Wild Larvae Research Articles

Observation of Aneurobracon philippinensis (Hymenoptera: Braconidae) Immatures Shows How Koinobiont Offspring Flexibly Adjust Their Development to Host Growth

AbstractKoinobionts are parasitoids that allow their hosts to grow after infection, and they finally kill their host individuals at parasitoid-specific host stages. Since fatal accidents of host organisms directly result in the deaths of parasitizing koinobionts, a longer parasitization period in vulnerable hosts is likely to increase the mortality of the koinobionts. However, for hosts inhabiting concealed environments in their later developmental stages, koinobionts should begin parasitization in early-stage hosts to make use of the grown hosts. A koinobiont parasitoid, Aneurobracon philippinensis (Muesebeck), mainly uses a leaf-mining moth, Acrocercops transecta Meyrick (Lepidoptera: Gracillariidae) as a host. Due to the three-dimensional structure of the mines constructed by later instars of A. transecta, females of A. philippinensis seldomly oviposit into later instar hosts, whereas feeding on final instar hosts is essential for A. philippinensis larvae. This implies that oviposition targets in the wild are shifted to early instars, though the final instar is the ideal target to shorten the parasitization period. The dissection of wild host larvae demonstrated that no eggs were observed in the final instar, supporting the above expectation. Laboratory parasitization experiments revealed that A. philippinensis eggs hatched approximately 80 h after oviposition, and hatched larvae stayed in the first instar until the host larvae completed making cocoons. These results suggest that the first-instar period of the parasitoid larvae functions as an adjusting period to synchronize the parasitoid and host developmental stages and that koinobiosis plays an important role in utilizing the final instar of A. transecta as a resource.

Host plant specificity and feeding preferences of Morpho epistrophus argentinus (Lepidoptera: Nymphalidae) larvae in coastal woodlands of Argentina

The selection of suitable host plants for larval development is critical for most herbivorous insects. In this study, we examined host plant preferences of larvae of the Epistrophus white morpho butterfly, Morpho epistrophus argentinus (H. Fruhstorfer), a specialized butterfly that oviposits on a few plant species. In many species, the selection of host plant species for larvae development is under the control of ovipositing females and the role of larvae in host selection is minimal. Through field observations, we characterized larval host plant use in coastal woodland patches of its southernmost distribution range at eastern Buenos Aires, Argentina. All sampled M.e. argentinus larvaegroups (n = 40) were found on Scutia buxifolia trees, although the number of larval groups did not correlate with cover of this plant at patch scale. To further examine host plant preferences we performed translocation experiments and feeding choice assays. M.e. argentinus larvae translocated to unsuitable host plants had a shorter residence time than those translocated to suitable (i.e. control) hosts. Choice assays also indicated strong feeding preferences of wild larvae for S. buxifolia leaves. Our results highlight the high degree of host specificity of this butterfly, and reveal that host preference of growing larvae matches that of ovipositing females.

Evaluation of optimum temperature for the early larval growth of Japanese eel in captivity

Early larval growth of the Japanese eel Anguilla japonica reared at five different water temperatures (19, 21, 23, 25, and 27 °C) is described for the first month post-hatching, a critical period of time in the culture of these sensitive larvae. After 3 weeks of culture, larvae reared at 27 °C attained the greatest total length and body height of all temperature treatments. Larvae reared at higher temperatures developed rapidly due to active feeding, despite higher metabolic costs. At lower temperatures of 19–21 °C, sudden mortality 12–16 days post-hatching coincides with the transition of larvae from endogenous to exogenous feeding. A linear relationship between water temperature and otolith diameter for cultured eel larvae otoliths suggests that the mean ambient water temperature experienced by wild larvae during their first month after hatching is about 23 °C. This temperature corresponds to mean water temperatures likely experienced by larvae during diel vertical migrations in the North Equatorial Current. We propose rearing early stage Japanese eel larvae at variable temperatures throughout the light/dark cycle to enhance their survival and growth in captivity.

