Octopamine is involved in TRP-induced thermopreference responses in American cockroach.

Insects' thermoregulatory processes depend on thermosensation and further processing of thermal information in the nervous system. It is commonly known that thermosensation involves thermoreceptors, including members of the TRP receptor family, but the involvement of neurotransmitters in thermoregulatory pathways remains unstudied. We conducted test to determine whether octopamine, a biogenic amine that acts as a neurotransmitter and neurohormone in insects, is involved in TRP-induced thermoregulatory responses in Periplaneta americana. We used capsaicin, an activator of the heat-sensitive TRP channel, Painless, to induce thermoregulatory response in cockroaches. Then, we evaluated the behavioural (thermal preferences and grooming), physiological (heart rate) and biochemical responses of insects to capsaicin, octopamine and phentolamine - octopaminergic receptor blocker. Capsaicin, similar to octopamine, increased cockroaches' grooming activity and heart rate. Moreover, octopamine level and protein kinase A (PKA) activity significantly increased after capsaicin treatment. Blocking octopaminergic receptors with phentolamine diminished cockroaches' response to capsaicin - thermoregulatory behaviour, grooming and heart rate were abolished. The results indicate that octopamine is a neurotransmitter secreted in insects after the activation of heat receptors.

Perfluorooctanoic acid induces behavioral impairment and oxidative injury in Nauphoeta cinerea nymphs.

Perfluorooctanoic acid (PFOA) is a persistent organic contaminant with potential health threats to both animals and humans. However, the impact of PFOA on insects, which play significant roles in ecosystems, is understudied. We evaluated the toxicological impact of ecologically relevant concentrations of PFOA (0, 25, 50, 100, and 200 µg L-1) on Nauphoeta cinerea nymphs following exposure for 42 consecutive days. We analyzed the behavior of the insects with automated video-tracking software and processed the head, midgut, and fat body for biochemical assays. PFOA-exposed insects exhibited significant reductions in locomotory abilities and an increase in freezing time. Furthermore, PFOA exposure reduced acetylcholinesterase activity in the insect head. PFOA exposure increased the activities of superoxide dismutase, glutathione peroxidase, and catalase in the head and midgut, but decreased them in the fat body. PFOA also significantly increased glutathione-S transferase activity, while decreasing glutathione levels in the head, midgut, and fat body. Additionally, PFOA exposure increased reactive oxygen and nitrogen species, nitric oxide, lipid peroxidation, and protein carbonyl contents in the head, midgut, and fat body of the insects. In conclusion, our findings indicate that PFOA exposure poses an ecological risk to Nauphoeta cinerea.

Metoprolol elicits neurobehavioral insufficiency and oxidative damage in nontarget Nauphoeta cinerea nymphs.

Metoprolol, a drug for hypertension and cardiovascular diseases, has become a contaminant of emerging concern because of its frequent detection in various environmental matrices globally. The dwindling in the biodiversity of useful insects owing to increasing presence of environmental chemicals is currently a great interest to the scientific community. In the current research, the toxicological impact of ecologically relevant concentrations of metoprolol at 0, 0.05, 0.1, 0.25, and 0.5 µg/L on Nauphoeta cinerea nymphs following exposure for 42 consecutive days was evaluated. The insects' behavior was analyzed with automated video-tracking software (ANY-maze, Stoelting Co, USA) while biochemical assays were done using the midgut, head and fat body. Metoprolol-exposed nymphs exhibited significant diminutions in the path efficiency, mobility time, distance traveled, body rotation, maximum speed and turn angle cum more episodes, and time of freezing. In addition, the heat maps and track plots confirmed the metoprolol-mediated wane in the exploratory and locomotor fitness of the insects. Compared with control, metoprolol exposure decreased acetylcholinesterase activity in insects head. Antioxidant enzymes activities and glutathione level were markedly decreased whereas indices of inflammation and oxidative injury to proteins and lipids were significantly increased in head, midgut and fat body of metoprolol-exposed insects. Taken together, metoprolol exposure induces neurobehavioral insufficiency and oxido-inflammatory injury in N. cinerea nymphs. These findings suggest the potential health effects of environmental contamination with metoprolol on ecologically and economically important nontarget insects.

