Larval Integument Research Articles

Polarization Microscopy and Infrared Microspectroscopy of Integument Coverings of Diapausing Larvae in Two Distantly Related Nonsocial Bees.

The larvae of the two distantly related nonsocial bees Ericrocis lata (Apidae) and Hesperapis (Carinapis) rhodocerata (Melittidae), which develop mostly under arid desert areas of North America, and that differ in that they either spin (E. lata) or do not spin (H. rhodocerata) protective cocoons before entering diapause, produce transparent films that cover the larval integument. To understand the nature of these films, their responses to topochemical tests and their characteristics when examined with fluorescence and high-performance polarization microscopy and microspectroscopy were studied. A positive staining by Sudan black B, birefringence of negative sign, and a Fourier transform-infrared (FT-IR) spectrum typical of lipids were detected for the integument covering of both species. The FT-IR signature, particularly, suggests a wax chemical composition for these lipid coverings, resembling the waxes that are used as construction materials in the honey cells produced by social bees. Considering the arid environmental conditions under which these larvae develop, we hypothesize that their covering films may have evolved as protection against water depletion. This hypothesis seems especially appropriate for H. rhodocerata larvae, which are capable of undergoing a long diapause period in the absence of a protective cocoon.

CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura

The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function of SlitBLOS2, CRISPR/Cas9 based target mutagenesis of SlitBLOS2 was achieved, displaying a mosaic translucent integument in 62.3–70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at the SlitBLOS2 specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function of SlitBLOS2 in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy in S. litura as well as other lepidopteran pests.

Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana.

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

CHITINASE: LOCALIZATION, ACTIVITY PATTERN AND CHANGES AFTER NOMOLT INSECTICIDE TREATMENT IN THE BLACK CUTWORM, AGROTIS EPSILON (HUFN.)

chitinase as endo-type of chitinolytic enzymes was studied in the 4th larval instar of the black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae) to detect some of its physiological aspects. Results showed that, the chitinase activity was low till 18 h before ecdysis to the 4th instar, but the activity started increasing at 10 h and reached a peak at 2 h before the ecdysis. This confirms that the appearance of chitinase activity is restricted to the molting period and the main function of chitinase in larvae is the digestion of old cuticle. On the other hand, the enzyme activity in pupal stage was 1.73-fold as compared to that present in larvae, might to share in the intensive physiological processes in this stage. Results of enzyme localization revealed that larval integument had the highest chitinase activity (72.3%), while hemolymph had the lowest one (1.9%). Hence, we infer that hemolymph plays a minor role in transporting the enzyme, and chitinase localized there in the integument. The results showed that hyperchitinase activity in larvae was not a sign of LC50 treatment by nomolt. This led to the assumption that nomolt acts by inhibition of chitin synthesis not by enhancing of chitin degradation. Future studies concerned with factors affecting chitinase must take into consideration the fact that larval chitinase activity is rhytmic, i.e. precaution that the sampling time is critical.

Density, Viability Conidia And Symptoms of Metarhizium anisopliae infection on Oryctes rhinoceros larvae

M. anisopliae is parasitic fungus on insect pests; it is used as a biocontrol agent. M. anisopliae can be propagated on maize or rice substrate. M. anisopliae is currently sold in the form of kaolin powder formulations. Before it is used to check the density, viability and pathogenicity of M. anisopliae. However the problem is the kaolin powder very soft, so it difficult to distinguish between kaolin and conidia. This article gives information on how to calculate conidia density, viability and symptoms of M. anisopliae infection on Oryctes rhinoceros larvae. The study was conducted in the laboratory to determine the density and viability. The pathogenicity testing was done using pots. The Pot is containing soil substrate mixed with M. Anispoliae and ten tails O. Rhinoceros larvae per pot. The results showed that the density of M. anisopliae conidia was 1.81 x 108 conidia mL-1 and the viability was 94% within 24 hours. The larval mortality began to emerge in the 1st week, and all larvae died at the sixth week. The symptom of M. anisopliae infection on Oryctes rhinoceros larvae, there was a black spot on the larval integument. The larvae movements become slow and poor appetite; it will die within 3-7 days. The larvae die hard, and the white hyphae grow on the body surface that turns green.

