TnaA,an SP-RING Protein,Interacts with Osa,a Subunit of the Chromatin Remodeling Complex BRAHMA and with the SUMOylation Pathway in Drosophila melanogaster

Tonalli A (TnaA) is a Drosophila melanogaster protein with an XSPRING domain. The XSPRING domain harbors an SP-RING zinc-finger, which is characteristic of proteins with SUMO E3 ligase activity. TnaA is required for homeotic gene expression and is presumably involved in the SUMOylation pathway. Here we analyzed some aspects of the TnaA location in embryo and larval stages and its genetic and biochemical interaction with SUMOylation pathway proteins. We describe that there are at least two TnaA proteins (TnaA₁₃₀ and TnaA₁₂₃) differentially expressed throughout development. We show that TnaA is chromatin-associated at discrete sites on polytene salivary gland chromosomes of third instar larvae and that tna mutant individuals do not survive to adulthood, with most dying as third instar larvae or pupae. The tna mutants that ultimately die as third instar larvae have an extended life span of at least 4 to 15 days as other SUMOylation pathway mutants. We show that TnaA physically interacts with the SUMO E2 conjugating enzyme Ubc9, and with the BRM complex subunit Osa. Furthermore, we show that tna and osa interact genetically with SUMOylation pathway components and individuals carrying mutations for these genes show a phenotype that can be the consequence of misexpression of developmental-related genes.     

Antennal Drumming,Trophallaxis, and Colony Development in the Social Wasp Polistes fuscatus (Hymenoptera: Vespidae)

Parental care is an important component of social behavior in both vertebrates and invertebrates. Social wasps are a useful system for investigating the interplay between behaviors associated with the feeding of larvae by adults and their role in the evolution and maintenance of sociality. Females of the primitively eusocial wasp genus Polistes perform conspicuous vibratory behaviors closely associated with adult–larva feeding interactions. Prior research strongly indicates that these signals are directed toward the larvae, but their function(s) remain unclear. Existing hypotheses on the function(s) have posited releaser effects on larvae, either stimulating or inhibiting release of larval saliva, a nutrient‐rich glandular secretion attractive to adults. Polistes fuscatus queens perform antennal drumming (AD), a behavior in which they rapidly beat their antennae synchronously on the rims of the nest cells during the feeding of larvae. We used radiolabeled prey to show that adults extract juice from the prey, which they subsequently regurgitate to larvae immediately following each AD burst. We also show that no saliva is imbibed by the queen during the contact. These results are consistent with the inhibition hypothesis on the function of AD, but not the stimulation hypothesis. We further demonstrate that AD is first performed on nests when the oldest larvae reach the third instar, and that the third instar is the first to produce measurable volumes of larval saliva. Removal of third‐, fourth‐, and/or fifth‐instar larvae from single‐foundress, pre‐pupal‐stage colonies did not cause a reduction in the queen’s AD rates compared with controls, suggesting that later‐stage larvae do not maintain AD behavior via an immediate releaser effect. We propose instead that third‐instar larvae, possibly via chemical components of the salivary secretion itself, modulate the physiology of queens so as to indirectly cause the onset and maintenance of AD behavior.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

Foraging by <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Larvae in a Patchy Environment

The food resources of Drosophila comprise decaying vegetable matter distributed in patches, yet foraging behavior has not been examined in larvae reared continuously in a patchy environment. Here, the extent and rate of inter-patch movement was studied in larvae of four wild strains of D. melanogaster inhabiting an experimental arena from the egg stage to the third larval instar. The hypotheses were that larvae would forage primarily in the third instar, that larvae would move from low-protein patches at higher rates than from high-protein patches, and that foraging rates would be higher on an agar substrate than on sand. Larvae hatching on a nutrient-poor food patch switched to a nutrient-rich patch during the first instar. The rate of interpatch switching increased with larval age, as did the number of larvae roving on the substrate between food patches. Inter-patch distance affected switching speed---the closer the patches, the faster the switching. High protein patches were preferred over low-protein patches, but there was a bias towards staying on the natal patch. Significant variation among strains in latency to forage, in proportion of larvae that switched patches, and in the rate of roving between patches suggests that there is natural genetic variation for these traits. Larvae switched food patches on a substrate of moist sand as quickly as on an agar substrate.     