Comparative analysis of adipokinetic hormones and their receptors in Blattodea reveals novel patterns of gene evolution.

Adipokinetic hormone (AKH) is a neuropeptide produced in the insect corpora cardiaca that plays an essential role in mobilising carbohydrates and lipids from the fat body to the haemolymph. AKH acts by binding to a rhodopsin-like G protein-coupled receptor (GPCR), the adipokinetic hormone receptor (AKHR). In this study, we tackle AKH ligand and receptor gene evolution as well as the evolutionary origins of AKH gene paralogues from the order Blattodea (termites and cockroaches). Phylogenetic analyses of AKH precursor sequences point to an ancient AKH gene duplication event in the common ancestor of Blaberoidea, yielding a new group of putative decapeptides. In total, 16 different AKH peptides from 90 species were obtained. Two octapeptides and seven putatively novel decapeptides are predicted for the first time. AKH receptor sequences from 18 species, spanning solitary cockroaches and subsocial wood roaches as well as lower and higher termites, were subsequently acquired using classical molecular methods and in silico approaches employing transcriptomic data. Aligned AKHR open reading frames revealed 7 highly conserved transmembrane regions, a typical arrangement for GPCRs. Phylogenetic analyses based on AKHR sequences support accepted relationships among termite, subsocial (Cryptocercus spp.) and solitary cockroach lineages to a large extent, while putative post-translational modification sites do not greatly differ between solitary and subsocial roaches and social termites. Our study provides important information not only for AKH and AKHR functional research but also for further analyses interested in their development as potential candidates for biorational pest control agents against invasive termites and cockroaches.

Different neuroendocrine cell types in the pars intercerebralis of Periplaneta americana produce their own specific IGF-related peptides.

Of the nine genes of the American cockroach, Periplaneta americana, coding for peptides related to insulin and insulin-like growth factor, seven show significant expression in the central nervous system as demonstrated by the polymerase chain reaction on reverse transcribed RNA. In situ hybridisation shows that five of those are expressed by cells in the pars intercerebralis. Antisera raised to the predicted peptides show that these cells are neuroendocrine in nature and project to the corpora cardiaca. Interestingly, there are at least three cell types that each express different genes. This contrasts with Drosophila where a single cell type expresses a number of genes expressing several such peptides. Whereas in Drosophila the neuroendocrine cells producing insulin-like peptides also express sulfakinins, the arthropod orthologs of gastrin and cholecystokinin, in Periplaneta the sulfakinins are produced by different cells. Other neuropeptides known to be produced by the pars intercerebralis in Periplaneta and other insect species, such as the CRF-like diuretic hormone, neuroparsin, leucokinin or myosuppressin, neither colocalize with an insulin-related peptide. The separate cellular localization of these peptides and the existence of multiple insulin receptors in this species implies a more complex regulation by insulin and IGF-related peptides in cockroaches than in the fruit fly.

Prodigiosin from Serratia Marcescens in Cockroach Inhibits the Proliferation of Hepatocellular Carcinoma Cells through Endoplasmic Reticulum Stress-Induced Apoptosis.