Establishment and seasonal activity in New Zealand of Mastrus ridens, a gregarious ectoparasitoid of codling moth Cydia pomonella

The establishment of Mastrus ridens Horstmann (Hym: Ichneumonidae) in New Zealand, from introductions in 2012–2015, was assessed in 2016. Two assessment techniques were used: (1) 10-cm wide corrugated cardboard trunk bands deployed throughout the summer and winter periods to catch wild codling moth larvae and their parasitoids; and (2) sentinel, laboratory reared, codling moth larvae, cocooned within narrow, 2-cm wide corrugated cardboard bands, deployed on a monthly rotation throughout the spring and summer. Trunk band recoveries showed at least a low rate of establishment of Mastrus ridens across the country. Mastrus ridens females attacked sentinel hosts from early spring (prior to pupation of wild hosts) until late autumn. This evidence for multi-voltinism (compared with one or two generations of their host) signals a potentially effective parasitoid. Four other codling moth parasitoids were also recovered frequently from either wild or sentinel codling moth larvae, with differences between regions. The extent to which they may disrupt or enhance biocontrol by M. ridens remains to be investigated.

The extraordinary larva of the White Admiral butterfly, Limenitis camilla (Linnaeus, 1764)

Our observations on the behaviour of the early instars of the White Admiral butterfly, Limenitis camilla are described from the study of wild and captive larvae. These are compared with other published sources.

Automated in vivo screen in zebrafish identifies Clotrimazole as targeting a metabolic vulnerability in a melanoma model

Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies.The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.

Key pollen host plants provide balanced diets for wild bee larvae: A lesson for planting flower strips and hedgerows

Abstract Bee‐friendly plants are defined by the quantity of food they produce and the visitation rates of adult insects foraging for nectar. However, it is pollen nutritional quality that enables proper larval development of bees, affecting their populations. Not all plants produce pollen that satisfies the nutritional requirements of bee larvae, and we lack an understanding of how different plant pollens impact bee nutritional demands. This study examined whether nutritionally desirable key plant species may promote wild bee larval development, which is essential if the population is to thrive. The generalist solitary mason bee Osmia bicornis L. was used as a model species to examine differences between bee larva nutritional demand and host plant nutrient supply; an ecological stoichiometry framework was applied. The stoichiometric ratios of 12 elements were investigated in bee bodies and cocoons (reflecting nutritional demand) and in the pollen supplied by the mother (nutritional supply; N = 15 × 2 sexes). Similarly, the stoichiometry of 62 pollen taxa, including native, alien, and garden plants and crops, was compared with the bee demand based on the literature. Compared to males, females had higher demands for P, Cu and Zn and were supplied with pollen richer in these elements. Therefore, when collecting pollen for their progeny, Osmia provides daughters and sons with different pollen mixtures, reflecting sex‐specific nutritional demand. Bees may be limited by the availability of P, Na, Mn, Mg, K, Fe, Ca, Zn and Cu, with high taxonomic variability in their concentrations in pollen. Female fitness may be particularly related to a high proportion of P in the diet. Synthesis and applications. Access to key plant species that allow nutritionally balanced larval diets may be essential for bee development, whether food is gathered intentionally or randomly. Such plant species—and not only those rich in nectar and pollen—should be promoted in wild bee conservation efforts, including planting flower strips and hedgerows. Bee‐friendly plants should not be defined and planted solely based on the quantities of food they produce and on the visitation rates of adult insects foraging for energy.

Hair Cell Development and Regeneration in Zebrafish Lacking the Cannabinoid Receptor2

Hair cells (HCs) are mechanosensory receptors found in sensory epithelia in the vertebrate inner ear that detect sound and head movements. HCs are fragile and prone to damage from various causes like disease, aging, and noise pollution. Unlike mammals, lower vertebrates including birds, amphibians and fish can regenerate HCs. To understand the underlying molecular mechanisms of HCs regeneration, we study the lateral line (LL) in a powerful genetic and regenerative animal model: the zebrafish. The LL is a skin‐deep fish sensory organ that is evolutionarily linked to the vertebrate sensory epithelia of the inner ear. It is composed of stereotypical distributed sensory patches called neuromasts (NMs) which contain HCs that are highly similar in function, cellular structure and gene expression to HCs of the inner ear. LL‐HCs are deflected and activate by surrounding water movements. Using CRISPR‐Cas9 genome editing technology, our laboratory has previously generated a null allele of the cnr2 gene (cnr2upr1) which is encoding the cannabinoid receptor 2 (CB2). We found that cnr2 mRNA is highly expressed in LL‐HCs. So, we hypothesized that CB2 has a role in the development and the regeneration of LL‐HCs. We counted LL‐HCs at different developmental stages in wild type and homozygote mutant larvae (cnr2upr1/upr1) to see if their number is affected in the absence of a functional CB2 protein. Next, to trigger vigorous HCs regeneration, we synchronously ablated LL‐HCs by copper treatment of 5‐day post‐fertilization (dpf) wild type and homozygote animals. We counted regenerating LL‐HCs for 3‐day post‐treatment. We will present our preliminary results that will ultimately allow us to conclude on the involvement of CB2 in development and in regeneration of the LL and on a potential link between endocannabinoid signaling and sensory epithelia development and regeneration.Support or Funding InformationCRESTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sequencing of an npc1L1 mutation in larval zebrafish.

