and less consumption of energy. Sudden warming would trigger molting and cause an extensive death, resulting in huge economic losses. Therefore, it is of great practical significance to study the molting mechanism of oriental river prawns. Molt-inhibiting hormone gene (MIH) plays a major role in regulating molting in crustaceans. In this study, a full length MIH cDNA of M. nipponense (Mn-MIH) was cloned from the eyestalk. The total length of the Mn-MIH was 925 bp, encoding a protein of 119 amino acids. Tissue distribution analysis showed that Mn-MIH was highly expressed in the eyestalk, and that it had relatively low expression in gill, ovary, and abdominal ganglion. Mn-MIH was detected in all developmental stages, and changed regularly in line with the molting cycle of the embryo and larva. Mn-MIH varied in response to the molting cycle, suggesting that Mn-MIH negatively regulates ecdysteroidogenesis. Mn-MIH inhibition by RNAi resulted in a significant acceleration of molting cycles in both males and females, confirming the inhibitory role of MIH in molting. After long-term RNAi males, but not females had significant weight gain, confirming that Mn-MIH plays an important role in growth of M. nipponense. Our work contributes to a better understanding of the role of Mn-MIH in crustacean molting and growth.

Bird molting and breeding in an area undergoing re-vegetation in the Atlantic Forest of southeastern Brazil

Molting and breeding cycles are high-energetic costs events, and usually synchronized and temporally well defined in tropical avian communities. However, in degraded and/or undergoing restoration areas, environmental stresses, such as forest fragmentation and restriction of food resources, could locally change these patterns. This study aims to analyze molting and breeding in a bird assembly in an undergoing re-vegetation area in the Atlantic Forest of southeastern Brazil. By mist netting, we searched for evidence of molting of fight feathers and presence of brood patch in captured birds. We also recorded three environmental variables (temperature, rainfall, and photoperiod) to test whether they affect those events. We sampled 85 individuals from 36 species. The breeding cycle started in July and peaked in November and December. The molting period was between July and April of the next year and peaked in January and February. Molting and breeding events were related to temperature. The overlap between these events was of 11% (n = 9). Our data suggest that molt and breeding times are in accordance to other studies where the climate is more seasonal, yet more studies are necessary to investigate those biological cycles and their possible alterations due to environmental degradation.

A chitinase with two catalytic domains is required for organization of the cuticular extracellular matrix of a beetle

Insect cuticle or exoskeleton is an extracellular matrix formed primarily from two different structural biopolymers, chitin and protein. During each molt cycle, a new cuticle is deposited simultaneously with degradation of the inner part of the chitinous procuticle of the overlying old exoskeleton by molting fluid enzymes including epidermal chitinases. In this study we report a novel role for an epidermal endochitinase containing two catalytic domains, TcCHT7, from the red flour beetle, Tribolium castaneum, in organizing chitin in the newly forming cuticle rather than in degrading chitin present in the prior one. Recombinant TcCHT7 expressed in insect cells is membrane-bound and capable of hydrolyzing an extracellular chitin substrate, whereas in vivo, this enzyme is also released from the plasma membrane and co-localizes with chitin in the entire procuticle. RNAi of TcCHT7 reveals that this enzyme is nonessential for any type of molt or degradation of the chitinous matrix in the old cuticle. In contrast, TcCHT7 is required for maintaining the integrity of the cuticle as a compact structure of alternating electron-dense and electron-lucent laminae. There is a reduction in thickness of elytral and leg cuticles after RNAi for TcCHT7. TcCHT7 is also required for formation of properly oriented long chitin fibers inside pore canals that are vertically oriented columnar structures, which contribute to the mechanical strength of a light-weight, yet rigid, adult cuticle. The conservation of CHT7-like proteins harboring such a unique domain configuration among many insect and other arthropod species indicates a critical role for the group III class of chitinases in the higher ordered organization of chitin fibers for development of the structural integrity of many invertebrate exoskeletons.

The relationship of blue crab (Callinectes sapidus) size class and molt stage to disease acquisition and intensity of Hematodinium perezi infections.

