Influence of chemical and visual stimuli in food-search behaviour of Procambarus clarkii under clear conditions

Chemical communication may play a major role in aquatic environments because of visibility limitations, but when turbidity is reduced do chemical and visual stimuli interact to elicit food-search behaviour by Procambarus clarkii? Does P. clarkii use visual cues to detect its prey? We conducted behavioural experiments in a Y maze, under non-turbid conditions. In the experiments P. clarkii was exposed to chemical plus visual (CV) and visual only (V) stimuli transmitted by Chironomus riparius. The results revealed that P. clarkii detected CV and V cues associated with the presence of C. riparius. In controls, with no prey, crayfish displayed only searching behaviour, whereas in treatments they exhibited searching, detection and attack behaviours. Further, there were no significant differences between CV and V stimuli in the mean number of attacks performed by crayfish. Thus, our findings indicate that under low turbidity, visual cues are sufficient to mediate prey detection by P. clarkii.     

Molecular cloning and localization of a cDNA encoding a crustacean hyperglycemic hormone from the South African spiny lobster,Jasus lalandii

A cDNA, encoding a crustacean hyperglycemic hormone (cHH) of the South African spiny lobster, Jasus lalandii has been cloned. The cDNA consists of 1773 bp with an open reading frame of 399 bp that encodes a preprohormone of 133 amino acid residues. The preprohormone consists of a 25 amino acid hydrophobic signal peptide, a 32 amino acid cHH precursor-related peptide (CPRP) and the cHH sequence of 72 amino acid residues. The cHH sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by Gly-Lys, where Gly serves as an amidation site. The deduced amino acid sequence of the CPRP is in complete agreement with a peptide previously elucidated from sinus glands of J. lalandii, code-named CPRP 2 and the sequence of the cHH peptide matches that of the minor cHH isoform of J. lalandii, i.e. crustacean hyperglycemic hormone-II (cHH-II), which was also previously obtained by peptide sequencing. In situ hybridization on eyestalks revealed strong cHH-II mRNA expression in a subset of neurosecretory cells of the X-organ.     

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.     

Shelter competition between resident male red swamp crayfish Procambarus clarkii (Girard) and conspecific intruders varying by sex and reproductive status

The shelter defense dynamics of reproductively active (Form I) male red swamp crayfish Procambarus clarkii (Girard) were investigated by staging serial intrusions by male, maternal or non-maternal female conspecifics after one day of residence. The male residents showed a direct contest advantage only against non-maternal females, and won a significantly higher proportion of the encounters against non-maternal females than against either male or maternal female intruders. However, there was no significant difference in success against male or maternal female intruders. Further experiments against male intruders showed that increasing male prior residence to either 2 or 4 days did not significantly improve the residents' proportion of successful encounters. A final experiment revealed extremely low shelter fidelity in male P. clarkii, the most likely reason why male shelter defense against conspecific males and maternal females is not successful. These results, combined with those of past research, suggest that non-maternal females, juveniles and recently molted conspecifics are most vulnerable to predation and cannibalism, since males and maternal females easily out-compete non-maternal females for shelter, and adults out-compete juveniles. These findings may have implications for P. clarkii aquaculture and general management.     

Habenariol, a freshwater feeding deterrent from the aquatic orchid Habenaria repens (Orchidaceae)

An uncommon ester, bis-p-hydroxybenzyl-2-isobutylmalate, habenariol, has been isolated, by bioassay-guided fractionation, from the organic extract of the freshwater orchid Habenaria repens. The structure of habenariol was determined by the interpretation of spectral data. Feeding bioassays showed that habenariol deters feeding by the common freshwater crayfish Procambarus clarkii. Habenariol is related to a bis-ester glycoside isolated from the non-aquatic orchid Galeola faberi.     

Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.     

Detoxification enzymes of freshwater crayfish Procambarus clarkii fed a diet enriched in selenium: Preliminary results

This study aimed at investigating the extent of accumulation of selenium in the hepatopancreas of the crayfish Procambarus clarkii and its effects on the enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase. Crayfish were fed for 15 days either a diet enriched in Se, ED (1.21 mg kg^-1) or a standard diet, SD (0.30 mg kg^-1). Results showed that Se accumulation is higher in the sample subject to ED rather than to SD. A sexual difference was found for the response of the analyzed enzymes in the ED sample only. Catalase activity and glutathione peroxidase activity decreased in females and males, respectively, whereas glutathione reductase activity showed significant reductions in both sexes. These preliminary data might provide information about prooxidant effects of Se on P. clarkii when fed a Se enriched diet.     

