Optimal hatching conditions of Zophobas atratus (Coleoptera: Tenebrionidae) eggs under various culture conditions

Supermealworm, Zophobas atratus (Coleoptera: Tenebrionidae) is a tropical species used for human consumption and as an ingredient in animal feed. We investigated the hatching characteristics of Z. atratus eggs under varying conditions to optimize artificial rearing conditions. Overall, the average egg weight, width, and length decreased by 42.3%, 3.7%, and 10.1%, respectively, on the 6th day post-oviposition. We varied either temperature, photoperiod, illuminance, or humidity, while the other conditions remained constant. Of the four experimental temperatures—25 °C, 27 °C, 30 °C, and 33 °C–30 °C was associated with the highest hatching rate (91.0%) during days 5–7. Although other experimental conditions did not have a significant effect on hatchability, we were able to elucidate the ideal conditions for improved hatchability. For photoperiod, hatchability was the highest (84%) at 12L/12D, followed by 81.0% at 15L/9D, and 70% at 9L/15D. Among the illuminance conditions tested—600, 1800, and 3000 lx—hatchability was the highest at 1800 lx (82.3% on days 4–6). Among the relative humidity (RH) levels tested—50%, 65%, and 80%—hatchability was the highest at 65% RH (88.5% on days 5–6). Thus, the ideal conditions for maximum hatchability were 30 °C, 12L/12D, 65% RH, and 1800 lx. Under these combined conditions, the hatching rate increased by 93.6%, and the hatching time was reduced by two days.     

Taxonomic revision of the genus Galictis (Carnivora: Mustelidae): species delimitation,morphological diagnosis,and refined mapping of geographical distribution

Although critical for enabling in‐depth evolutionary, ecological, or conservation‐orientated studies, taxonomic knowledge is still scarce for many groups of organisms, including mammals of the order Carnivora. For some of these taxa, even basic aspects such as species limits and geographical distribution are still uncertain. This is the case for the Neotropical mustelid genus Galictis, considered one of the least studied carnivoran genera in the Americas. To address this issue, we performed a comprehensive assessment of morphological and molecular characters to test the number of species within Galictis, and to characterize their distinctiveness and evolutionary history. In addition, we reviewed and consolidated the available information on the taxonomy of this genus, so as to provide a historical framework upon which we could interpret our data. Our analyses demonstrated that two Galictis species can be clearly delimited and diagnosed using metric and nonmetric morphological characters as well as DNA sequences from mitochondrial and nuclear gene segments. On the basis of this clarified species‐level delimitation, we reassessed the geographical range of each Galictis taxon, identifying possible areas of sympatry between them. These results provide a solid taxonomic framework for Galictis, enabling the development of additional studies focusing on this poorly known taxon. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 167 , 449–472.     

Detectability of lesser prairie-chicken leks: A comparison of surveys from aircraft

Lesser prairie-chickens (Tympanuchus pallidicinctus) are traditionally monitored by spring road-based lek surveys and counts of males attending leks. Several weaknesses exist with ground-based monitoring methods such as the bias of restricting surveys to roads, unknown probability of lek detection, and man-hours required to survey large tracts of habitat. We evaluated aerial surveys to locate lesser prairie-chicken leks in Texas and New Mexico using a Cessna 172 airplane (C172), R-22 Beta II helicopter (R-22), and R-44 Raven II helicopter (R-44) during spring 2007–2008. We determined lek activity during surveys with remote cameras placed on leks and cross-referenced time on the photo frame to time on our Global Positioning System flight log. From remote cameras we found that 305 leks were available for detection during survey flights. We determined lek detectability was 32.7% (95% CI = 20.3–47.1%) in the C172, 72.3% (64.50–79.14%) in the R-22, and 89.8% (82.0–95.0%) in the R-44. We created 16 a priori logistic regression models incorporating aircraft platform, distance to lek, survey date, lek size, and lek type to explain lek detection from aerial surveys. Our top ranked model included platform, distance, and lek type (model weight; w_i = 0.288). We had four competitive models and model averaged to draw inferences. Model averaging showed that detectability was generally greatest with the R-44, followed by the R-22, and lowest with the C172, with a slight deviation from this ranking at increased distances. Within our transect width, model averaging also suggested that detectability decreased as distance from the transect to the lek increased during helicopter surveys, and detectability increased as distance from the transect to the lek increased during C172 surveys. Furthermore, man-made leks were more likely to be detected than natural leks and large leks were more likely to be detected than medium or small leks. Aerial surveys effectively locate new leks and monitor lek density, and alleviate weaknesses associated with ground-based monitoring. We recommend using the R-44 to conduct lek surveys while flying at an altitude of 15 m at a speed of 60 km/hr on sunny mornings. © 2011 The Wildlife Society.     