Hepatocellular carcinoma (HCC) is the most common primary liver malignant tumor, and the targeted therapy for HCC is very limited. Our previous study demonstrated that prodigiosin(PG), a secondary metabolite from Serratia marcescens found in the intestinal flora of cockroaches, inhibits the proliferation of HCC and increases the expression of CHOP, a marker protein for endoplasmic reticulum stress (ERS)-mediated apoptosis, in a dose-dependent manner. However, the mechanisms underlying the activity of PG in vivo and in vitro are unclear. This study explored the molecular mechanisms of PG-induced ERS against liver cancer in vitro and in vivo. The apoptosis of hepatocellular carcinoma cells induced by PG through endoplasmic reticulum stress was observed by flow cytometry, colony formation assay, cell viability assay, immunoblot analysis, and TUNEL assay. The localization of PG in cells was observed using laser confocal fluorescence microscopy. Flow cytometry was used to detect the intracellular Ca2+ concentration after PG treatment. We found that PG could promote apoptosis and inhibit the proliferation of HCC. It was localized in the endoplasmic reticulum of HepG2 cells, where it induces the release of Ca2+. PG also upregulated the expression of key unfolded response proteins, including PERK, IRE1α, Bip, and CHOP, and related apoptotic proteins, including caspase3, caspase9, and Bax, but down-regulated the expression of anti-apoptotic protein Bcl-2 in liver cancer. Alleviating ERS reversed the above phenomenon. PG had no obvious negative effects on the functioning of the liver, kidney, and other main organs in nude mice, but the growth of liver cancer cells was inhibited by inducing ERS in vivo. The findings of this study showed that PG promotes apoptosis of HCC by inducing ERS.

Burkholderia pseudomallei JW270 Is Lethal in the Madagascar Hissing Cockroach Infection Model and Can Be Utilized at Biosafety Level 2 to Identify Putative Virulence Factors.

Burkholderia pseudomallei, the causative agent of melioidosis, is classified by the CDC as a tier 1 select agent, and work involving it must be performed in a biosafety level 3 (BSL-3) laboratory. Three BSL-2 surrogate strains derived from B. pseudomallei 1026b, a virulent clinical isolate, have been removed from the CDC select agent list. These strains, Bp82, B0011, and JW270, are highly attenuated in rodent models of melioidosis and cannot be utilized to identify virulence determinants because of their high 50% lethal dose (LD50). We previously demonstrated that the Madagascar hissing cockroach (MHC) is a tractable surrogate host to study the innate immune response against Burkholderia. In this study, we found that JW270 maintains its virulence in MHCs. This surprising result indicates that it may be possible to identify potential virulence genes in JW270 by using MHCs at BSL-2. We tested this hypothesis by constructing JW270 mutations in genes that are required (hcp1) or dispensable (hcp2) for B. pseudomallei virulence in rodents. JW270 Δhcp1 was avirulent in MHCs and JW270 Δhcp2 was virulent, suggesting that MHCs can be used at BSL-2 for the discovery of important virulence factors. JW270 ΔBPSS2185, a strain harboring a mutation in a type IV pilin locus (TFP8) required for full virulence in BALB/c mice, was also found to be attenuated in MHCs. Finally, we demonstrate that the hmqA-G locus, which encodes the production of a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines, is important for JW270 virulence in MHCs and may represent a novel virulence determinant.

An embryonic cell line from the American cockroach Periplaneta americana L. (Blattaria: Blattidae) exhibits susceptibility to AcMNPV.

Although the baculovirus expression vector system (BEVS) is widely used in the production of recombinant proteins, only a few lepidopteran insect cell lines have been successfully used so far. This study aimed at evaluating the characteristics of an embryonic cell line from the American cockroach Periplaneta americana L., RIRI-PA1, and determining whether it could be used in recombinant protein expression. Wild type Autographa californica multiple nucleopolyhedrovirus (AcMNPV-wt) and green fluorescent protein (GFP)-replicating recombinant baculoviruses (AcMNPV-GFP) were used to infect RIRI-PA1 respectively, demonstrating that RIRI-PA1 cells could be infected by AcMNPV and express recombinant proteins. Within 24 h of infection with AcMNPV-GFP, the GFP expression was higher than that in Sf21 cells. Furthermore, the infection of RIRI-PA1 cells increased gradually (multiplicity of infection, 10) within 24 h, while in Sf21 cells, the infection only began to increase within 48 h. However, after exposure for 96-168 h, the virus progeny and recombinant protein production of RIRI-PA1 cells was lower than those of Sf21 cells. Western blotting revealed that RIRI-PA1 cells could express recombinant GFP, and the protein expression level positively correlated with the multiplicity of infection. In conclusion, this is the first report that a cell line from P. americana has shown susceptibility to infection from a baculovirus and likewise express recombinant protein. Although the yield of recombinant GFP was not as high as that of Sf21 cells, the results nonetheless showed that RIRI-PA1 had an infection rate advantage in the short term (within 24 h of infection), which is of great value for further development and utilization.