The time--mortality response of mosquito larvae infected with the fungus Culicinomyces

In laboratory experiments with Culicinomyces, the time of death of infected Anopheles hilli larvae decreased as the concentration of conidia to which they were exposed increased. At a concentration of 10³ conidia/ml, most first instar and third instar larvae died later than 2 days after exposure and the majority of dead specimens developed external sporulation on the exterior cuticle. On the other hand, at a concentration of 10⁵ conidia/ml, more than 60% of first and third instar larvae died within 2 days of exposure. Very few of these larvae developed external sporulation and it appears that most of them died after penetration by the fungus through the gut cuticle but before the hemocoel was colonized by mycelium. The reason for this rapid death at high concentrations of inoculum is not known, but it is thought that it may be caused by toxic substances associated with the invading hyphae which only attain a lethal titer when massive invasion originates from large numbers of conidia.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Messenger RNA sequence complexity and homology in developmental stages of Drosophila

Total polysomal RNA and polyadenylated mRNA from third instar larvae, pupae, and adults of D. melanogaster were hybridized in vast excess to labeled single-copy DNA in order to measure the sequence complexity of each RNA population. Then, to measure the sequence homology between the populations, each was hybridized to DNA enriched for messenger coding sequences in third instar larvae and to DNA depleted of these sequences. Our results show that a similar number of genes, approximately 16,000, is expressed in larvae, pupae, and adults, and that only one-third of these is expressed as polyadenylated mRNA. Further, the composition of both polyadenylated and nonpolyadenylated mRNA classes is shown to change very little between these three stages of development. Finally, the head of adult Drosophila is shown to contain 11,000 RNA species, approximately 70% of the number contained in the entire adult.     

Alterations in the production of hemocytes due to a neoplastic mutation of Drosophila melanogaster

The hemocytes of a genetically induced, temperature-sensitive lethal mutation of Drosophila, Tum¹, were examined both quantitatively and qualitatively during the third larval instar. At the tumor-permissive temperature, 29°C, there was a fourfold increase in the concentration of circulating hemocytes in mutant larvae as compared to control. Additionally, the relative frequency of lamellocytes was 30 times greater in Tum¹ larvae than Basc in the early third instar. However, the severity of this abnormality gradually diminished as Tum¹ approached pupariation; though high frequencies of lamellocytes were always present. At the tumor-restrictive temperature (15°C) the concentration of circulating hemocytes was over twice that found at 29°C for Tum¹ larvae, and did not change during the course of third instar. However, in contrast to 29°C there was no abnormal increase in the frequency of lamellocytes at the tumor-restrictive temperature. Control larvae had equivalent concentrations of hemocytes at both temperatures. In one of two temperature shift experiments, Tum¹ larvae shifted from 15° to 29°C at the beginning of third instar expressed the abnormal hemocyte concentration and differentiation associated with larvae raised continuously at 29°C. In addition, Tum¹ larvae shifted from 29° to 15°C expressed reduced abnormalities of hemocyte differentiation, e.g., with fewer lamellocytes in circulation. The possibility of a temperature-sensitive period for the activation of the Tum¹ gene is discussed.     

Induced antibacterial proteins in the haemolymph of Phormia terranovae (Diptera): Purification and possible origin of one protein

Injection of live bacteria into third instar larvae of the dipteran Phormia terranovae results in the appearance in the haemolymph of at least five heat-stable, more or less basic proteins, showing antibacterial activity with Escherichia coli. One of these proteins has been partially purified and characterized as a non-lysozymic peptide of 9 kD, with an isoelectric point of 7.8. Fat body tissue has been shown to be responsible for the synthesis of the antibacterial proteins in Phormia terranovae.     

