The mitogenome of Hydropsyche pellucidula (Hydropsychidae): first gene arrangement in the insect order Trichoptera.

We describe the mitochondrial genome of Hydropsyche pellucidula Curtis 1834, which is first described for the suborder Annulipalpia and the first in the order Trichoptera to show a non-canonical gene order. The mitogenome was obtained by de novo assembly of shotgun sequenced total genomic DNA using Illumina Miseq technology, which produced an average coverage of 115× and a minimum coverage of 48×. The mitochondrial genome includes 13 protein-coding genes, 2 rRNAs and 22 tRNAs. The genome is characterized by a rearrangement in the relative position of protein-coding and ribosomal genes. This mitogenome sequence will be useful for studying the family Hydropsychidae, which is commonly used for freshwater pollution biomonitoring.

Lessons from genome skimming of arthropod-preserving ethanol.

Field-collected specimens of invertebrates are regularly killed and preserved in ethanol, prior to DNA extraction from the specimens, while the ethanol fraction is usually discarded. However, DNA may be released from the specimens into the ethanol, which can potentially be exploited to study species diversity in the sample without the need for DNA extraction from tissue. We used shallow shotgun sequencing of the total DNA to characterize the preservative ethanol from two pools of insects (from a freshwater habitat and terrestrial habitat) to evaluate the efficiency of DNA transfer from the specimens to the ethanol. In parallel, the specimens themselves were subjected to bulk DNA extraction and shotgun sequencing, followed by assembly of mitochondrial genomes for 39 of 40 species in the two pools. Shotgun sequencing from the ethanol fraction and read-matching to the mitogenomes detected ~40% of the arthropod species in the ethanol, confirming the transfer of DNA whose quantity was correlated to the biomass of specimens. The comparison of diversity profiles of microbiota in specimen and ethanol samples showed that 'closed association' (internal tissue) bacterial species tend to be more abundant in DNA extracted from the specimens, while 'open association' symbionts were enriched in the preservative fluid. The vomiting reflex of many insects also ensures that gut content is released into the ethanol, which provides easy access to DNA from prey items. Shotgun sequencing of DNA from preservative ethanol provides novel opportunities for characterizing the functional or ecological components of an ecosystem and their trophic interactions.

Comprehensive analysis of the frequency of recognition of melanoma-associated antigen (MAA) by CD8 melanoma infiltrating lymphocytes (TIL): implications for immunotherapy.

Fifty-nine tumor-infiltrating lymphocyte (TIL) cultures established from melanoma-invaded lymph nodes were screened for recognition of 28 melanoma-associated antigens (MAA) in association with31 HLA molecules. Twenty-three (39%) TIL lines reacted to at least one melanoma antigen. Melanosomal proteins were recognized by 19 TIL populations and the most prominent responses against these proteins were directed against Melan-A/MART-1 (mainly in association with HLA-A*0201) and gp100 (in association with diverse HLA contexts). Ten TIL populations reacted against 10 tumor-specific antigens, in association with 8 different HLA molecules. HLA-A*0201 and B*3501-restricted responses were the most frequent with, respectively, 17 and 7 responses directed against 5 distinct antigens. Unexpectedly, the recognition by TIL of different MAA was frequently restricted by a single HLA in individual tumors, and there was no evidence for the existence of dominant MAA epitopes between tumors,except for Melan-A/MART-1 antigen. This analysis also led to the detection of 21 new HLA-peptide complexes recognized by melanoma TIL. This study, which is to our knowledge the most comprehensive analysis of TIL specificity to tumor antigens, has several implications for the design of immunotherapeutic strategies based on immunization against selected tumor epitopes.

Cloning of a cDNA coding for active tyrosine hydroxylase in the rainbow trout (Oncorhynchus mykiss): comparison with other hydroxylases and enzymatic expression.

Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout (Oncorhynchus mykiss). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate L-tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of approximately 65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.

Release of pituitary gonadotrophins GtH I and GtH II in the rainbow trout (Oncorhynchus mykiss): modulation by estradiol and catecholamines.

The present study focused on the role of catecholaminergic neurons and estrogens on the release of gonadotropins I and II in immature and early vitellogenic female rainbow trout. The ovariectomy-induced increase of GtH I blood levels (from about 10 to 15 ng/ml) was prevented in vitellogenic fish by E2 supplementation. E2 implantation of immature fish decreased blood GtH I levels (from about 6 to 1 ng/ml). Blood levels of GtH II were low (about 0.5 ng/ml) and not altered by ovariectomy and E2 treatment. These data demonstrate that estrogens exert a negative feedback on the release of GtH I in trout. A treatment with alpha-methyl-p-tyrosine (MPT), an inhibitor of catecholamine synthesis, increased blood GtH II levels of sham-operated vitellogenic fish and ovariectomized fish implanted with E2, but had no effects in ovariectomized fish. MPT did not modify blood GtH I levels in any experimental group. A treatment of E2-implanted immature or vitellogenic fish with the dopamine antagonist pimozide also increased blood GtH II levels, but did not significantly change blood GtH I levels. These data demonstrate that release of GtH II, but not of GtH I, depends on an E2-activated DA inhibitory tone.

Estrogen receptors are expressed in a subset of tyrosine hydroxylase-positive neurons of the anterior preoptic region in the rainbow trout.

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

Tyrosine hydroxylase activity and dopamine turnover of rainbow trout (Oncorhynchus mykiss) brain: the special status of the hypothalamus.

The dynamics of catecholamine (CA)-synthesis enzymes have been poorly studied in fish. Tyrosine hydroxylase (TH), the rate-limiting enzyme of CA synthesis has been only studied inin vitro conditions. In the present report thein vivo CA synthesis and the CA metabolism were studied in different regions of the forebrain of the rainbow trout. Levels of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) were determined by HPLC following a treatment with hydroxybenzylhydrazine (NSD), a potential inhibitor of DOPA decarboxylase. Kinetics of the accumulation of DOPA and of the decline of DOPAC were in agreement with those found in rat, evidencing that the accumulation of DOPA following NSD can be used in trout to quantify thein vivo enzymatic activity of tyrosine hydroxylase. Experiments using treatment with NSD or with methyl-p-tyrosine reached a same conclusion: the DA neuronal activity in trout is much higher than NE neuronal activity. However, the hypothalamus had high DA levelsvs. lowin vitro andin vivo TH activities and exhibited a low CA turnover.

Involvement of estradiol in a catecholamine inhibitory tone of gonadotropin release in the rainbow trout (Oncorhynchus mykiss).

The present study aimed to examine the inhibitory action of catecholaminergic neurons on the release of gonadotropin II (GtH2) in female rainbow trout at different stages of the reproductive cycle. Estradiol (E2) implants in sexually immature female increased blood E2 levels (from 0.55 to about 9 ng/ml) and pituitary GtH2 contents (from 15 to about 8500 ng/pit), but did not modify blood GtH2 levels or pituitary dopamine (DA) and norepinephrine contents. Subsequent treatment by alpha-methyl-p-tyrosine (MPT) increased blood GtH2 levels (from 0.5 to about 3 ng/ml) and decreased pituitary DA levels, only in E2-implanted fish. MPT also increased blood GtH2 levels of sexually recrudescent (about threefold) and sexually mature (about twofold) fish. In the periovulatory period, there was a highly significant positive correlation between blood E2 levels--which fall up until ovulation--and ability of MPT to increase GtH2 release. GtH2 release in rainbow trout is concluded to be inhibited by a brain-pituitary DA tone and there are relationships between this inhibitory tone and the level of estradiol.