Through the eDNA looking glass: Responses of fjord benthic foraminiferal communities to contrasting environmental conditions.

The health of coastal marine environments is severely declining with global changes. Proxies, such as those based on microeukaryote communities, can record biodiversity and ecosystem responses. However, conventional studies rely on microscopic observations of limited taxonomic range and size fraction, missing putatively ecologically informative community components. Here, we tested molecular tools to survey foraminiferal biodiversity in a fjord system (Sweden) on spatial and temporal scales: Alpha and beta diversity responses to natural and anthropogenic environmental trends were assessed and variability of foraminiferal environmental DNA (eDNA) compared to morphology-based data. The identification of eDNA-obtained taxonomic units was aided by single-cell barcoding. Our study revealed wide diversity, including typical morphospecies recognized in the fjords, and so-far unrecognized taxa. DNA extraction method impacted community composition outputs significantly. DNA extractions of 10 g sediment more reliably represented present diversity than of 0.5-g samples and, thus, are preferred for environmental assessments in this region. Alpha- and beta diversity of 10-g extracts correlated with bottom-water salinity similar to morpho-assemblage diversity changes. Sub-annual environmental variability resolved only partially, indicating damped sensitivity of foraminiferal communities on short timescales using established metabarcoding techniques. Systematically addressing the current limitations of morphology-based and metabarcoding studies may strongly improve future biodiversity and environmental assessments.

Ca²âº/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²âº signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

Unlocking foraminiferal genetic diversity on estuarine mudflats with eDNA metabarcoding.

Environmental biomonitoring is a prerequisite for efficient evaluation and remediation of ecosystem degradation due to anthropogenic pressure or climate change. Estuaries are key habitats subject to multiple anthropogenic and natural stressors. Due to these multiple stressors, the detection of anthropogenic pressure is challenging. The fact that abundant natural stressors often lead to negative quality assessments has been coined the "estuarine quality paradox". To solve this issue, the application of molecular approaches with successful bioindicators like foraminifera is promising. However, sampling protocols, molecular procedures and data analyses need to be validated before such tools can be routinely applied. We conducted an environmental DNA survey of estuarine mudflats along the French Atlantic coast, using a metabarcoding approach targeting foraminifera. Our results demonstrate that estuarine environments have only a few active OTUs dominating the community composition and a large stock of dormant or propagule stages. This last genetic diversity components constitute an important reservoir, with different species which can potentially develop in response to the temporal variability of the multiple stressors. In fact, different OTUs were dominant in the studied estuaries. Our statistical model shows that the physical and chemical characteristics of the sediment and the climatic conditions explain only 43 % of the community composition variance. This suggests that other, less easily quantifiable factors, such as the history and use of the estuaries or the ecological drift could play an important role as well. Environmental DNA biomonitoring opens new perspectives to better characterize the genetic diversity in estuaries.

Trophic strategies of intertidal foraminifera explored with single-cell microbiome metabarcoding and morphological methods: What is on the menu?

In mudflats, interactions and transfers of nutrients and secondary metabolites may drive ecosystems and biodiversity. Foraminifera have complex trophic strategies as they often rely on bacteria and eukaryotes or on potential symbionts for carbon and nitrogen resources. The capacity of these protists to use a wide range of adaptive mechanisms requires clarifying the relationships between them and their microbial associates. Here, we investigate the interactions of three foraminiferal species with nearby organisms in situ, by coupling molecular (cloning/Sanger and high-throughput sequencing) and direct counting and morphological identification with microscopy. This coupling allows the identification of the organisms found in or around three foraminiferal species through molecular tools combined with a direct counting of foraminifera and diatoms present in situ through microscopy methods. Depending on foraminiferal species, and in addition to diatom biomass, diatom frustule shape, size and species are key factors driving the abundance and diversity of foraminifera in mudflat habitats. Three different trophic strategies were deduced for the foraminifera investigated in this study: Ammonia sp. T6 has an opportunistic strategy and is feeding on bacteria, nematoda, fungi, and diatoms when abundant; Elphidium oceanense is feeding mainly on diatoms, mixed with other preys when they are less abundant; and Haynesina germanica is feeding almost solely on medium-large pennate diatoms. Although there are limitations due to the lack of species coverage in DNA sequence databases and to the difficulty to compare morphological and molecular data, this study highlights the relevance of combining molecular with morphological tools to study trophic interactions and microbiome communities of protists at the single-cell scale.

Molecular and functional characterization of a novel sodium channel TipE-like auxiliary subunit from the American cockroach Periplaneta americana.

In Drosophila melanogaster, the functions of voltage-gated sodium (Nav) channels are modulated by TipE and its orthologs. Here, we describe a novel TipE homolog of the American cockroach, Periplaneta americana, called PaTipE. Like DmTipE, PaTipE mRNAs are ubiquitously expressed. Surprisingly, PaTipE mRNA was undetectable in neurosecretory cells identified as dorsal unpaired median neurons. Phylogenetic analysis placed this new sequence in TipE clade, indicating an independent evolution from a common ancestor. Contrary to previous reports, our data indicate that the auxiliary subunits of insect Nav channels are very distant from the mammalian BKCa auxiliary subunits. To decipher the functional roles of PaTipE, we characterized the gating properties of DmNav1-1 channels co-expressed with DmTipE or PaTipE, in Xenopus oocytes. Compared to DmTipE, PaTipE increased Na(+) currents by a 4.2-fold. The voltage-dependence of steady-state fast inactivation of DmNav1-1/PaTipE channels was shifted by 5.8 mV to more negative potentials than that of DmNav1-1/DmTipE channels. DmNav1-1/PaTipE channels recovered 3.2-fold slower from the fast-inactivated state than DmNav1-1/DmTipE channels. In conclusion, this study supports that the insect Nav auxiliary subunits share functional features with their mammalian counterparts, although structurally and phylogenetically distant.

Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumß1 expressions levels, whereas Apisumß2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

Calcium pathways such as cAMP modulate clothianidin action through activation of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors.

Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via α-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current-voltage curves were dependent to clothianidin concentrations. At 10 µM clothianidin, increasing cAMP concentration induced a linear current-voltage curve. Clothianidin effects were blocked by 0.5 µM α-bungarotoxin suggesting that cAMP modulation occurred through α-bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to α-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 µM mecamylamine and 20 µM d-tubocurarine. In addition, we found that application of 1mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides.

Characterization of tyramine ß-hydroxylase, an enzyme upregulated by stress in Periplaneta americana.

Octopamine (OA) is an important neuroactive substance that modulates several physiological functions and behaviors of various invertebrate species. This biogenic monoamine, structurally related to noradrenaline, acts as a neurotransmitter, a neuromodulator, or a neurohormone in insects. The tyramine ß-hydroxylase (TBH) catalyzes the last step in OA biosynthesis and thus plays a key role in the regulation of synthesis and secretion of OA in neurons. The aim of this study was to characterize TBH in the cockroach Periplaneta americana and to get a better understanding of its regulation under stress conditions in this insect. First of all, five full-length cDNAs encoding TBH isoforms were cloned from the nerve cord of the physiological model P. americana. PaTBH transcripts were found mainly expressed in nervous tissues and in octopaminergic dorsal unpaired median neurons. In addition, a new ELISA assay was developed so as to allow determination of both OA level and TBH activity in stressed cockroaches. Mechanical stressful stimulation led to a significant increase in TBH activity after 1 and 24  h, with a higher induction after 1  h than after 24  h. Thus, TBH could be considered as a promising biomarker of stress in insects rather than OA.

Pretreatment of the cockroach cercal afferent/giant interneuron synapses with nicotinoids and neonicotinoids differently affects acetylcholine and nicotine-induced ganglionic depolarizations.

We have recently demonstrated that neonicotinoid insecticides were able to act as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) expressed at the synapse between the cercal nerve XI and the giant interneurons, in the sixth abdominal ganglion. In this work, we demonstrated that nicotinoids such as nornicotine acted as an agonist of nicotinic acetylcholine receptors expressed at cercal afferent/giant interneurons while cotinine was a poor agonist. Indeed, nornicotine induced a ganglionic depolarization which was blocked by the nicotinic antagonist mecamylamine. In addition, we found that pretreatment of the sixth abdominal ganglion with 1 and 10 µM nornicotine and cotinine had no significant effect on acetylcholine and nicotine-induced depolarization. But pretreatment with 1 and 10 µM acetamiprid and imidacloprid had a strong effect. 1 and 10 µM acetamiprid completely blocked acetylcholine-induced depolarization, whereas imidacloprid had a partial effect. The present work therefore suggests, in agreement with previous studies, that nornicotine and cotinine bind to distinct cockroach postsynaptic nAChRs, whereas acetamiprid and imidacloprid have competitive effects with acetylcholine and nicotine on ganglionic depolarization.

Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+) currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.

Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells.

Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil.

The discovery of a novel sodium channel in the cockroach Periplaneta americana: evidence for an early duplication of the para-like gene.

Voltage-gated sodium channels (Na(v) channels) belong to a superfamily of ion channels which play an essential role in membrane excitability. Only one gene encoding Na(v) channels has been characterized so far in insects. Here, we have cloned one full-length cDNA encoding a conventional insect Na(v) channel (PaNa(v)1) and two full-length cDNAs encoding putative insect Na(v) channels (PaFPC1 and PaFPC2) in Periplaneta americana, a model insect for neurophysiological studies. The ORFs of PaFPC1 and PaFPC2 contained 4662 bp and encoded 1553 amino acid residues, and the ORF of PaNa(v)1 contained 6153 bp and encoded 2051 amino acid residues. PaFPC1 and PaFPC2 are two isoforms, which differ by eight single amino acid substitutions. PaFPC1 shares 37.5-55% protein identities with known insect Na(v) channels, while PaNa(v)1 shares 70-97.5% protein identities with these latter. Both PaFPC1 and PaFPC2 possess the molecular hallmarks of Na(v) channels except the motif involved in fast inactivation. Contrary to PaNa(v)1 transcripts which are expressed mainly in the central nervous system, those ones of PaFPC are also expressed in non-neuronal tissues (muscles, gut and mushroom-shaped accessory glands). A detailed phylogenetic analysis confirmed that PaNa(v)1 and PaFPC are evolutionarily closely related to insect Na(v) channel genes.