How the intestinal absorption of lipids impact dyslipidemias such as obesity and diabetes is currently under debate. NPC1L1 is a critical transport protein in enterocyte cholesterol absorption. To understand the mechanism of intestinal NPC1L1 uptake within the enterocyte, we are creating a npc1l1 -/- (knockout) line in zebrafish. We hypothesize that the npc1l1 -/- larval zebrafish will show reduced intestinal cholesterol absorption when challenged to a high fat diet compared to wild type larvae. To do this, we are screening two genomic mutations within the NPC1L1 sequence. These zebrafish lines were obtained from the Zebrafish International Resource Center. We have amplified the DNA fragments containing the mutation. We will then sequence the DNA fragments at the Marshall Genomics Core. Sequences from point mutations will be compared with wild type sequences. Identified carriers of the mutation will be outcrossed to eliminate background mutations and intercrossed to breed for homozygous carriers. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.

The epg5 knockout zebrafish line: a model to study Vici syndrome

ABSTRACT The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5 -/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5 -/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5 -/- mutants. By aging, epg5 -/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5 -/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Effects of releasing two Diachasmimorpha longicaudata population lines for the control of Ceratitis capitata infesting three key host fruit species

Exotic plants favor persistence and spread of the invasive medfly, Ceratitis capitata. Peach and orange are key host plants for medfly proliferation in Argentina. Consequently, actions to suppress medfly populations are taken, especially those performing augmentative releases of parasitoids. This study provides information on the capability of two population lines of the parasitoid Diachasmimorpha longicaudata to control medfly infesting the fruits of peach, as well as sour and sweet orange. One parasitoid line comes from non-irradiated larvae of wild medfly. The other comes from irradiated larvae of the Temperature Sensitive Lethal Vienna-8 medfly strain. The parasitoid host-finding ability in each aforementioned fruit species, the effectiveness of females to kill medfly larvae, the fruit height level preference for parasitoid foraging activity, and the influence of environmental conditions on parasitoid performance were compared and assessed. Parasitoids foraged for 48 h on fruits artificially inoculated with wild medfly larvae in field cages. Females of both parasitoid lines showed a similar effectiveness pattern, foraged efficiently on fruit at ground and canopy levels, and were able to overcome local climate conditions and to develop at least one new generation under natural environmental conditions. These outcomes may provide relevant information for the implementation of augmentative biological control against medfly.

The genetic population structure ofThunnus thynnus(Linnaeus, 1758) in the Mediterranean Sea, a controversial issue

The Atlantic bluefin tuna (ABFT), Thunnus thynnus (Linnaeus, 1758), is an important commercial species managed as two different stocks, western and eastern Atlantic, with their spawning grounds in the Gulf of Mexico and in the Mediterranean Sea, respectively. The eastern Atlantic stock has been overexploited in the last decades, leading to the application of specific management measures introduced by the International Commission for the Atlantic Tuna (ICCAT). A clear understanding of the genetic structure of ABFT Mediterranean population should be pursued in order to support management decisions. To date the genetic studies on the Mediterranean ABFT, carried out with different molecular markers and sampling procedures, have produced unclear results. Here, we analysed ABFT samples from central and western Mediterranean Sea with mitochondrial sequences and 11 microsatellite loci to investigate, among the others, the area of the Strait of Messina, where environmental conditions seem to support a resident population of ABFT. Furthermore, genetic analyses of mitochondrial sequences were carried out including nucleotide sequences of Adriatic ABFT wild larvae retrieved from GenBank. Among the investigated areas a genetic differentiation was detected between the Strait of Messina and the Tyrrhenian Sea with microsatellite loci according to the exact G test, but not to the Bayesian analyses carried out with STRUCTURE. The analyses with mitochondrial sequences do not reveal any differentiation among sampled areas, however, a highly significant genetic divergence was observed between the Adriatic mitochondrial sequences retrieved from GenBank and the central‐western Mediterranean sequences obtained in the present work. Our results provide some evidence of population structure of Mediterranean ABFT adding pieces to a still unclear picture.