Prophenoloxidase of Odontotermes formosanus (Shiraki) (Blattodea: Termitidae) Is a Key Gene in Melanization and Has a Defensive Role during Bacterial Infection.

Melanization mediated by the prophenoloxidase (PPO)-activating system is an important innate immunity to fight pathogens in insects. In this study, the in vitro time-dependent increase in the intensity of melanization and phenoloxidase (PO) activity from the hemolymph of Odontotermes formosanus (Shiraki) challenged by pathogenic bacteria was detected. PPO is one of the key genes in melanization pathway, whereas the molecular characteristics and functions of O. formosanus PPO are unclear. The OfPPO gene was cloned and characterized. The open reading frame of OfPPO is 2085 bp in length and encodes a 79.497 kDa protein with 694 amino acids. A BLASTx search and phylogenetic analyses revealed that OfPPO shares a high degree of homology to the Blattodea PPOs. Moreover, real-time fluorescent quantitative PCR analysis showed that OfPPO is ubiquitously expressed in all castes and tissues examined, with the highest expression in workers and variable expression patterns in tissues of different termite castes. Furthermore, the expression of OfPPO was significantly induced in O. formosanus infected by pathogenic bacteria. Intriguingly, in combination with silencing of OfPPO expression, pathogenic bacteria challenge caused greatly increased mortality of O. formosanus. These results suggest that OfPPO plays a role in defense against bacteria and highlight the novel termite control strategy combining pathogenic bacteria application with termite PPO silencing.

Distribution and daily oscillation of GABA in the circadian system of the cockroach Rhyparobia maderae.

Gamma-aminobutyric acid (GABA) is the prevalent inhibitory neurotransmitter in nervous systems promoting sleep in both mammals and insects. In the Madeira cockroach, sleep-wake cycles are controlled by a circadian clock network in the brain's optic lobes, centered in the accessory medulla (AME) with its innervating pigment-dispersing factor (PDF) expressing clock neurons at the anterior-ventral rim of the medulla. GABA is present in cell clusters that innervate different circuits of the cockroach's AME clock, without colocalizing in PDF clock neurons. Physiological, immunohistochemical, and behavioral assays provided evidence for a role of GABA in light entrainment, possibly via the distal tract that connects the AME's glomeruli to the medulla. Furthermore, GABA was implemented in clock outputs to multiple effector systems in optic lobe and midbrain. Here, GABAergic brain circuits were analyzed further, focusing on the circadian system in search for sleep/wake controlling brain circuits. All GABA-immunoreactive neurons of the cockroach brain were also stained with an antiserum against the GABA-synthesizing enzyme glutamic acid decarboxylase. We found strong overlap of the distribution of GABA-immunoreactive networks with PDF clock networks in optic lobes and midbrain. Neurons in five of the six soma groups that innervate the clock exhibited GABA immunoreactivity. The intensity of GABA immunoreactivity in the distal tract showed daily fluctuations with maximum staining intensity in the middle of the day and weakest staining at the end of the day. Quantification via enzyme-linked immunosorbent assay and quantitative liquid chromatography coupled to electrospray ionization tandem mass spectrometry, likewise, showed higher GABA levels in the optic lobe during the inactivity phase of the cockroach during the day and lower levels during its activity phase at dusk. Our data further support the hypothesis that light- and PDF-dependently the circadian clock network of the cockroach controls GABA levels and thereby promotes sleep during the day.