In situ patterns of nuclear replication in brain ganglia ofl(2) gl 4 mutant larvae ofDrosophila melanogaster

The1(2) gl ⁴ mutation (a deletion mutation of a recessive oncogene) ofDwsophila melanogaster causes neuroblastoma in the optic centres of brain of late third instar larvae. We have studied thein situ patterns of DNA synthesis in these brains by immunocytochemical detection of cells incorporating 5-bromodeoxyuridine. It was seen that1 (2)gl ⁴ brains from younger 3rd instar larvae had fewer replicating cells than in wild type larvae of comparable age but in brain ganglia of olderl (2) gl ⁴ larvae the number of replicating cells was much higher. The spatial distribution of replicating cells in optic lobes of brain ganglia of1 (2) gl ⁴ larvae was disturbed from early 3rd instar stage, much before the tumourous growth was morphologically detectable. The stereotyped pattern of asymmetrical cell divisions of the neuroblasts and their progeny cells was also not seen in1 (2) gl ⁴ brain ganglia. Therefore, it appears that thel (2)gl ⁴ product has an important role early in development to determine the temporal and spatial patterns of neuroblast cell division in developing brains.     

MASS PREPARATION OF NUCLEI FROM THE LARVAL SALIVARY GLANDS OF DROSOPHILA HYDEI

A method has been developed for isolating gram quantities of salivary glands from late third instar larvae of Drosophila hydei. The isolated glands have a normal appearance and incorporate RNA and DNA precursors normally. Nuclei can be isolated from these glands in 90% yield with the use of detergents. These nuclei contain morphologically normal giant polytene chromosomes.     

Competence of Macrotermes michaelseni (Isoptera: Macrotermitinae) larvae to differentiate into soldiers under the influence of juvenile hormone analogue (ZR-515, methoprene)

It has been shown that only third instar larvae of Macrotermes michaelseni have the competence to differentiate into presoldiers under the influence of juvenile hormone analogue (JHA). The timing of events leading to presoldier formation was independent of JHA dose above the threshold. Further studies with homogeneous groups of third instar larvae of different ages showed that only larvae of a certain age (0–6 days) could respond to topically applied JHA to produce presoldiers and intercastes (intermediate forms). Older larvae did not respond, hence, 0–6 days interval is the competence period for presoldier differentiation in this species. It seems also that the corpora allata of those individuals which differentiate into presoldiers become activated during the competence period, possibly by the parents or other means.     

Developmental cytology of the midgut in the flesh-fly, Sarcophaga bullata (Parker)

The cytological comparisons of the midgut in Sarcophaga bullata (Parker) between the second instar, the third instar larvae and the adult are made. The adult midgut differs from that of the larvae in the following ways: (1) the peritrophic membrane is thicker than in the larvae and has become multi-layered; (2) epithelial cells are smaller; (3) branched microvilli are present throughout the entire midgut instead of being present only in the posterior region as in the larval midgut; (4) nuclear pores are less frequent; (5) lysosome-type structures occur less frequently; (6) the basal membrane is thicker; (7) the z-bands in the surrounding muscle fibers are more distinct in adults. The possible function and the significance of these structures related to previous observations in Sarcophaga and other Diptera are discussed.     

Development of Microplitis bicoloratus on Spodoptera litura and implications for biological control

