Dark Cuticle Research Articles

Atypical strategies for cuticle pigmentation in the blood-feeding hemipteran Rhodnius prolixus.

Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ATP-binding cassette proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here, we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode N-beta-alanyl dopamine hydroxylase branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.

Arylalkalamine N-acetyltransferase-1 functions on cuticle pigmentation in the yellow fever mosquito, Aedes aegypti.

Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the acetylation of dopamine, 5-hydroxy-tryptamine, tryptamine, octopamine, norepinephrine and other arylalkylamines to form respective N-acetyl-arylalkylamines. Depending on the products formed, aaNATs are involved in a variety of physiological functions. In the yellow fever mosquito, Aedes aegypti, a number of aaNATs and aaNAT-like proteins have been reported. However, the primary function of each individual aaNAT is yet to be identified. In this study we investigated the function of Ae. aegypti aaNAT1 (Ae-aaNAT1) in cuticle pigmentation and development of morphology. Ae-aaNAT1 transcripts were detected at all stages of development with highest expressions after pupation and right before adult eclosion. Ae-aaNAT1 mutant mosquitoes generated using clustered regularly interspaced palindromic repeats (CRISPR) - CRISPR-associated protein 9 had no obvious effect on larval and pupal development. However, the mutant mosquitoes exhibited a roughened exoskeletal surface, darker cuticles, and color pattern changes suggesting that Ae-aaNAT1 plays a role in development of the morphology and pigmentation of Ae. aegypti adult cuticles. The mutant also showed less blood feeding efficiency and lower fecundity when compared with the wild-type. The mutation of Ae-aaNAT1 influenced expression of genes involved in cuticle formation. In summary, Ae-aaNAT1 mainly functions on cuticular pigmentation and also affects blood feeding efficiency and fecundity.

Cuticle darkening correlates with increased body copper content in Drosophila melanogaster

Insect epidermal cells secrete a cuticle that serves as an exoskeleton providing mechanical rigidity to each individual, but also insulation, camouflage or communication within their environment. Cuticle deposition and hardening (sclerotization) and pigment synthesis are parallel processes requiring tyrosinase activity, which depends on an unidentified copper-dependent enzyme component in Drosophila melanogaster. We determined the metallomes of fly strains selected for lighter or darker cuticles in a laboratory evolution experiment, asking whether any specific element changed in abundance in concert with pigment deposition. The results showed a correlation between total iron content and strength of pigmentation, which was further corroborated by ferritin iron quantification. To ask if the observed increase in iron body content along with increased pigment deposition could be generalizable, we crossed yellow and ebony alleles causing light and dark pigmentation, respectively, into similar genetic backgrounds and measured their metallomes. Iron remained unaffected in the various mutants providing no support for a causative link between pigmentation and iron content. In contrast, the combined analysis of both experiments suggested instead a correlation between pigment deposition and total copper body content, possibly due to increased demand for epidermal tyrosinase activity.

The role of sex and temperature in melanin-based immune function

Sex differences in immunity have been observed across a wide range of species. Still, it remains unclear how sex-specific interactions with the environment are linked to sex differences in immunity. We studied the plasticity of immunological sex differences by focusing on melanin-based traits in the Pacific field cricket (Teleogryllus oceanicus (Le Guillou, 1841)). Insects rely on the pigment melanin for both immune function and coloration of the cuticle; therefore, changes in melanin production for one of these traits may indirectly affect the other. Male crickets use melanized wing structures to chirp. These cuticular structures are missing in females and a songless male morph. Given that the thermal environment influences cuticle melanization, we investigated the interactive effects of sex and developmental temperature on melanin-based immunity. Both immunity and wing cuticle melanism were reduced in individuals that developed under warmer temperatures. Rearing temperature also mediated the extent to which the sexes differed in immune traits. Males had darker cuticles, whereas females expressed greater immune activity, suggesting that sex-specific investment in melanin corresponds with sex differences in immunity. However, the lack of immunological differences between the two male morphs does not support the hypothesis that investment in cuticle melanism affects investment in immunity.