Evidence of density-dependent cannibalism in the diet of wild Atlantic bluefin tuna larvae (Thunnus thynnus) of the Balearic Sea (NW-Mediterranean)

The heavy exploitation rates of Atlantic bluefin tuna (ABFT) during the nineties propitiated research into the larval ecology of ABFT and its associated species. The transition from a planktivorous to a piscivorous diet is considered a major bottleneck in the survival of ABFT larvae observed in aquaculture experiments. Although larval piscivory was reported in the Gulf of Mexico (GOM), the most important spawning grounds of this species in the W Atlantic, trophic studies have not been able to reveal piscivory in Mediterranean ABFT larvae. This study analyzes the trophic behavior of Mediterranean ABFT larvae by stomach content analysis. The results show that more than 90% of the larvae had at least one prey in their stomachs. The diet shifted from copepods and cladocerans to gastropod larvae in pre-flexion stages and to ABFT larvae in post-flexion stages. This is the first time that cannibalism is reported for wild ABFT larvae from the Mediterranean Sea. Intracohort cannibalistic feeding was observed when the requisite density-dependent processes aligned, namely the spatio/temporal overlap of a wide range of ABFT larval cohorts of different size class. Moreover, stomach contents of ABFT larvae revealed the ingestion of microplastic fibres. Whether these plastic contaminants were passively or actively ingested, they may affect the condition of larvae. The presence of microplastic strands in fish larvae undoubtedly raises concern because its impact on the survival of ABFT larvae still remains uncertain and is open to scientific experimentation.

Development of a PCR-RFLP assay to identify Drosophila melanogaster among field-collected larvae.

The fruit fly Drosophila melanogaster is a model organism to study several aspects of metazoan biology. Most of the work has been conducted in adult fruit flies, including laboratory and field‐derived specimens, but Drosophila melanogaster larvae recently became a valuable model to better understand animal physiology, development, or host–microbe interactions. While adult flies can be easily assigned to a given Drosophila species based on morphological characteristics, such visual identification is more intricate at the larval stage. This could explain the limited number of studies focusing on larvae, especially field‐derived samples. Here, we developed a polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay that discriminates D. melanogaster from other ecologically relevant Drosophila species at the larval stage. The method, which targets the cytochrome oxidase I (COI) gene, was validated using laboratory‐derived larvae from seven D. melanogaster populations originating from different geographic areas as well as six Drosophila species. We further validated this PCR‐RFLP assay in a natural context, by identifying wild larvae collected in two locations in France. Notably, among all PCR‐RFLP profiles that matched the D. melanogaster species, 100% were correctly identified, as confirmed by COI sequencing. In summary, our work provides a rapid, simple, and accurate molecular tool to identify D. melanogaster from field‐collected larvae.

A Simplified Gas Chromatographic Fatty-Acid Analysis by the Direct Saponification/Methylation Procedure and Its Application on Wild Tuna Larvae.