Chronic ciprofloxacin and atrazine co-exposure aggravates locomotor and exploratory deficits in non-target detritivore speckled cockroach (Nauphoeta cinerea).

The global detection of ciprofloxacin and atrazine in soil is linked to intensive anthropogenic activities in agriculture and inadvertent discharge of industrial wastes to the environment. Nauphoeta cinerea is a terrestrial insect with cosmopolitan distribution and great environmental function. The current study probed the neurobehavioral and cellular responses of N. cinerea singly and jointly exposed to atrazine (1.0 and 0.5 µg g-1 feed) and ciprofloxacin (0.5 and 0.25 µg g-1 feed) for 63 days. Results demonstrated that the reductions in the body rotation, maximum speed, turn angle, path efficiency, distance traveled, episodes, and time of mobility induced by atrazine or ciprofloxacin per se was exacerbated in the co-exposure group. The altered exploratory and locomotor in insects singly and jointly exposed to ciprofloxacin and atrazine were verified by track plots and heat maps. Furthermore, we observed a decrease in acetylcholinesterase and anti-oxidative enzyme activities with concomitant elevation in the levels of lipid peroxidation, nitric oxide, and reactive oxygen and nitrogen species were significantly intensified in the midgut, hemolymph, and head of insects co-exposed to ciprofloxacin and atrazine. In conclusion, exposure to binary mixtures of ciprofloxacin and atrazine elicited greater locomotor and exploratory deficits than upon exposure to the individual compound by inhibiting acetylcholinesterase activity and induction of oxido-inflammatory stress responses in the insects. N. cinerea may be a usable model insect for checking contaminants of ecological risks.

Streptozotocin induces brain glucose metabolic changes and alters glucose transporter expression in the Lobster cockroach; Nauphoeta cinerea (Blattodea: Blaberidae).

The development of new models to study diabetes in invertebrates is important to ensure adherence to the 3R's principle and to expedite knowledge of the complex molecular events underlying glucose toxicity. Streptozotocin (STZ)-an alkylating and highly toxic agent that has tropism to mammalian beta cells-is used as a model of type 1 diabetes in rodents, but little is known about STZ effects in insects. Here, the cockroach; Nauphoeta cinerea was used to determine the acute toxicity of 74 and 740 nmol of STZ injection per cockroach. STZ increased the glucose content, mRNA expression of glucose transporter 1 (GLUT1) and markers of oxidative stress in the head. Fat body glycogen, insect survival, acetylcholinesterase activity, triglyceride content and viable cells in head homogenate were reduced, which may indicate a disruption in glucose utilization by the head and fat body of insects after injection of 74 and 740 nmol STZ per nymph. The glutathione S-transferase (GST) activity and reduced glutathione levels (GSH) were increased, possibly via activation of nuclear factor erythroid 2 related factor as a compensatory response against the increase in reactive oxygen species. Our data present the potential for metabolic disruption in N. cinerea by glucose analogues and opens paths for the study of brain energy metabolism in insects. We further phylogenetically demonstrated conservation between N. cinerea glucose transporter 1 and the GLUT of other insects in the Neoptera infra-class.

DNA methylation patterns respond to thermal stress in the viviparous cockroach Diploptera punctata.

It is increasingly recognized that epigenetic mechanisms play a key role in acclimatization and adaptation to thermal stress in invertebrates. DNA methylation and its response to temperature variation has been poorly studied in insects. Here, we investigated DNA methylation and hydroxymethylation patterns in the viviparous cockroach Diploptera punctata at a global and gene specific level in response to variation in temperature. We specifically studied methylation percentage in the heat shock protein 70 (Hsp70), whose function is linked to thermal plasticity and resistance. We found high levels of DNA methylation in several tissues but only low levels of DNA hydroxymethylation in the brain. Hsp70 methylation patterns showed significant differences in response to temperature. We further found that global DNA methylation variation was considerably lower at 28°C compared to higher or lower temperatures, which may be indicative of the optimal temperature for this species. Our results demonstrate that DNA methylation could provide a mechanism for insects to dynamically respond to changing temperature conditions in their environment.