Cuticular colour reflects underlying architecture and is affected by a limiting resource

Central to the basis of ecological immunology are the ideas of costs and trade-offs between immunity and life history traits. As a physical barrier, the insect cuticle provides a key resistance trait, and Tenebrio molitor shows phenotypic variation in cuticular colour that correlates with resistance to the entomopathogenic fungus Metarhizium anisopliae. Here we first examined whether there is a relationship between cuticular colour variation and two aspects of cuticular architecture that we hypothesised may influence resistance to fungal invasion through the cuticle: its thickness and its porosity. Second, we tested the hypothesis that tyrosine, a semi-essential amino acid required for immune defence and cuticular melanisation and sclerotisation, can act as a limiting resource by supplementing the larval diet and subsequently examining adult cuticular colouration and thickness. We found that stock beetles and beetles artificially selected for extremes of cuticular colour had thicker less porous cuticles when they were darker, and thinner more porous cuticles when they were lighter, showing that colour co-varies with two architectural cuticular features. Experimental supplementation of the larval diet with tyrosine led to the development of darker adult cuticle and affected thickness in a sex-specific manner. However, it did not affect two immune traits. The results of this study provide a mechanism for maintenance of cuticular colour variation in this species of beetle; darker cuticles are thicker, but their production is potentially limited by resource constraints and differential investments in resistance mechanisms between the sexes.

An Experimental Evolution Test of the Relationship between Melanism and Desiccation Survival in Insects.

We used experimental evolution to test the ‘melanism-desiccation’ hypothesis, which proposes that dark cuticle in several Drosophila species is an adaptation for increased desiccation tolerance. We selected for dark and light body pigmentation in replicated populations of D. melanogaster and assayed several traits related to water balance. We also scored pigmentation and desiccation tolerance in populations selected for desiccation survival. Populations in both selection regimes showed large differences in the traits directly under selection. However, after over 40 generations of pigmentation selection, dark-selected populations were not more desiccation-tolerant than light-selected and control populations, nor did we find significant changes in mass or carbohydrate amounts that could affect desiccation resistance. Body pigmentation of desiccation-selected populations did not differ from control populations after over 140 generations of selection, although selected populations lost water less rapidly. Our results do not support an important role for melanization in Drosophila water balance.

A dark cuticle allows higher investment in immunity, longevity and fecundity in a beetle upon a simulated parasite attack.

Cuticle melanism in insects is linked to a number of life history traits: a positive relationship is hypothesized between melanism, immune function, fecundity and lifespan. However, it is not clear how activation of the immune system affects trade-offs between life history traits in female mealworm beetles (Tenebrio molitor) differing in cuticle melanization. The females with tan, brown and black cuticles examined in the present study did not differ in the intensity of encapsulation response, fecundity and longevity when their immune system was not activated. However, we found that immune activation and cuticle melanization have a significant effect on life history traits. Offspring number and lifespan decreased in females with tan and brown cuticles, while the fecundity and lifespan of black females were not affected. Importantly, we inserted the implants again and found a significant decrease in the strength of encapsulation response in females with tan and brown cuticles. In contrast, black females increased melanotic reactions against the nylon implant, suggesting immunological priming. The results show that cuticle melanization plays an important adaptive role under the risk of being infected, while the lack of these benefits before the insertion of nylon monofilaments suggests that there are costs associated with an activated immunity system.

Reproductive Incompatibility Involving Senegalese Aedes aegypti (L) Is Associated with Chromosome Rearrangements

Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane’s rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane’s rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was used to identify AT-rich regions, chromomycin A3 following pretreatment with barium hydroxide stained for GC-rich regions and stained the ribosomal RNA locus and YOYO-1 was used to test for differential staining. Chromosome patterns in SenAae strains revealed by these three stains differed from those in IB12. For FISH, 40 BAC clones previously physically mapped on Aaa chromosomes were used to test for chromosome rearrangements in SenAae relative to IB12. Differences in the order of markers identified two chromosomal rearrangements between IB12 and SenAae strains. The first rearrangement involves two overlapping pericentric (containing the centromere) inversions in chromosome 3 or an insertion of a large fragment into the 3q arm. The second rearrangement is close to the centromere on the p arm of chromosome 2. Linkage analysis of the SDL and the white-eye locus identified a likely chromosomal rearrangement on chromosome 1. The reproductive incompatibility observed within SenAae and between SenAae and Aaa may be generally associated with chromosome rearrangements on all three chromosomes and specifically caused by pericentric inversions on chromosomes 2 and 3.

An Opposite Pattern to the Conventional Thermal Hypothesis: Temperature-Dependent Variation in Coloration of Adults of Saccharosydne procerus (Homoptera: Delphacidae).