A method for the direct preparation of fatty-acid methyl esters (FAME) was simplified for fatty-acid analysis of a single fish larva using gas chromatography (GC). The method included the isolation of a larval trunk and drying in a glass vial, followed by saponification of all the contents without prior lipid extraction. Thereafter, the fatty acids released were methylated by trimethylsilyldiazomethane. This method has advantages over another method, direct acid-catalyzed transesterification, because both the saponification and methylation at room temperature can reduce loss of unsaturated fatty acids and formation of artifacts unavoidable in acidic reaction at high temperature. GC of the products showed that the simplified method can yield methyl esters without artifacts interfering analysis. More than 50 fatty acids were determined, which are twice as many as those previously analyzed using high-performance liquid chromatography. Observation of consistent small impurities in GC of blank tests allowed the accurate determination of fatty acids by correcting the peak areas. Dry matter weights (<3 mg) and the total fatty-acid contents displayed a linear relationship. Fatty-acid analysis of wild larvae of bluefin tuna, yellowfin tuna, and skipjack tuna collected from the waters around Japan (n = 100) revealed that the eicosapentaenoic acid (EPA) level in bluefin tuna collected from the Japan Sea was significantly higher than that in the three species collected from Nansei Islands. The simplified direct saponification/methylation method will be a powerful tool for investigating growth and survival of individual larval tuna and other fish species.

Daily growth patterns and age-at-recruitment of the anchoveta Engraulis ringens as indicated by a multi-annual analysis of otolith microstructure across developmental stages.

The anchoveta (Engraulis ringens) plays a key role in the ecology of the Humboldt Current System and is of major economic importance; however, many aspects of its early life history are still poorly understood. In this study, an analysis of daily age and length patterns was carried out using the sagittal otoliths from wild larvae (0-0.2 cm standard length, LS ), pre-recruits (3-6 cm total length, LT ), recruits (7-12 cm LT ) and young adults (12-15 cm LT ). Additionally, variability in growth and age at recruitment (AR ) were evaluated for recruits caught in northern Chile in 1973, 1982, 2009, 2010, 2012, 2013, 2014 and 2015. The age-length relationship showed four allometric patterns that were well described by Laird-Gompertz models. The absolute growth rates at the inflexion point (GAR ) were 0.56, 0.75, 1.22 and 1.16 mm d-1 for larvae, pre-recruits, recruits and young adults, respectively. At the interannual scale, GAR values were always >1 mm d-1 (mean ± S.D. 1.37 ± 0.21 mm d-1 ; range 1.12-1.64 mm d-1 ), irrespective of the season of hatching (i.e. winter v. spring); additionally, in most cases, GAR values were reached before the second month of life (mean ± S.D. 50.47 ± 9.73 days) at c. 4 cm LT (mean ± S.D. 4.22 ± 0.29 cm). Mean AR was < 150 days (112 ± 29 days; range 75-149 days); in contrast, estimates of AR were higher and growth rates were lower in 1973, 1983 and 2000. These results demonstrate very fast growth and early AR of anchoveta in northern Chile, suggesting most fish are removed by the fisheries at very early ages. An evaluation of the implications of these results on stock assessment and management of this species is highly recommended.

Parasitoids associated with codling moth (Cydia pomonella) in apple-growing regions in New Zealand

Codling moth (Cydia pomonella) (CM) is a major pest of apples in New Zealand. Several biocontrol agents introduced in the past to control CM have only been partially successful at reducing CM populations, so a parasitoid wasp, Mastrus ridens (Hymenoptera: Ichneumonidae), was recently released into apple-growing regions. This study sought evidence of the establishment of CM parasitoids. Corrugated cardboard bands (2-cm wide bands with sentinel CM larvae and 10-cm wide empty bands to trap wild CM larvae) were used to assess the presence of M. ridens and other CM parasitoids in Hawke’s Bay, Nelson, Central Otago and Waikato regions. Five CM parasitoid species, including M. ridens, were recovered from sentinel and wide bands. Liotryphon caudatus (Hymenoptera: Ichneumonidae, released to control CM in 1906) was found in Hawke’s Bay and Waikato. Ascogaster quadridentata (Hymenoptera: Braconidae, released to control CM in the 1930s), Glabridorsum stokesii (Hymenoptera: Ichneumonidae, released in 1967 to control light brown apple moth), Dibrachys microgastri (Hymenoptera: Chalcidoidea: Pteromalidae, an accidental arrival) and M. ridens were found in all regions. The interspecific competition between M. ridens and other parasitoid species remains to be investigated.