The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus.

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.

Modified expression of antioxidant genes in lobster cockroach, Nauphoeta cinerea exposed to methylmercury and monosodium glutamate.

Methylmercury (MeHg) is a neurotoxicant that poses risk to human health and the environment, while glutamate homeostasis is necessary for the proper functioning of the brain. We have previously shown an increase in oxidative stress after cockroach exposure to diet containing monosodium glutamate (MSG), both separately and combined with a low dose of methylmercury. We herein seek to corroborate these findings by quantifying the expression levels of certain antioxidant genes in Nauphoeta cinerea exposed to MeHg and MSG. Cockroaches were fed with the basal diet alone, basal diet +2% NaCl, basal diet +2% MSG; basal diet +0.125 mg/g MeHg, basal diet +0.125 mg/g MeHg +2% NaCl; and basal diet +0.125 mg/g MeHg +2% MSG for 21 days and mRNA from head homogenate was used to quantify the expression of antioxidant genes such as glutathione-s-transferase (GstS, GstT, GstD), thioredoxin (Trx1, Trx2, Trx5), peroxiredoxin (prx4), superoxide dismutase (Sod), catalase (Cat). MeHg, NaCl and MSG alone downregulated mRNA levels of GstS and Trx5, in contrast, co-exposure of MeHg + MSG, upregulated these genes. MeHg + NaCl upregulated the mRNA levels of Cat and Sod but these genes were downregulated by NaCl alone. MeHg + NaCl and MeHg + MSG upregulated GstD and GstT. MeHg alone upregulated the transcription levels of Trx1, Trx2 and Prx4. The disruptions in the transcription levels of various genes by MeHg and MSG, reinforce the toxicity of these neurotoxicants. In general, the data suggest their additive effects and support the use of N. cinerea as a model for toxicological studies.

Optimal conditions for the storage of German cockroach extract.

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease­rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18­26˚C, RT) or refrigerated (2­8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol NS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease­rich cockroach extract.

The extremely low energy cost of biosynthesis in holometabolous insect larvae.

The metabolic cost of growth, which quantifies the amount of energy required to synthesize a unit of biomass, is an important component of an animal's ontogenetic energy budget. Here we investigated this quantity as well as other energy budget variables of the larvae of a holometabolous insect species, Vanessa cardui (painted lady). We found that the high growth rate of this caterpillar cannot be explained by its metabolic rate and the percentage of the metabolic energy allocated to growth; the key to understanding its fast growth is the extremely low cost of growth, 336 Joules/gram of dry mass. The metabolic cost of growth in caterpillars is 15-65 times lower than that of the endothermic and ectothermic species investigated in previous studies. Our results suggest that the low cost cannot be attributed to its body composition, diet composition, or body size. To explain the "cheap price" of growth in caterpillars, we assumed that a high metabolic cost for biosynthesis resulted in a high "quality" of cells, which have fewer errors during biosynthesis and higher resistance to stressors. Considering the life history of the caterpillars, i.e., tissue disintegration during metamorphosis and a short developmental period and lifespan, we hypothesized that an energy budget that allocates a large amount of energy to biosynthesizing high quality cells would be selected against in this species. As a preliminary test of this hypothesis, we estimated the metabolic cost of growth in larvae of Manduca sexta (tobacco hornworm) and nymphs of Blatta lateralis (Turkestan cockroach). The preliminary data supported our hypothesis.

The power of neuropeptide precursor sequences to reveal phylogenetic relationships in insects: A case study on Blattodea.