In the blue crab, Callinectes sapidus, early studies suggested a relationship between smaller crabs, which molt more frequently, and higher rates of infection by the dinoflagellate parasite, Hematodinium perezi. In order to better explore the influence of size and molting on infections, blue crabs were collected from the Maryland coastal bays and screened for the presence of H. perezi in hemolymph samples using a quantitative PCR assay. Molt stage was determined by a radioimmunoassay which measured ecdysteroid concentrations in blue crab hemolymph. Differences were seen in infection prevalence between size classes, with the medium size class (crabs 61 to 90 mm carapace width) and juvenile crabs (≤ 30 mm carapace width) having the highest infection prevalence at 47.2% and 46.7%, respectively. All size classes were susceptible to infection, although fall months favored disease acquisition by juveniles, whereas mid-sized animals (31–90 mm carapace width) acquired infection predominantly in summer. Disease intensity was also most pronounced in the summer, with blue crabs > 61 mm being primary sources of proliferation. Molt status appeared to be influenced by infection, with infected crabs having significantly lower concentrations of ecdysteroids than uninfected crabs in the spring and the fall. We hypothesize that infection by H. perezi may increase molt intervals, with a delay in the spring molt cycle as an evolutionary adaptation functioning to coincide with increased host metabolism, providing optimal conditions for H. perezi propagation. Regardless of season, postmolt crabs harbored significantly higher proportions of moderate and heavy infections, suggesting that the process of ecdysis, and the postmolt recovery period, has a positive effect on parasite proliferation.

Development of calcium bodies in Hylonsicus riparius (Crustacea: Isopoda)

Calcium bodies are internal epithelial sacs found in terrestrial isopods of the family Trichoniscidae that contain a mineralized extracellular matrix that is deposited and resorbed in relation to the molt cycle. Calcium bodies in several trichoniscids are filled with bacteria, the function of which is currently unknown. The woodlouse Hyloniscus riparius differs from other trichoniscids in that it possesses two different pairs of calcium bodies, the posterior pair being filled with bacteria and the anterior pair being devoid of bacteria. We explored the development of these organs and bacterial colonization of their lumen during the postmarsupial development with the use of optical clearing and whole-body confocal imaging of larval and juvenile stages. Our results show that calcium bodies are formed as invaginations of the epidermis in the region of intersegmental membranes during the postmarsupial development. The anterior pair of calcium bodies is generated during the first postmarsupial manca stage, whereas the posterior calcium bodies first appear in juveniles and are immediately colonized by bacteria, likely through a connection between the calcium body lumen and the body surface. Mineral is deposited in calcium bodies as soon as they are present.

Evaluation of the Potential Side-Effects of Novaluron on the Shrimp <i>Palaemon adspersus</i>: Moulting Hormone Profile, Cuticle Secretion and Chitin Contents

The leaching of a large amount of pollutants derived from agricultural and domestic activities (fertilizers, pesticides, detergents) might contaminate especially the aquatic environments affecting several non-target aquatic organisms such as crustacean species. The current study aimed to evaluate under laboratory conditions the potential side-effects of novaluron (20% Wettable Powder), a potent benzoylurea derivative insecticide on mosquito larvae, against a non-target shrimp, <i>Palaemon adspersus</i> Rathke, 1837 (Decapoda, Palaemonidae). This species is abundant in the lagoon El-Mellah (Northeast Algeria) and a relatively important species for the local fishery industry. The compound was tested at two concentrations (0.91 mg/L and 4.30 mg/L) corresponding respectively to the LC₅₀ and LC₉₀ determined against fourth-instar larvae of <i>Culiseta longiareolata</i> (Diptera, Culicidae). The newly ecdysed adult shrimps were exposed for 15 days, i.e. stage A until D during a moult cycle. Under normal conditions, changes in hemolymphatic ecdysteroid concentrations during the molting cycle presented a peak at stage D, just before the ecdysis while in the treated series, we note an increase in hemolymphatic ecdysteroid concentrations at stages C and D and an absence of the peak as compared to the controls. Histological observations of integuments revealed that novaluron caused a significant reduction in thickness of the new cuticle at its LC₅₀ and an inhibition of the new cuticle secretion at its LC₅₀. The determination of chitin amounts, showed that exposure of shrimps to novaluron resulted in a significant decrease of values at all molting stages with a dose-response manner in comparison to controls. Thus, the overall data confirm the primary mode of action of novaluron on chitin. This insecticide can present secondary effects on this non-target shrimp species commercially important for the local economy.

Do arthropods feel anxious during molts?

The molting process of arthropods, chiefly controlled by ecdysteroids, is generally considered very stressful. Our previous investigations have shown that crayfish, after having experienced stressful situations, display anxiety-like behavior (ALB), characterized by aversion to light in a dark/light plus-maze (DLPM). In the present experiments, the spontaneous exploratory behavior of isolated crayfish was analyzed in a DLPM at different stages of their molt cycle. All tested animals displayed transitory aversion to light similar to ALB, before and, mostly, after molting, but not during inter-molt. Injection of ecdysteroids into inter-molt animals elicited ALB after a delay of 4 days, suggesting a long-term, possibly indirect, hormonal effect. Importantly, ecdysteroid-induced ALB was suppressed by the injection of an anxiolytic benzodiazepine. Thus, molts and their hormonal control impose internal stress on crayfish, leading to aversion behavior that has the main characteristics of anxiety. These observations are possibly generalizable to many other arthropods.