Penetration-enhancement underlies synergy of plant essential oil terpenoids as insecticides in the cabbage looper, Trichoplusia ni

Many plant essential oils and their terpenoid constituents possess bioactivities including insecticidal activity, and they sometimes act synergistically when mixed. Although several hypotheses for this have been proposed, the underlying mechanism has not been fully elucidated thus far. In the present study, we report that in larvae of the cabbage looper, Trichoplusia ni, most synergistic or antagonistic insecticidal activities among mixtures of plant essential oil constituents are pharmacokinetic effects, owing to changes in solubility as well as spreadability on a wax layer. Among the major constituents of rosemary (Rosmarinus officinalis) oil, in vitro analysis revealed up to a 19-fold increase in penetration of camphor in a binary mixture with 1,8-cineole through the larval integument, suggesting increased penetration as the major mechanism for synergy. A total of 138 synergistic or antagonistic interactions among 39 compounds were identified in binary mixtures via topical application, and these were highly correlated to changes in surface tension as measured by contact angle of the mixtures on a beeswax layer. Among compounds tested, trans-anethole alone showed evidence of internal synergy, whereas most of remaining synergistic or antagonistic combinations among the three most active compounds were identified as penetration-related interactions, confirmed via a divided-application bioassay.

Distribution and Metabolism of Bt-Cry1Ac Toxin in Tissues and Organs of the Cotton Bollworm, Helicoverpa armigera

Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have been widely used in transgenic crops due to their toxicity against insect pests. However, the distribution and metabolism of these toxins in insect tissues and organs have remained obscure because the target insects do not ingest much toxin. In this study, several Cry1Ac-resistant strains of Helicoverpa armigera, fed artificial diets containing high doses of Cry1Ac toxin, were used to investigate the distribution and metabolism of Cry1Ac in their bodies. Cry1Ac was only detected in larvae, not in pupae or adults. Also, Cry1Ac passed through the midgut into other tissues, such as the hemolymph and fat body, but did not reach the larval integument. Metabolic tests revealed that Cry1Ac degraded most rapidly in the fat body, followed by the hemolymph, peritrophic membrane and its contents. The toxin was metabolized slowly in the midgut, but was degraded in all locations within 48 h. These findings will improve understanding of the functional mechanism of Bt toxins in target insects and the biotransfer and the bioaccumulation of Bt toxins in arthropod food webs in the Bt crop ecosystem.

Molybdenum cofactor deficiency causes translucent integument, male-biased lethality, and flaccid paralysis in the silkworm Bombyx mori

Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants.

In search of pathogens: transcriptome-based identification of viral sequences from the pine processionary moth (Thaumetopoea pityocampa).

Thaumetopoea pityocampa (pine processionary moth) is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East and North Africa. Apart from causing significant damage to pinewoods, T. pityocampa occurrence is also an issue for public and animal health, as it is responsible for dermatological reactions in humans and animals by contact with its irritating hairs. High throughput sequencing technologies have allowed the fast and cost-effective generation of genetic information of interest to understand different biological aspects of non-model organisms as well as the identification of potential pathogens. Using these technologies, we have obtained and characterized the transcriptome of T. pityocampa larvae collected in 12 different geographical locations in Turkey. cDNA libraries for Illumina sequencing were prepared from four larval tissues, head, gut, fat body and integument. By pooling the sequences from Illumina platform with those previously published using the Roche 454-FLX and Sanger methods we generated the largest reference transcriptome of T. pityocampa. In addition, this study has also allowed identification of possible viral pathogens with potential application in future biocontrol strategies.

Chemical defense balanced by sequestration and de novo biosynthesis in a lepidopteran specialist.

The evolution of sequestration (uptake and accumulation) relative to de novo biosynthesis of chemical defense compounds is poorly understood, as is the interplay between these two strategies. The Burnet moth Zygaena filipendulae (Lepidoptera) and its food-plant Lotus corniculatus (Fabaceae) poses an exemplary case study of these questions, as Z. filipendulae belongs to the only insect family known to both de novo biosynthesize and sequester the same defense compounds directly from its food-plant. Z. filipendulae and L. corniculatus both contain the two cyanogenic glucosides linamarin and lotaustralin, which are defense compounds that can be hydrolyzed to liberate toxic hydrogen cyanide. The overall amounts and ratios of linamarin and lotaustralin in Z. filipendulae are tightly regulated, and only to a low extent reflect the ratio in the ingested food-plant. We demonstrate that Z. filipendulae adjusts the de novo biosynthesis of CNglcs by regulation at both the transcriptional and protein level depending on food plant composition. Ultimately this ensures that the larva saves energy and nitrogen while maintaining an effective defense system to fend off predators. By using in situ PCR and immunolocalization, the biosynthetic pathway was resolved to the larval fat body and integument, which infers rapid replenishment of defense compounds following an encounter with a predator. Our study supports the hypothesis that de novo biosynthesis of CNglcs in Z. filipendulae preceded the ability to sequester, and facilitated a food-plant switch to cyanogenic plants, after which sequestration could evolve. Preservation of de novo biosynthesis allows fine-tuning of the amount and composition of CNglcs in Z. filipendulae.