Recent state-of-the-art analyses in insect phylogeny have exclusively used very large datasets to elucidate higher-level phylogenies. We have tested an alternative and novel approach by evaluating the potential phylogenetic signals of identified and relatively short neuropeptide precursor sequences with highly conserved functional units. For that purpose, we examined available transcriptomes of 40 blattodean species for the translated amino acid sequences of 17 neuropeptide precursors. Recently proposed intra-ordinal relationships of Blattodea, based on the analysis of 2370 protein-coding nuclear single-copy genes (Evangelista et al., 2019), were corroborated with maximum support. The functionally different precursor units were analyzed separately for their phylogenetic information. Although the degree of information was different in the different sequence motifs, all precursor units contained phylogenetic informative data at the ordinal level, and their separate analysis did not reveal contradictory topologies. This study is the first comprehensive exploitation of complete neuropeptide precursor sequences of arthropods in such a context and demonstrates the applicability of these rather short but conserved sequences for an alternative, fast and simple analysis of phylogenetic relationships.

Real-time telemetry monitoring of oxygen in the central complex of freely-walking Gromphadorhina portentosa.

A new telemetric system for the electrochemical monitoring of dissolved oxygen is showed. The device, connected with two amperometric sensors, has been successfully applied to the wireless detection of the extracellular oxygen in the central complex of freely-walking Gromphadorhina portentosa. The unit was composed of a potentiostat, a two-channel sensor conditioning circuit, a microprocessor module, and a wireless serial transceiver. The amperometric signals were digitalized and sent to a notebook using a 2.4 GHz transceiver while a serial-to-USB converter was connected to a second transceiver for completing the communication bridge. The software, running on the laptop, allowed to save and graph the oxygen signals. The electronics showed excellent stability and the acquired data was linear in a range comprised between 0 and -165 nA, covering the entire range of oxygen concentrations. A series of experiments were performed to explore the dynamics of dissolved oxygen by exposing the animals to different gases (nitrogen, oxygen and carbon dioxide), to low temperature and anesthetic agents (chloroform and triethylamine). The resulting data are in agreement with previous O2 changes recorded in the brain of awake rats and mice. The proposed system, based on simple and inexpensive components, can constitute a new experimental model for the exploration of central complex neurochemistry and it can also work with oxidizing sensors and amperometric biosensors.

Photoperiod-dependent release of suppression pheromone in the male lobster cockroach Nauphoeta cinerea.

The complex agonistic repertoire between male lobster cockroaches (Nauphoeta cinerea) makes this species an excellent model for aggression studies. During the establishment of dominance hierarchies, 3-hydroxy-2-butanone (3H-2B) functions as a suppression pheromone, keeping the rivals in a submissive state. In the present study, we evaluated the release of 3H-2B by dominant individuals across four different time phases within the 24-h photoperiod, i.e., early scotophase (ES), late scotophase (LS), early photophase (EP), and late photophase (LP). For each time phase, we collected volatile pheromones during a 60-min first-encounter fight to measure the level of released 3H-2B. Subsequently, the amount of 3H-2B remaining in the sternal glands of dominant and subordinate individuals was measured and compared to socially naïve male controls. Release of 3H-2B was relatively high during ES or LP first-encounter fights, compared to LS or EP encounters. The attack duration and aggressive posture intensity in dominant males were positively correlated with the amount of 3H-2B release in all four phases. A similar statistical distribution was found between the amount of 3H-2B released by dominant males and the amount of 3H-2B in the sternal glands of naïve male sternal during LS, EP, and LP. However, during ES, the statistical distribution of 3H-2B released by the dominant was significantly greater than the distribution of 3H-2B content in socially naïve male sternal glands. The observed phase-dependence of 3H-2B release might be due to variations in 3H-2B biosynthesis or the scotophase-specific behavior of naïve males, wherein an aggressive posture is spontaneously adopted with concomitant 3H-2B release.