Melanism is a common polymorphism in many insect species that also influences immune function. According to the thermal melanin hypothesis, ectothermic individuals from cooler environments have darker cuticles and higher polyphenol oxidase (PO) levels, which represent a better immunocompetence. In this study, the links among environmental temperature, melanism, and PO activity of Saccharosydne procerus (Matsumura) were examined. Most S. procerus have a black spot on their forewings at high temperatures in the field and in the laboratory. In PO activity assay, a positive association between PO level and temperature was found. Our results showed that a diversification of melanism occurred under different temperatures and that melanism in S. procerus presented an opposite pattern to the one proposed by the thermal hypothesis.

How Old are Colonizing Hypothenemus hampei (Ferrari) Females When They Leave the Native Coffee Fruit?

The age of colonizing Hypothenemus hampei (Ferrari) females when they leave the native coffee fruit was determined under laboratory conditions. This biological parameter might be useful for planning experiments with this species because certain physiological statuses are expressed from or until a determined age, which in turn may determine the response of individuals to different treatments. An experimental device was used to simulate the conditions inside a coffee berry and to permit the observation of the abandonment behavior of the beetles. Virgin and mated females with or without melanized cuticles were used in the experiments. On average, colonizing coffee berry borers were 15-days-old at the moment of host abandonment. Females at this age were mated, had full dark cuticles, and were able to display flight and lay viable eggs. Interestingly, H. hampei females that mated before they acquire a fully dark cuticle abandoned the host 1.7 times faster than females that mated after they reach this physiological status. Further studies into food conditions and their impacts on the pre- abandonment of H. hampei females are encouraged.

Altricial Development in Subsocial Wood-Feeding Cockroaches

Species in the wood-feeding genus Salganea within the cockroach subfamily Panesthiinae (Blaberidae) typically live in biparental families; their first instars suffer high mortality when removed from adults, and in at least one species, adults are known to feed neonates on oral liquids. In the closely related gregarious wood-feeding genus Panesthia , no parental interaction with offspring is known. We compared the external morphology of first instars of these two genera and found that eye development and cuticular pigmentation at hatching are correlated with social structure. First instars of Panesthia have a dark cuticle and well-developed eyes. First instars of Salganea have a pale, transparent cuticle, and eyes significantly smaller than those of Panesthia relative to adult eye size. The body size of the first-instar of Salganea esakii is significantly smaller than that of Panesthia angustipennis spadica , relative to adult body size (24.0 and 27.4% of adult head-capsule width, respectively), but a more general survey suggests that, overall, neonate size may be similar in the two genera. We compared the first instars of these two taxa of Panesthiinae to those in the biparental, wood-feeding cockroach Cryptocercus (Cryptocercidae) and discuss how decreased investment in both integumentary and ocular development in subsocial cockroaches parallels that seen in altricial vertebrates.

Analysis of Pyrimidine Catabolism inDrosophila melanogasterUsing Epistatic Interactions With Mutations of Pyrimidine Biosynthesis and β-Alanine Metabolism

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.

Greater cuticular melanism is not associated with greater immunogenic response in adults of the polymorphic mountain stone weta, Hemideina maori

Abstract. 1. Greater immune function is associated with the high‐density melanic phase of polyphenic insects, appearing to compensate for density‐dependent increases in susceptibility to parasites and/or pathogens. Other types of discrete variation in cuticular colour occur in insects (which may or may not be associated with melanin pigmentation), but whether this variation is predictive of immune ability has not been investigated.2. In the mountain stone weta Hemideina maori, a black morph and yellow banded morph occur. These morphs are not seasonally polyphenic and have discrete haplotype genetic markers. Black individuals are typically found at lower local densities than yellow individuals, contrary to relations between cuticular melanism and density seen in polyphenic insects.3. Yellow males and females had greater melanotic encapsulation responses upon immune challenge than did black males and females, but these differences were not associated with differences in temperature selection between morphs. Morph differences in melanotic encapsulation responses were somewhat related to differences between morphs in haemocyte concentrations.4. These results indicate that a common form of immune expression is not heightened with dark coloration in the mountain stone weta. Thus, earlier findings of greater immunity associated with darker cuticles in phase polyphenic insects cannot be extended to insects with other forms of discrete colour variation. These findings will help in elucidating causes and consequences of such colour polymorphism, which is widespread in several insect orders.

Susceptibility to insect growth regulators and cuticle deposition of the cat flea (Siphonaptera: Pulicidae) as a function of age.