The ecology of yeasts in the bark beetle holobiont: a century of research revisited.

Yeasts are extremely common associates of scolytine bark beetles, yet the basic ecology of yeasts in the bark beetle holobiont remains poorly understood. Yeasts are present in all beetle life stages and consistently isolated from adult, larval, and pupal integuments and mycangial structures, but yeasts are also found in oviposition galleries, pupal chambers, larval and adult digestive tracts, as well as phloem and xylem tissues. Yeasts in the Saccharomycetaceae family are the most prevalent associates, and most individual beetles are associated with only one or several yeast species. Kuraishia capsulata and Ogataea pini are the most commonly encountered yeast species in surveys of Dendroctonus and Ips beetles; most beetles that have been surveyed are vectors for one or both yeasts. Yeasts have significant but often overlooked functional roles in bark beetle ecology. Infochemicals resulting from volatile production by yeast have wide-ranging bioactivity for arthropods: Yeast emissions attract beetles at low concentrations but repel beetles at high concentrations, and yeast emissions can also serve as cues to predators and parasites of bark beetles. In some cases, yeasts can modify tree chemistry over time or metabolize toxic terpenoids, though potential consequences for beetle performance or the growth of nutritional fungi remain to be demonstrated. Also, the presence of yeast species can restrict or promote the establishment and growth of filamentous fungi, including mutualists, entomopathogens, and opportunistic saprophytes. The role of yeasts as nutritional symbionts has received mixed support, though a nutritional hypothesis has not been extensively tested. Continued research on the functional ecology of bark beetle-yeast associations is needed to better understand the emergent properties of these complex symbiont assemblages.

Hiding in plain sight: cuticular compound profile matching conceals a larval tortoise beetle in its host chemical cloud.

Larvae of tortoise beetles are postulated to have fecal shields as the main defensive strategy against predators. Such a device protects beetles both physically and chemically. In order to examine how larvae Chelymorpha reimoseri are protected against predatory ants, which frequently visit extrafloral nectaries in their host plant, the morning glory Ipomoea carnea, we conducted anti-predation bioassays with live 5th instars. In the field, larvae in contact with ants had survival between 40 and 73%, independently of shield presence. In the laboratory, when exposed to Camponotus crassus, larvae with shields had significantly higher survival (85%) than those without shields (64%). In both scenarios, larval survival was significantly higher when compared with palatable Spodoptera frugiperda larvae, as the latter were all consumed. We also observed that when C. reimoseri larvae showed no movement, the ants walked on them without attacking. We hypothesized that if the larval integument has a pattern of cuticular compounds (CCs) similar to that of its host plant, larvae would be rendered chemically camouflaged. In the field and laboratory, the freeze-dried palatable larvae of S. frugiperda treated with CCs of 5th instar C. reimoseri and left on I. carnea leaves were significantly less removed by ants than controls without these compounds. We also found a similarity of approximately 50% between the CCs in C. reimoseri larvae and I. carnea host leaves. Both findings provide evidence in support of the hypothesis that chemical camouflage plays an important role in larval defense, which is reported for the first time in an ectophagous leaf beetle larva.

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.

Deletion ofv-chiAfrom a Baculovirus Reduces Horizontal Transmission in the Field

Nucleopolyhedroviruses (NPVs) can initiate devastating disease outbreaks in populations of defoliating Lepidoptera, a fact that has been exploited for the purposes of biological control of some pest insects. A key part of the horizontal transmission process of NPVs is the degradation of the larval integument by virus-coded proteins called chitinases, such as V-CHIA produced by the v-chiA genes. We used recombinant and naturally occurring strains of the Lymantria dispar NPV (LdMNPV) to test horizontal transmission in the field, release of virus from dead larvae under laboratory conditions, and cell lysis and virus release in cell culture. In the field, strains of LdMNPV lacking functional v-chiA genes showed reduced horizontal transmission compared to wild-type or repaired strains. These findings were mirrored by a marked reduction in released virus in laboratory tests and cell culture when the same strains were used to infect larvae or cells. Thus, this study tests the pivotal role of liquefaction and the v-chiA gene in field transmission for the first time and uses complementary laboratory data to provide a likely explanation for our findings.