Transient gut retention and persistence of Salmonella through metamorphosis in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)

Aims: This study was undertaken to determine the retention of Salmonella through Alphitobius diaperinus metamorphosis and its contribution, through defecation, to external contamination. Methods and Results: Insects were exposed to a tagged Salmonella enterica and evaluated for external elimination. (i) Each day for 3 weeks, a filter collected frass from a restrained insect for analysis. (ii) Exposed larvae in a closed container were followed through pupation, and newly emerged adults were examined for their retention of marker bacteria. Conclusions: Exposed adults and larvae produced Salmonella‐positive frass for an average of 8 days, ranging from 6 to 11 days and 6 to 12 days, respectively. Nineteen per cent of the larvae carried Salmonella through metamorphosis and eclosion, with 5% of the pupal exuviae being positive as well. Significance and Impact of the Study: Many sources of foodborne pathogens within the poultry production facilities, including reservoir populations, currently go unrecognized. This diminishes the ability of producers to mitigate the transfer of pathogens between animals, humans and the environment. Poultry management standards accept the reutilization of litter. Alphitobius diaperinus survive between flock rotations on the reutilized litter, and it was demonstrated in this study that the Salmonella they carry can survive with them.     

Agonistic action of synthetic analogues of quisqualic acid at the insect neuromuscular junction

One hundred twenty analogues of quisqualic acid were synthesized and assayed on the neuromuscular junction of larva of the mealworm, Tenebrio molitor. Two new agonists for amino acid receptors, L-glutamic acid N-thiocarboxyanhydride (L-GANTA) and DL-hydantoinpropionic acid (DL-HPA), were discovered in this study. L-GANTA and DL-HPA produced muscle membrane depolarization, accompanied by a reduction of the muscle input resistance. The amplitude of excitatory postsynaptic potentials was decreased in the presence of L-GANTA and DL-HPA. The apparent dissociation constants obtained from dose-depolarization plots were 7 x 10^?4 M for L-GANTA and 9 x 10^?4 M for DL-HPA. Some structural constraints imposed on agonists at amino acid receptors on insect muscle were discussed.     

Selection of Beauveria bassiana isolates to control Alphitobius diaperinus

This paper presents results on isolates of the entomopathogenic fungus Beauveria bassiana for Alphitobius diaperinus (Coleoptera: Tenebrionidae) control. Of the 30 isolates tested, four (CG 71, CG 152, UNIOESTE 4, and UNIOESTE 40) resulted in mortality rates >or= 40% within 10 days. These mortality rates could be considered high because of the resistance of these species to B. bassiana. Tests were conducted using these isolates to estimate LC(50), mortality rate over time, vegetative growth, and conidial production on artificial medium and on insects. Isolates CG 71, CG 152, and UNIOESTE 4 showed the best performance and great potential to be used in an integrated management program in poultry farms to control A. diaperinus. Also, the molecular profiles of 12 isolates were analyzed using the RAPD technique. The high-virulence isolates presented a more homogeneous RAPD pattern than the others. Genetic sequencing of the ITS region was performed for one of the virulent isolates (UNIOESTE 4) and compared with sequences deposited at the NCBI database, confirming its taxonomical position as belonging to B. bassiana Clade A.     

Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains

Two cDNAs encoding different acetylcholinesterase (AChE) genes (AdAce1 and AdAce2) were sequenced and analyzed from the lesser mealworm, Alphitobius diaperinus. Both AdAce1 and AdAce2 were highly similar (95 and 93% amino acid identity, respectively) with the Ace genes of Tribolium castaneum. Both AdAce1 and AdAce2 have the conserved residues characteristic of AChE (catalytic triad, intra-disulfide bonds, and so on). Partial cDNA sequences of the Alphitobius Ace genes were compared between two tetrachlorvinphos resistant (Kennebec and Waycross) and one susceptible strain of beetles. Several single nucleotide polymorphisms (SNPs) were detected, but only one non-synonymous mutation was found (A271S in AdAce2). No SNPs were exclusively found in the resistant strains, the A271S mutation does not correspond to any mutations previously reported to alter sensitivity of AChE to organophosphates or carbamates, and the A271S was found only as a heterozygote in one individual from one of the resistant A. diaperinus strains. This suggests that tetrachlorvinphos resistance in the Kennebec and Waycross strains of A. diaperinus is not due to mutations in either AChE gene. The sequences of AdAce1 and AdAce2 provide new information about the evolution of these important genes in insects.     