Changes in the ultrastructure of the carapace during the molt cycle of Macrobrachium nipponense

Changes in the ultrastructure of the carapace during the molt cycle of <i>Macrobrachium nipponense</i>

What Kills the Hindgut Flagellates of Lower Termites during the Host Molting Cycle?

Subsocial wood feeding cockroaches in the genus Cryptocercus, the sister group of termites, retain their symbiotic gut flagellates during the host molting cycle, but in lower termites, closely related flagellates die prior to host ecdysis. Although the prevalent view is that termite flagellates die because of conditions of starvation and desiccation in the gut during the host molting cycle, the work of L.R. Cleveland in the 1930s through the 1960s provides a strong alternate hypothesis: it was the changed hormonal environment associated with the origin of eusociality and its concomitant shift in termite developmental ontogeny that instigates the death of the flagellates in termites. Although the research on termite gut microbial communities has exploded since the advent of modern molecular techniques, the role of the host hormonal environment on the life cycle of its gut flagellates has been neglected. Here Cleveland’s studies are revisited to provide a basis for re-examination of the problem, and the results framed in the context of two alternate hypotheses: the flagellate symbionts are victims of the change in host social status, or the flagellates have become incorporated into the life cycle of the eusocial termite colony. Recent work on parasitic protists suggests clear paths for exploring these hypotheses and for resolving long standing issues regarding sexual-encystment cycles in flagellates of the Cryptocercus-termite lineage using molecular methodologies, bringing the problem into the modern era.

Molecular characterization, RNA interference and recombinant protein approach to study the function of the putative Molt Inhibiting Hormone (FmMIH1) gene from the shrimp Fenneropenaeus merguiensis

The Molt Inhibiting Hormone gene and cDNA of the banana shrimp Fenneropenaeus merguiensis (FmMIH1) has been cloned and characterized. FmMIH1 possesses most of the characteristics of the eyestalk CHH/MIH/GIH family subtype-II neuropeptides. FmMIH1 open reading frame consists of 315 bp encoding for 105 amino acid residues. The mature peptide of FmMIH1 consists of 76 amino acid residues, a glycine residue at position 11 of the mature peptide and 6 cysteine residues located in the conserved position. In addition to eyestalk, high levels of FmMIH1 transcript could also be detected in the intestine. FmMIH1 transcript level is low throughout the post-molt, early to mid-intermolt and premolt. However, a sharp increase could be observed in late intermolt (C3 stage). Both alignment and phylogenetic analysis reveal that FmMIH1 is most similar to the MIH1 of other shrimps. For functional assay, RNA interference results show that a significant 2.3 days (P < 0.05) reduction in molt cycle duration could be observed in shrimp receiving dsFmMIH1 injection. Surprisingly, injection of recombinant FmMIH1 could also cause a significant reduction of the molt cycle (average 1.9 days, P < 0.05). We hypothesize that the recombinant protein is biological inactive but it competes with the endogenous MIH for carrier protein binding and consequently reduces the amount of biological MIH that could reach the targets. In conclusion, the result of this study will provide us new insight in molting/growth control in crustacean.

Molting drives mortality in foraging populations of Formosan subterranean termites (Isoptera: Rhinotermitidae) baited with a chitin synthesis inhibitor, noviflumuron.

The lethal time of a chitin synthesis inhibitor (CSI)-baited colony of Coptotermes formosanus Shiraki largely depends on the time taken for workers to molt after consuming a lethal dose. Given that termite molting biology can regulate the time to elimination of a baited termite colony, the objective of this study was to determine how the fasting period (an important element of the worker molting cycle) and acquisition of the lethal dose can impact the total elimination period for a colony. Noviflumuron eliminated the foraging population over a 30 m-long foraging arena within 9 weeks. However, mortality in treatment groups did not begin until day 11. The 10-day lag post treatment corresponds to the fasting period during which workers to prepare for ecdysis on the 11th day, at which time mortality occurs due to the effect of CSI. This study shows that, with the exception of successful molting events during the first 10 days after baiting, mortality among workers is inevitable in the next molt with lethal doses of noviflumuron. The 10-day fasting period can comprise at least 22% of a molt cycle (45 days for a fourth worker instar or lower), and can provide a window during which a worker escapes the effect of CSI bait, thereby extending the total time needed for elimination of a colony. © 2017 Society of Chemical Industry.