Possible roles of Juvenile Hormone and Juvenile Hormone binding protein on changes in the integument during termination of larval diapause in the bamboo borer Omphisa fuscidentalis

AbstractTo understand the role of Juvenile Hormone (JH) on changes in the integument of Omphisa fuscidentalis Hampson (Lepidoptera: Pyralidae) during larval–pupal development, the expression patterns of transcripts of the O. fuscidentalis JH binding protein gene (OfJHBP) are determined in the integument of diapausing larvae. Quantitative real‐time polymerase chain reaction (qRT‐PCR) studies show that the relative levels OfJHBP mRNA in the integument do not differ significantly in different segments of the body. Application of the JH analogue (JHA) methoprene to diapausing larvae results in a relatively low expression of OfJHBP mRNA from days 0 to 8 after JHA application and reaches maximal levels in the pupal stage. Changes in OfJHBP expression after 20‐hydroxyecdysone (20E) injection also show low levels of OfJHBP mRNA from days 0 to 8 and show a peak of expression in the pupal stage. Incubation of the integument in vitro in the presence of JHA (3.22 µm) induces high levels of OfJHBP expression within 120 min, whereas incubation with 20E (2.08 µm) induces gene expression at 150 min. To study the cooperative effect on OfJHBP expression of these two hormones, dose–response experiments are performed. Larval integument is maintained in the presence of 0.32 µm JHA or 2.08 µm 20E or a combination of both hormones in vitro for 120 min. Induction of OfJHBP expression by JHA and 20E in combination is significantly higher than that of either hormone alone. The results impact not only on our understanding of how methoprene (and hence JH) terminates larval diapause through an increase in ecdysteroid titre in the haemolymph, but also on how JH acts directly on the integument to stimulate the expression of a JH binding protein gene.

MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM, BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p(B) ), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p(S) ) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA-induced damage.

Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori

Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different al alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al2), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al2 larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (lem) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by lem (lem/leml) females. We found that lem/lem, al2/al2 larvae produced by lem (lem/lem) females were viable during the first instar stage, suggesting that al2/al2 embryo could synthesis BH4 by using maternally transmitted BmPTS.

Laboratory and field evaluations of the susceptibility of immature Choristoneura rosaceana (Lepidoptera: Tortricidae) to Beauveria bassiana (Hypocreales: Cordycipitaceae)

The susceptibility of immature Choristoneura rosaceana (Harris) to Beauveria bassiana-GHA (BotaniGard® 22WP) was evaluated under laboratory and field conditions. Although egg masses ≤24 h old were susceptible to infection by topically sprayed B. bassiana spores in the laboratory and resulting mycosis significantly reduced the percent of neonates able to emerge, no significant egg mass infections resulted from orchard treatment of ≤24–48 h eggs. Exposure to high levels of the B. bassiana product on apple leaf surfaces in the laboratory caused significant dose-related mortality in first to fourth instar C. rosaceana. First instar C. rosaceana were the most susceptible of the larval stages assayed in the laboratory although only 36% of neonates introduced into the orchard prior to Beauveria treatment and 27% of the neonates emerging from orchard treated egg masses became infected. Fourth instar mortalities were similar but moderate when treated with 1×108 spores mL−1 in the orchard and on leaf surfaces in the laboratory. Beauveria bassiana-induced larval mortalities were significantly higher when the spores were applied directly to the larval integument as opposed to the leaf surface where the insect would encounter the pathogen in a treated orchard. Feeding of C. rosaceana larvae on antibiotic containing meridic diet prior to their use in trials did not impact the susceptibility of the larvae to B. bassiana. Antibiotic containing meridic diet significantly reduced larval C. rosaceana mortality when treated larvae or the spores were placed directly on the diet as opposed to leaf tissue.

A homolog of the human Hermansky–Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori

Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ( 2 ), and oa ( v )), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.

A review of the North American genus Epimartyria (Lepidoptera, Micropterigidae) with a discussion of the larval plastron.

The indigenous North American micropterigid genus Epimartyria Walsingham,1898 is revised. Three species are recognized, including Epimartyria auricrinella Walsingham, 1898 which occurs widely over much of the northeastern United States and Canada, a new species, Epimartyria bimaculella Davis & Landry from northwestern United States and Canada, and Epimartyria pardella (Walsingham, 1880) from northern California to northern Oregon. The larva of Epimartyria auricrinella is described in detail, supplemented with illustrations of the external structure of the larval integument. The larval plastron is described and illustrated for Epimartyria, and this is compared with the plastrons of Neomicropteryx Issiki, 1931 and Micropterix Hübner, 1825. COI barcode sequences show that the three species are genetically distinct, congruent with morphological differences. Marked haplotype divergence within some Epimartyria auricrinella populations appears to be unrelated to morphology, geography or phenology.