Pollination biology of two chiropterophilous agaves in Arizona

I studied the pollination biology of two closely related species of agave, Agave palmeri and A. chrysantha (Agavaceae), which exhibit several chiropterophilous (bat-pollinated) traits. Floral studies, floral visitor observations, and pollination studies were conducted over four summers at six different sites to examine floral traits and determine the relative importance of diurnal vs. nocturnal pollinators. Agave chrysantha appears to have developed minor shifts in several floral characters that enhance diurnal pollination. Although floral shifts towards diurnal pollination were fewer in A. palmeri, stigmas were diurnally receptive and copious floral rewards were available in the morning, indicating that some adaptations exist to allow for multiple pollinators. Differences in fruit and seed set between naturally day- and night-pollinated umbels for both species were either not significant or significantly higher in day-pollinated plants. Bats were not important pollinators of A. chrysantha, and the mutualistic relationship between A. palmeri and the lesser long-nosed bat was found to be asymmetric. "Bat-adapted" floral traits appear to be flexible enough to respond to the climatic and pollinator unpredictability experienced by agaves at the northern edge of their distribution. This variability may be a more important factor affecting evolution of floral characters than a particular pollinator.     

Extraction of chitin and chitosan from larval exuvium and whole body of edible mealworm,Tenebrio molitor

The purpose of this study was to investigate the production of chitin and chitosan from both the exuvium and whole body of mealworm (Tenebrio molitor) larvae. Chitin from the exuvium and whole body of T. molitor larvae was chemically extracted with acid and alkali solutions to achieve demineralization (DM) and deproteinization (DP), respectively. The average DM (%) and DP (%) on a dry weight (DW) basis was 32.56 and 73.16% from larval exuvium, and 41.68 and 91.53% from whole body, respectively. To obtain chitosan, chitin particles from the exuvium and whole body of T. molitor larva were heated at various temperatures in different concentrations of NaOH. Average chitin yields were 18.01% and 4.92% of DW from the exuvium and whole body, respectively. The relative average yield of chitosan from whole body was 3.65% of DW. On average, over 90% of chitosan derived from whole body was deacetylated. The viscosity of chitosan from whole body was ranged from 48.0 cP to 54.0 cP. The chitin content of dry and wet byproducts from whole body were 17.32% and 16.94% respectively, compared to dry weight. The chitosan contents of byproducts on a DW basis were 14.48% in dry and 13.07% in wet byproduct. These results indicate that the exuvium and whole body of T. molitor larva may serve as a source of chitin and chitosan for use in domestic animal feed.     

Trehalase in the spermatophore from the bean-shaped accessory gland of the male mealworm beetle,Tenebrio molitor: purification,kinetic properties and localization of the enzyme

Trehalase from the bean-shaped accessory glands of the male mealworm beetle, Tenebrio molitor, was purified by acid treatment, with subsequent chromatography on columns of DEAE-cellulofine and Sephacryl S-300. The molecular masses of the native and the denatured forms were estimated to be 43 and 62 kDa by gel filtration and SDS-PAGE, respectively, an indication that the trehalase may be composed of a single polypeptide. The optimum pH of the reaction catalyzed by trehalase was 5.6–5.8. The K _m for trehalose was 4.4 mmol·l^–1. Immunohistochemical experiments with trehalase-specific antiserum showed that the enzyme was localized in one specific type of secretory cell in the bean-shaped accessory gland epithelium and within the semisolid secretory mass that was a precursor to the wall of spermatophore. SDS-PAGE and immunoblotting analysis revealed the presence of a polypeptide of about 62 kDa in the spermatophore, Immunohistochemical observations showed that the trehalase was located at the outgrowth in the anterior portion of the spermatophore. When a fresh spermatophore was immersed in phosphate-buffered saline it discharged sperm in the same manner as in the bursa copulatrix of the female. Before the rupture of the expanded bulb of the spermatophore, almost all of the trehalase had dissolved in the phosphate-buffered saline. The addition of validoxylamine A to the saline, a specific inhibitor of trehalase, did not affect the expansion and evacuation of the spermatophore. These results demonstrate that trehalase, synthesized by a specific type of secretory cell in the bean-shaped accessory gland epithelium, is actively passed into the lumen of the bean-shaped accessory gland and then incorporated into the spermatophore. Trehalase appears to be one of the structural proteins of the spermatophore, although the possibility can not yet be completely ruled out that the trehalase-trehalose system functions for the nourishment and/or activation of the sperm in the bursa copulatrix of the female.Abbreviations BAG bean-shaped accessory gland(s) - DEAE diethylaminoethyl - Kpi buffer K₂HPO₄/KH₂PO₄ buffer (pH 7.0) - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SDS sodium dodecy sulphate - Spph spermatophore(s) - TAG tubular accessory gland(s)