Syndecan Affects Odor Response as well as Learning and Memory in Drosophila melanogaster

Syndecan (Sdc) is a transmembrane heparan sulfate proteoglycan that plays a crucial role in axon guidance and synapse formation during CNS development in Drosophila melanogaster. To further examine the effect of syndecan on CNS function, Sdc23 mutant D. melanogaster larvae were used to examine odor preference and the capacity for learning and memory. A series of olfaction assays in both wild type and mutant larvae were performed to characterize naive odor responses before adding a training period to identify the capacity for associative learning. These results showed that Sdc23 larvae prefer odors that wild type larvae do not respond to, suggesting a difference in odor receptor pathways and wiring. In addition, associative learning has been documented in wild type larvae, yet no evidence of associative learning in Sdc23 larvae was found, suggesting that the syndecan also plays a role in learning and memory in D. melanogaster larvae. KEYWORDS: Syndecan; Proteoglycans; Neurodevelopment; Axon Guidance; Olfaction; Attraction Index; Associative Learning; Drosophila

Near full-length 16S rRNA gene next-generation sequencing revealed Asaia as a common midgut bacterium of wild and domesticated Queensland fruit fly larvae

BackgroundGut microbiota affects tephritid (Diptera: Tephritidae) fruit fly development, physiology, behavior, and thus the quality of flies mass-reared for the sterile insect technique (SIT), a target-specific, sustainable, environmentally benign form of pest management. The Queensland fruit fly, Bactrocera tryoni (Tephritidae), is a significant horticultural pest in Australia and can be managed with SIT. Little is known about the impacts that laboratory-adaptation (domestication) and mass-rearing have on the tephritid larval gut microbiome. Read lengths of previous fruit fly next-generation sequencing (NGS) studies have limited the resolution of microbiome studies, and the diversity within populations is often overlooked. In this study, we used a new near full-length (> 1300 nt) 16S rRNA gene amplicon NGS approach to characterize gut bacterial communities of individual B. tryoni larvae from two field populations (developing in peaches) and three domesticated populations (mass- or laboratory-reared on artificial diets).ResultsNear full-length 16S rRNA gene sequences were obtained for 56 B. tryoni larvae. OTU clustering at 99% similarity revealed that gut bacterial diversity was low and significantly lower in domesticated larvae. Bacteria commonly associated with fruit (Acetobacteraceae, Enterobacteriaceae, and Leuconostocaceae) were detected in wild larvae, but were largely absent from domesticated larvae. However, Asaia, an acetic acid bacterium not frequently detected within adult tephritid species, was detected in larvae of both wild and domesticated populations (55 out of 56 larval gut samples). Larvae from the same single peach shared a similar gut bacterial profile, whereas larvae from different peaches collected from the same tree had different gut bacterial profiles. Clustering of the Asaia near full-length sequences at 100% similarity showed that the wild flies from different locations had different Asaia strains.ConclusionsVariation in the gut bacterial communities of B. tryoni larvae depends on diet, domestication, and horizontal acquisition. Bacterial variation in wild larvae suggests that more than one bacterial species can perform the same functional role; however, Asaia could be an important gut bacterium in larvae and warrants further study. A greater understanding of the functions of the bacteria detected in larvae could lead to increased fly quality and performance as part of the SIT.

A Host-Pathogen Interaction Screen Identifies ada2 as a Mediator of Candida glabrata Defenses Against Reactive Oxygen Species.

Candida glabrata (C. glabrata) forms part of the normal human gut microbiota but can cause life-threatening invasive infections in immune-compromised individuals. C. glabrata displays high resistance to common azole antifungals, which necessitates new treatments. In this investigation, we identified five C. glabrata deletion mutants (∆ada2, ∆bas1, ∆hir3, ∆ino2 and ∆met31) from a library of 196 transcription factor mutants that were unable to grow and activate an immune response in Drosophila larvae. This highlighted the importance of these transcription factors in C. glabrata infectivity. Further ex vivo investigation into these mutants revealed the requirement of C. glabrata ADA2 for oxidative stress tolerance. We confirmed this observation in vivo whereby growth of the C. glabrata Δada2 strain was permitted only in flies with suppressed production of reactive oxygen species (ROS). Conversely, overexpression of ADA2 promoted C. glabrata replication in infected wild type larvae resulting in larval killing. We propose that ADA2 orchestrates the response of C. glabrata against ROS-mediated immune defenses during infection. With the need to find alternative antifungal treatment for C. glabrata infections, genes required for survival in the host environment, such as ADA2, provide promising potential targets.