Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Nitric oxide production in blowfly hemolymph after yeast inoculation

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses that include both cellular and humoral components. Cellular responses are mediated by hemocytes and Immoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In this work, we determined NO production in Chrysomya megaccphala hemolymph and hemocytes after yeast inoculation. Assays were performed with non-infected controls (NIL), saline-injected larvae (SIL) or larvae injected with Saccharomyces cerevisiae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24 or 48 h post-injection. NO levels in SIL were comparable to those measured in NIL until 12 h, which might be considered the basal production, increasing at 24 and 48 h post-injection, probably in response to the increased larval fragility after cuticle rupture. YIL exhibited significantly higher levels of NO than were found in other groups, peaking at 24 h. L-NAME and EDTA caused a significant reduction of NO production in YIL at this time, suggesting the activity of a Ca2+ -dependent NOS. Plasmatocytes and granular cells phagocytosed the yeasts. Plasmatocytes initiated the nodule formation and granular cells were the only hemocyte type to produce NO. These results permit us to conclude that yeasts induced augmented NO production in C. megacephala hemolymph and granular cells are the hemocyte type involved with the generation of this molecule. (c) 2005 Elsevier B.V. All rights reserved.

TRADE-OFF BETWEEN IMMUNE STIMULATION AND EXPRESSION OF STORAGE PROTEIN GENES

Proteins stored in insect hemolymph may serve (is a source of amino acids and energy for metabolism, and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCH and Western blot.. After ensuring that. the immune system had, been activated by measuring the ensuing expression (, the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (pro PO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein. were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLP-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLP-III transcripts. Our findings are consistent with a down-regulation, of the express and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon. represents a strategy to redirect resources to combat injury or infection. (C) 2009 Wiley Periodicals, Inc.

Life cycle of Triatoma ryckmani (Hemiptera: Reduviidae) in the laboratory, feeding patterns in nature and experimental infection with Trypanosoma cruzi

A cohort initiated with 121 eggs, yielding 105 first instar nymphs (eclosion rate: 86.78%), allowed us to observe the entire life cycle of Triatoma ryckmani under laboratory conditions (24ºC and 62% relative humidity), by feeding them on anesthetized hamsters. It was possible to obtain 62 adults and the cycle from egg to adult took a mean of 359.69 days with a range of 176-529 days (mortality rate of nymphs: 40.95%). Mean life span of adults was of 81 days for females and 148 days for males. The developmental periods of 4th and 5th nymphs were longer than those of the other instars. This suggests that young siblings have a better chance of taking a hemolymph meal from older ones, in order to survive during fasting periods during prolonged absences of vertebrate hosts from natural ecotopes. The stomach contents of 37 insects showed blood from rodents (15 cases), lizards (7 cases), birds (6 cases) and insect hemolymph (7 cases). Out of 10 insects fed by xenodiagnosis on a Trypanosoma cruzi infected mouse, all but one became infected with the parasite.

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Tricho-grammatidae) was successfully used to rear Trichogramm pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace's insect medium, a common component of artificial diets, was found to markedly improve the development of T pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts. (c) 2005 Elsevier Inc. All rights reserved.

Recognition and inactivation of LPS by lipophorin particles

Lipophorin is the major lipid carrier in insects, but various observations indicate that lipophorin is also involved in immune reactions. To examine a possible role of lipophorin in defence reactions, we mixed hemolymph plasma from Galleria mellonella with LPS and noticed that lipophorin forms detergent-insoluble aggregates, while most other plasma proteins are not affected. Lipophorin particles isolated by low-density gradient centrifugation retained LPS-induced aggregation properties, which suggested to us that these immune-reactive particles are able to recognise LPS and respond by forming insoluble aggregates. Antibodies against LPS-binding proteins, such as immulectin-2 and beta-1,3-glucan binding protein, cross-reacted with proteins associated with purified lipophorin particles. To examine whether LPS-mediated aggregates inactivate LPS, we added LPS-lipophorin mixtures to purified lipophorin particles and monitored aggregate formation. Under these conditions lipophorin did not form insoluble aggregates, which indicates that lipophorin particles sequester LPS into non-toxic aggregates. (c) 2005 Elsevier Ltd. All rights reserved.

Changes in the hemolymph and fat body metabolites of Diatraea saccharalis (Fabricius) (Lepidoptera : Crambidae) parasitized by Cotesia flavipes (Cameron) (Hymenoptera : Braconidae)

The koinobiont Cotesia flavipes responds to and is influenced by biochemical changes in the host hemolymph composition, Diatraea saccharalis. Changes in the composition of macronutrients may occur due to the hosts own development or by changes induced after parasitization. These changes occur to facilitate parasitoid invasion and to make the host internal environment suitable to parasitoid immature development. Therefore, changes in the availability of stored and circulating nutrients may correlate with the nutritional requirements of specific parasitoid immature stages. In here, we describe changes in the biochemical composition of parasitized and control larvae at different stages of parasitoid development to gain information on C flavipes host regulation and on its quantitative immature nutritional requirements. Total proteins, lipids and carbohydrates free in the hemolymph or stored in host fat bodies, and the SDS-PAGE protein profile of the hemolymph were evaluated in control and parasitized 6th instar during the whole parasitoid development. Changes in the total protein available in the host hemolymph were detected soon after parasitization, but carbohydrate and lipids were observed to differ only towards parasitoid larvae egression. Although C. flavipes affected the availability of all macronutrients observed in the host hemolymph, lipids and proteins stored in the host fat bodies were unaffected. However, carbohydrate concentration at the end of parasitoid larval development was much lower in parasitized than in control larvae at the same stage of development. SDS-PAGE analysis indicated C flavipes up-regulated two host proteins (125 and 48 kDa) and released two parasitism-specific proteins towards the end of parasitoid larval development. We provide a discussion on the role these changes may have on the process of host regulation and their possible requirement to sustain parasitoid development. (C) 2007 Elsevier Inc. All rights reserved.