Microplitis bicoloratus Chen (Hymenoptera:Braconidae:Microgastrinae), a new species of Microplitis Förster from China, is a solitary endoparasitoid of the larvae of the cotton leafworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). This parasitoid is the first to be successfully reared and evaluated in the laboratory as a potential agent for the biological control of S. litura in China. Oviposition, immature development, and the effects of parasitism on the development of S. litura were studied. In long-term oviposition trials, females laid eggs on S. litura larvae for up to 10 days; oviposition was heavily skewed toward the first few days, with approximately one third of the eggs laid on day 1 and over 50% laid by day 3. This rapid oviposition rate increases the potential for biological suppression of host populations because the likelihood of mortality for the parasites from exposure to detrimental environmental factors or generalist predators increases with time. Immature development of the parasitoid in its host only required 7 days: eggs hatched within 24 h, the first instar larva required 2 days, the second instar larva needed 3 days, and the third instar larvae exited the host and pupated in 1 day, at 27±1°C, 60–80% relative humidity and a 12:12-h (long day) photoperiod. The development of the parasitized hosts was disrupted. When the parasitoid larvae finished development, the body weights of host larvae were significantly reduced regardless of which host instar was parasitized. Our results suggest that M. bicoloratus has considerable potential as a biological control agent for S. litura.     

The effects of pH,phenol, and sodium chloride on survival and caloric,lipid, and nitrogen content of a laboratory population of chironomus attenuatus (Walk.)

Continuous flow, laboratory microcosms were used to measure the effects of pH, phenol, and NaCl on the survival of the life stages of Chironomus attenuatus, the caloric content of third and fourth instar larvae and adults, the lipid and protein-nitrogen content of fourth instar larvae, and the interaction among the various responses. The metabolism of phenol was studied using uniformly labeled phenol-C¹⁴. pH had a significant effect on the survival of all life stages of C. attenuatus. Survival was higher for all larval instars at pH 7.2 while adult emergence was higher at pH 6.2. Increasing phenol levels resulted in a nearly linear increase in caloric content. Sodium chloride affected the lipid content of fourth instar larvae. The lipid content was higher with NaCl present in the media than without NaCl. Interaction had a significant effect on all responses except survival of third and fourth instar larvae. Phenol-C¹⁴ was metabolized by bacteria, but not by C. attenuatus.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

Biosteres longicaudatus: Developmental dependence on host (Anastrepha suspensa) physiology

Larvae of Anastrepha suspensa that were in the first day of the third instar were parasitized by females of the solitary endoparasitoid, Biosteres longicaudatus. At the end of the 6-hr oviposition period, larvae were ligated posterior to the ring gland so that some larvae had parasitoids anterior to the ligature while in others, the parasitoids were in the abdomen, posterior to the ligature. Ninety-two percent of the parasitoids anterior to the ligature hatched to the first through third instars. Parasitoids posterior to the ligature had a 75% egg hatch to the first instar only. No larval molts to the second or subsequent instars occurred in these parasitoids. Upon parabiosis to 3-day-old, unparasitized host pupae, the ligated larvae pupated and 97% of the first-instar parasitoids in these parabiosed larval abdomens molted to the second instar. Newly laid parasitoid eggs transplanted to 3-day-old pupal hosts had less than one-third of the egg hatch of those transplanted to first-day third-instar hosts. The data implicate the physiological state of the host (vis-a-vis pupation and associated events) as being an important factor in the development of the endoparasitoid.     

Predation capacity and prey preference of <Emphasis Type="Italic">Chrysoperla carnea</Emphasis> on <Emphasis Type="Italic">Pieris brassicae</Emphasis>

The predation capacity and prey preference of larvae of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) on eggs or larvae of Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) in the absence and presence of cabbage aphids as an alternative prey were evaluated in laboratory experiments at 25°C. Both instars preyed upon butterfly eggs and larvae as well as on cabbage aphids with the third instar being the most voracious. The lacewings had a strong preference for caterpillars to butterfly eggs. In the presence of the aphids the predation on P. brassicae eggs or larvae was either completely abandoned or reduced by about 70%, respectively, by second instar lacewings and either reduced by about 80% or maintained, respectively, by third instar lacewings. Both instars thus had a clear preference for aphids compared to eggs of P. brassicae. However, second instar lacewings preferred aphids to caterpillars whereas the opposite was the case for third instar lacewings. The results indicate that 3rd instar C. carnea has a potential as biocontrol agent against P. brassicae.