Habitat use and dispersal of the invasive crayfish Procambarus clarkii in ephemeral water bodies of Portugal

We used radio-telemetry to analyze habitat use and dispersal of the invasive crayfish Procambarus clarkii in the extreme environmental conditions of a temporary river in southern Portugal. The rationale of this study was that an understanding of the properties that make this species a successful invader can be of help for the conservation of freshwater biodiversity. The results showed that: (1) crayfish displaced at a similar rate (ranging 2.5-38 m d^-1) as other European populations; (2) movement was faster at night; (3) crayfish movement had no clear environmental correlates, except for water temperature; (4) peaks of more intense locomotion were intercalated with longer periods of slow or null speed; and (5) P. clarkii did not excavate burrows in the study habitat, but took refuge under boulders and most often occupied complex microhabitats (e.g., vegetated sections of the river). The conclusion was that, notwithstanding their extreme conditions, ephemeral water bodies in southern Europe are highly susceptible to invasion by P. clarkii.     

Novel protocol for the chemical synthesis of crustacean hyperglycemic hormone analogues--an efficient experimental tool for studying their functions

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe3 and (Glp1, D-Phe3) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe3 modifies the cHH functionality, contributing to the diversification of the hormone pool.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.     

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.     

基于生态位模型的外来入侵种克氏原螯虾在中国的适生区预测

克氏原螯虾在20世纪初作为重要的水产品引入中国,但因其繁殖能力强、生长迅速、适应性强、喜掘洞穴,对农作物、池埂及农田水利有一定破坏作用,降低入侵地区当地物种多样性,对当地生态系统造成严重危害。因此,研究未来气候情景下克氏原螯虾适生区的变化,可为其监控和管理措施提供关键信息,有效预防和控制其蔓延。本研究基于克氏原螯虾的分布点,应用最大熵(MaxEnt)模型和规则集遗传算法(GARP)模型模拟了当前气候条件下克氏原螯虾在中国的潜在适生区,并预测了2041—2060年和2061—2080年克氏原螯虾在4种气候变化情景下(RCP 2.6、RCP 4.5、RCP 6.0、RCP 8.5)的分布,采用ROC曲线对预测结果进行检验和评价。结果表明: 在当前气候条件下克氏原螯虾集中分布在上海、江苏、浙江、安徽等长江沿岸地区;最冷季平均温度、最冷月最低温度对克氏原螯虾分布影响最大,其次是温度季节性变化、最暖月最高温度和最干月降水量。在未来气候情景下,2061—2080年克氏原螯虾的适生区面积有不同程度的变化,在RCP 2.6和RCP 4.5情景下总适生面积增加,但在RCP 8.5情景下呈先增后减趋势,而在RCP 6.0情景下无明显变化;克氏原螯虾适生区在空间分布上不仅有纬度方向上的扩散,也有向海拔较高地区迁移的趋势。     

Crayfish invading Europe: the case study of Procambarus clarkii

The red swamp crayfish, Procambarus clarkii, native to northeastern Mexico and southcentral USA, is today the dominant macroinvertebrate in several European countries. While the first introduction of this species into Spain is well-documented, little is known about its pathways of invasion and the reason for its rapid spread in several European countries. Study of the biology of the species has revealed a number of properties that makes this crayfish a successful invader. Procambarus clarkii exhibits properties characteristic of an r-selected species, including early maturity at small body size, rapid growth rates, large numbers of offspring at a given parental size, and relatively short life spans. It is also plastic in its life cycle, able to disperse widely in the habitat and to tolerate environmental extremes. It displays generalist and opportunistic feeding habits, consuming macrophytes and preying on amphibians. Procambarus clarkii can also replace indigenous crayfish by a combination of mechanisms, including competitive exclusion and transmission of the fungus-like Aphanomyces astaci, responsible for the crayfish plague. Finally, this species shows a wide behavioral flexibility when coping with new types of predators. The results of these studies, combined with the increasing information available in the scientific literature on this and other crayfish species, will help us understand invasions in this taxon and make predictions about the identity of future crayfish invaders.     

安徽三大水系入侵物种克氏原螯虾的种群遗传格局

克氏原螯虾(Procambarus clarkii)是长江中下游地区较为常见的外来物种, 目前已经扩散到安徽境内的主要水系。我们采集了安徽境内长江、淮河和新安江三大流域的4个地区9个地点的182个样本, 利用9对微卫星分子标记, 通过PCR扩增和微卫星分型, 分析了其种群遗传多样性水平和遗传结构, 构建了地方种群的遗传距离格局。结果表明本研究区域克氏原螯虾种群遗传多样性处于较高水平, 总的期望杂合度(H_E)和观测杂合度(H_O)分别为0.78和0.36。望江种群遗传多样性最高(期望杂合度为0.76), 肥西种群遗传多样性最低(期望杂合度为0.56)。克氏原螯虾种群显示了极强的杂合子缺失, 人为捕捞可能是造成其种群私有等位基因数目较多和杂合子缺失的主要原因。地方种群分化系数(F_st)与地理距离呈显著正相关(r= 0.33, P< 0.05), 望江种群和城东湖种群存在明显的跳跃性扩散(jump dispersal)。因此, 水系间的交流是种群扩散的主要途径, 人为贸易促使克氏原螯虾种群在不同地区的交流, 对其种群扩散也有一定的作用。     

Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells

Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O₂) to severe (1% O₂) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways.     