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton break-down. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Metabolic Post-feeding Changes in Fat Body and Hemolymph of Dipetalogaster maximus (Hemiptera:Reduviidae)

Lipids and glycogen in fat body as well as the modifications in the wet weight of this organ were evaluated in an unfed insect, Dipetalogaster maximus, on day 5 after adult ecdysis (time 0) and during a 30-day period after ingestion of blood meal. Total lipids, high density lipophorin (HDLp), carbohydrates, total proteins and uric acid were determined in the hemolymph during the same period. Fat body wet weight was maximum on day 10 post-feeding and represented on day 30 only 42% of the maximum weight. Lipids stored in the fat body increased up to day 15 reaching 24% of the total weight of tissue. Glycogen was maximum on day 20, representing approximately 3% of the fat body weight. HDLp represented at all times between 17-24% of the total proteins, whose levels ranged between 35 and 47 mg/ml. Uric acid showed at 20, 25 and 30 days similar levels and significantly higher than the ones shown at days 10 and 15. Hemolymphatic lipids fluctuated during starvation between 3-4.4 mg/ml and carbohydrates showed a maximum on day 15 after a blood meal, decreasing up to 0.26 mg/ml on day 25. The above results suggest that during physiological events such as starvation, the availability of nutrients is affected, involving principally the fat body reserves

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Rhodnius prolixus is the main Trypanosoma rangeli vector in several Latin-American countries and is susceptible to infection with KP1(+) strains; however, it presents an invasion-resistant response to KP1(-) strains. The present work has identified a trypanolytic protein against T. rangeli KP1(-) in the R. prolixus hemolymph which was fractioned with ammonium sulfate (following dialysis). The results revealed a protein component which did not depend on divalent cations for its biological function whilst keeping its trypanolytic activity at temperatures ranging from -20ºC to 37ºC, at 7.0 to 10.5 pH. The protein was partially purified by gel filtration chromatography and ionic exchange chromatography. The major component presented a molecular weight of around 79 kDa and an isoelectric point between 4.9 and 6.3 and may be directly related to hemolymph trypanolytic activity against T. rangeli KP1(-) populations.

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.

Chemometric analysis of Hymenoptera toxins and defensins: A model for predicting the biological activity of novel peptides from venoms and hemolymph

When searching for prospective novel peptides, it is difficult to determine the biological activity of a peptide based only on its sequence. The trial and error approach is generally laborious, expensive and time consuming due to the large number of different experimental setups required to cover a reasonable number of biological assays. To simulate a virtual model for Hymenoptera insects, 166 peptides were selected from the venoms and hemolymphs of wasps, bees and ants and applied to a mathematical model of multivariate analysis, with nine different chemometric components: GRAVY, aliphaticity index, number of disulfide bonds, total residues, net charge, pI value, Boman index, percentage of alpha helix, and flexibility prediction. Principal component analysis (PCA) with non-linear iterative projections by alternating least-squares (NIPALS) algorithm was performed, without including any information about the biological activity of the peptides. This analysis permitted the grouping of peptides in a way that strongly correlated to the biological function of the peptides. Six different groupings were observed, which seemed to correspond to the following groups: chemotactic peptides, mastoparans, tachykinins, kinins, antibiotic peptides, and a group of long peptides with one or two disulfide bonds and with biological activities that are not yet clearly defined. The partial overlap between the mastoparans group and the chemotactic peptides, tachykinins, kinins and antibiotic peptides in the PCA score plot may be used to explain the frequent reports in the literature about the multifunctionality of some of these peptides. The mathematical model used in the present investigation can be used to predict the biological activities of novel peptides in this system, and it may also be easily applied to other biological systems. © 2011 Elsevier Inc.

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

Cryptocyanin, a crustacean molting protein: Evolutionary link with arthropod hemocyanins and insect hexamerins

Cryptocyanin, a copper-free hexameric protein in crab (Cancer magister) hemolymph, has been characterized and the amino acid sequence has been deduced from its cDNA. It is markedly similar in sequence, size, and structure to hemocyanin, the copper-containing oxygen-transport protein found in many arthropods. Cryptocyanin does not bind oxygen, however, and lacks three of the six highly conserved copper-binding histidine residues of hemocyanin. Cryptocyanin has no phenoloxidase activity, although a phenoloxidase is present in the hemolymph. The concentration of cryptocyanin in the hemolymph is closely coordinated with the molt cycle and reaches levels higher than hemocyanin during premolt. Cryptocyanin resembles insect hexamerins in the lack of copper, molt cycle patterns of biosynthesis, and potential contributions to the new exoskeleton. Phylogenetic analysis of sequence similarities between cryptocyanin and other members of the hemocyanin gene family shows that cryptocyanin is closely associated with crustacean hemocyanins and suggests that cryptocyanin arose as a result of a hemocyanin gene duplication. The presence of both hemocyanin and cryptocyanin in one animal provides an example of how insect hexamerins might have evolved from hemocyanin. Our results suggest that multiple members of the hemocyanin gene family—hemocyanin, cryptocyanin, phenoloxidase, and hexamerins—may participate in two vital functions of molting animals, oxygen binding and molting. Cryptocyanin may provide important molecular data to further investigate evolutionary relationships among all molting animals.