Wandering larval, pharate pupal, pupal, and pharate adult stages of the cat flea, Ctenocephalides felis (Bouché), were identified by examination of cuticular cross sections via scanning electron microscopy. Visible morphological characteristics of each stage were identified and described. Various stages of the flea throughout metamorphosis were exposed to juvenile hormone mimics and insect developmental inhibitors. Wandering larvae treated with juvenile hormone mimics were unable to pupate, whereas treated pharate pupae ecdysed to the pupal stage but were unable to enclose. Pupae and pharate adults did not succumb to juvenile hormone mimic exposure, even at concentrations 3 orders of magnitude greater than those that killed 100% of the wandering larvae and the pharate pupae. Adult females were more susceptible to juvenile hormone mimics than males. Possible explanations for the variation in tolerance to juvenile hormone mimics of the differing stadia are discussed. None of the stages were susceptible to insect developmental inhibitor exposure. Analysis of catecholamines that are precursors of cuticle sclerotization and melanization were measured in the wandering larval through the pharate adult stages of the cat flea. N-acetyldopamine concentration was highest in the pupa stage when the flea had a hard, clear cuticle; N-beta-alanyldopamine concentration was highest during the production of the hard, dark cuticle of the pharate adult; and dopamine fluctuated throughout development but was highest in the pupal stage.

Catecholamines in haemolymph and cuticle during larval, pupal and adult development of Manduca sexta (L.)

Catecholamines in haemolymph and cuticle of the tobacco hornworm, Manduca sexta (L.), were analyzed by liquid chromatography with electrochemical detection. During pupal tanning, N- β-alanyldopamine (NBAD) was the major dopamine derivative in haemolymph. It increased to maximal titres by the time of larval-pupal ecdysis, and subsequently decreased as pupal cuticle tanned. NBAD progressively increased in cuticle for several days after ecdysis. N-acetyldopamine (NADA), although present in only minor amounts in pupae, was the primary catecholamine in haemolymph of both larvae and adults shortly before and after ecdysis. NADA predominated in the hard colourless cuticle of the larval head capsule, and in the wings and abdominal tergites of the adult. NBAD, however, was predominant in the dark cuticle of larval mandibles, and in the thin soft larval abdominal cuticle after stabilization. Dopamine, the precursor of NBAD and NADA, was usually the second or third most abundant catecholamine in both haemolymph and cuticle. The kinds of sclerotins produced may depend in part upon the relative abundance of the different catecholamines which are selectively incorporated into the various regions of the exoskeleton. The catecholamines in haemolymph occurred mainly as ring hydroxyl conjugates that were probably β-glucosides. The relative percentage of free or unconjugated compounds increased after ecdysis and accumulated in cuticle. Conjugation, therefore, appears to be important for sequestration of tanning precursors in haemolymph, followed by hydrolysis and uptake of free catecholamines for stabilization and pigmentation of new cuticle.

Properties of Cuticle and Insect Ecology

THERE is no doubt that the physiological significance of insect coloration has not yet been fully realized by ecologists, while its direct survival value has often been overstressed. From this point of view Dr. Kalmus's article in NATURE1 is to be welcomed. His treatment of the problem, however, is such that it may bring undeserved discredit to essentially sound ideas. Starting with references to some recent investigations on the physical properties of insect cuticle, Dr. Kalmus proceeds to formulate thirteen “Rules”, based on the still scanty results of those investigations, on a number of assumptions (for example, “a dark cuticle is mechanically the stronger”, which he admits to be an assumption not yet proved, but on which no less than four “Rules” are based), and on teleological speculation. The evidence quoted in support of each “Rule” is exceedingly meagre and often inaccurate, as a few examples will show. The mole cricket quoted in Rule 3 is not “entirely dark” but paler than most of its relatives. Rule 4 (“giant forms are usually black ; bright colours are more frequent among small insects”) is not supported by any evidence, and its correctness is highly doubtful. Mayflies, on the sole support of which Rule 9 rests, are not necessarily pale and some are even black. Eggs of Phasmodea (Rule 10) are not “dark and hard”, but always hard and only sometimes dark. Rule 11, stating that “most non-black insects are found in the tropics” has since been presented2 in a more intelligible way by saying that “the proportion of non-black insects is greater in the tropics”, but it is legitimate to ask on what evidence this statement, implying the existence of some numerical data, is based. Similar statements of alleged quantitative character constitute many of the “Rules”, and it is surprising that they should emanate from a University Department of Biometry where more respect for statistical data would be expected.