Structure-activity relationships of an inhibitory nematode FMRFamide-related peptide,SDPNFLRFamide (PF1), on Ascaris suum muscle

FMRFamide-related peptides are widespread among the Nematoda. Among them is a family of extended PNFLRFamide peptides encoded on the flp-1 peptide precursor gene in Caenorhabditis elegans. The most studied peptide from this series is SDPNFLRFamide (PF1). Each residue in this peptide was sequentially substituted with either alanine or the corresponding d-isomer of the native amino acid in order to define structure–function relationships in this peptide using an Ascaris suum muscle tension assay. In general, substitutions in the N-terminal tetrapeptide had only minor consequences for efficacy, while substitutions in the C-terminal tetrapeptide caused more dramatic changes. Such substitutions typically markedly diminished efficacy, but d-isomer substitution at either position 5 (Phe) or 6 (Leu) converted the inhibitory activity of the prototype into excitation. In addition, it has been evident that KPNFLRFamide and SDPNFLRFamide, though encoded on flp-1 and sharing a PNFLRFamide hexapeptide, act through different receptors. KPNFLRFamide directly gates a chloride channel in A. suum muscle cells, while SDPNFLRFamide acts through nitric oxide synthase to open K⁺ channels in the same tissue. The use of K⁺ channel blockers and nitric oxide synthase inhibitors in electrophysiological experiments employing A. suum muscle membranes allowed the unambiguous conclusion that the N-terminal lysine is absolutely required for activation of the chloride channel and excludes interaction with the SDPNFLRFamide receptor.     

中国海梨甲藻科几种甲藻的分类与形态鉴定

由于生活史特殊以及分类学历史复杂, 国内外有关梨甲藻科物种的分类学研究比较少。本研究通过形态分类学方法, 详细阐述了梨甲藻物种的分类学历史沿革, 对我国海域的11种梨甲藻科物种的孢囊进行了分类学研究。我们认为其中的1种应隶属于球甲藻属(Dissodinium), 其余10种可归为梨甲藻属(Pyrocystis)(包括2个变种, 2个变型), 对每个物种都进行了详细的形态学描述, 认为拟新月球甲藻(Dissodinium pseudolunula)与新月梨甲藻(Pyrocystis lunula)、优美梨甲藻(Pyrocystis elegans)与粗梨甲藻(Pyrocystis robusta)的分类学关系还需要进一步研究。     

Laboratory conditioning to still or flowing water does not affect the responses to a food stimulus by red swamp crayfish (Procambarus clarkii) in flowing water habitats

We tested the role of hydrodynamics in the use of chemical signals by the red swamp crayfish (Procambarus clarkii). We conditioned the crayfish in either a still water or flowing water habitat for 2 weeks. We then observed the crayfish in control (no stimulus) and experimental (food stimulus) trials in one of two flowing water habitats-laminar or turbulent. Habitat use was significantly different between control and experimental observations, but there was no effect of conditioning habitat or experimental habitat on the responses to the food stimulus. This study adds to a growing body of literature consistent with the idea that gross responses to chemical stimuli in crayfish are not affected by the hydrodynamic context in which they are encountered.     

Circadian rhythm does not affect responses to chemical cues in the red swamp crayfish, Procambarus clarkii

Studies of crayfish chemical ecology have been conducted in both day and night conditions. This variation may hinder the comparison of data among studies, if the responses by crayfish to chemical cues are dependent upon the time at which the cues are encountered. We tested the hypothesis that responses to chemical cues are dependent on observation time using the red swamp crayfish, Procambarus clarkii. Procambarus clarkii is known to exhibit a light-regulated circadian rhythm, with nocturnal activity peaks. Habitat use differed significantly between non-stimulated periods and periods of exposure to a food stimulus, but no effects of photoperiod (normal vs. reversed) or laboratory conditions (dark vs. light) were observed. The results suggest that, all else being equal, (1) studies of crayfish chemical ecology can be successfully conducted in a variety of experimental conditions, and (2) previous studies conducted at various times of the day should have comparable results.