Methods to determine resistance to anthelmintics when continuing larval development occurs

The in vivo faecal egg count reduction test (FECRT) is the most commonly used test to detect anthelmintic resistance (AR) in gastrointestinal nematodes (GIN) of ruminants in pasture based systems. However, there are several variations on the method, some more appropriate than others in specific circumstances. While in some cases labour and time can be saved by just collecting post-drench faecal worm egg counts (FEC) of treatment groups with controls, or pre- and post-drench FEC of a treatment group with no controls, there are circumstances when pre- and post-drench FEC of an untreated control group as well as from the treatment groups are necessary. Computer simulation techniques were used to determine the most appropriate of several methods for calculating AR when there is continuing larval development during the testing period, as often occurs when anthelmintic treatments against genera of GIN with high biotic potential or high re-infection rates, such as Haemonchus contortus of sheep and Cooperia punctata of cattle, are less than 100% efficacious. Three field FECRT experimental designs were investigated: (I) post-drench FEC of treatment and controls groups, (II) pre- and post-drench FEC of a treatment group only and (III) pre- and post-drench FEC of treatment and control groups. To investigate the performance of methods of indicating AR for each of these designs, simulated animal FEC were generated from negative binominal distributions with subsequent sampling from the binomial distributions to account for drench effect, with varying parameters for worm burden, larval development and drench resistance. Calculations of percent reductions and confidence limits were based on those of the Standing Committee for Agriculture (SCA) guidelines. For the two field methods with pre-drench FEC, confidence limits were also determined from cumulative inverse Beta distributions of FEC, for eggs per gram (epg) and the number of eggs counted at detection levels of 50 and 25. Two rules for determining AR: (1) %reduction (%R) < 95% and lower confidence limit <90%; and (2) upper confidence limit <95%, were also assessed. For each combination of worm burden, larval development and drench resistance parameters, 1000 simulations were run to determine the number of times the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been declared. When continuing larval development occurs during the testing period of the FECRT, the simulations showed AR should be calculated from pre- and post-drench worm egg counts of an untreated control group as well as from the treatment group. If the widely used resistance rule 1 is used to assess resistance, rule 2 should also be applied, especially when %R is in the range 90 to 95% and resistance is suspected.

Increased attractiveness of honeybee hive product volatiles to adult small hive beetle, Aethina tumida, resulting from small hive beetle larval infestation

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a recent but significant pest of honeybee Apis mellifera L. (Hymenoptera: Apidae) hives in various regions throughout the world, including Eastern Australia. The larval stage of this beetle damages hives when they feed on brood, pollen, and honeycomb, leaving behind fermented wastes. In cases of extreme damage, hives collapse and are turned to an odorous mass of larvae in fermenting hive products. The yeast Kodamaea ohmeri (Etchells & Bell) Yamada et al. (Ascomycota) has been consistently isolated from the fermenting material as well as each life stage of this beetle. Various studies have noted that the small hive beetle is attracted to volatiles from hive products and those of the yeast K. ohmeri, although earlier studies have not used naturally occurring hive products as their source of fermentation. This study investigated changes through time in the attractiveness of natural honeybee hive products to the small hive beetle as the hive products were altered by the action of beetle larvae and fermentation by K. ohmeri. We used gas chromatography-mass spectrometry and choice-test behavioural assays to investigate these changes using products sampled from three apiaries. Attractiveness of the fermenting hive products (‘slime’) increased as fermentation progressed, and volatile profiles became more complex. Fermenting hive products remained extremely attractive for more than 30 days, significantly longer than previous reports. These results have strong implications for the development of an external attractant trap to assist in the management of this invasive pest.

Characterisation of the DNA-polymerase-alpha-primase complex from the silk glands of Bombyx mori

Silk gland cells ofBombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle silk gland nuclei increased by 300000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase with no detectable level of polymerase . DNA polymerase from silk gland extracts was purified to homogeneity (using a series of columns involving ionexchange, gel-filtration and affintiy chromatography), resulting in a 4000-fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180-kDa subunit. The enzyme shows a PI of 6.2 and theKm values for the dNTP vary over 5-16 . The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single-stranded DNA template. The primase activity is resident in the 45-kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase α in silk glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

The role of larval nutrition in pre-imaginal biasing of caste in the primitively eusocial wasp Ropalidia marginata (Hymenoptera: Vespidae)

1. When freshly eclosed females of the primitively eusocial wasp Ropalidia marginata (Lep.) are isolated, only about 50% of them build nests and lay eggs thereby suggesting a pre-imaginal biasing of caste. 2. Wasps that lay eggs take a very variable amount of time after eclosion to start doing so. 3. Females eclosing from nests where larvae are fed at a relatively higher rate are more likely to become egg-layers and are likely to take less time after eclosion to begin to lay eggs. 4. Thus, both forms of pre-imaginal biasing of caste, namely, differences in egg laying capacity and differences in the time taken to attain reproductive maturity, appear to be influenced by larval nutrition.

DNA polymerase alpha-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini.

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.

Identification of larval sea basses (Centropristis spp.) using ribosomal DNA-specific molecular assays

The identification of sea bass (Centropristis) larvae to species is difficult because of similar morphological characters, spawning times, and overlapping species ranges. Black sea bass (Centropristis striata) is an important fishery species and is currently considered to be overfished south of Cape Hatteras, North Carolina. We describe methods for identifying three species of sea bass larvae using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays based on species-specific amplification of rDNA internal transcribed spacer regions. The assays were tested against DNA of ten other co-occurring reef fish species to ensure the assay's specificity. Centropristis larvae were collected on three cruises during cross-shelf transects and were used to validate the assays. Seventy-six Centropristis larva were assayed and 69 (91%) were identified successfully. DNA was not amplified from 5% of the larvae and identification was inconclusive for 3% of the larvae. Those assays can be used to identify sea bass eggs and larvae and will help to assess spawning locations, spawning times, and larval dispersal.

Relationships between Larval and Juvenile Abundance of Winter-Spawned Fishes in North Carolina, USA

We analyzed the relationships between the larval and juvenile abundances of selected estuarine-dependent fishes that spawn during the winter in continental shelf waters of the U.S. Atlantic coast. Six species were included in the analysis based on their ecological and economic importance and relative abundance in available surveys: spot Leiostomus xanthurus, pinfish Lagodon rhomboides, southern flounder Paralichthys lethostigma, summer flounder Paralichthys dentatus, Atlantic croaker Micropogonias undulatus, and Atlantic menhaden Brevoortia tyrannus. Cross-correlation analysis was used to examine the relationships between the larval and juvenile abundances within species. Tests of synchrony across species were used to find similarities in recruitment dynamics for species with similar winter shelf-spawning life-history strategies. Positive correlations were found between the larval and juvenile abundances for three of the six selected species (spot, pinfish, and southern flounder). These three species have similar geographic ranges that primarily lie south of Cape Hatteras. There were no significant correlations between the larval and juvenile abundances for the other three species (summer flounder, Atlantic croaker, and Atlantic menhaden); we suggest several factors that could account for the lack of a relationship. Synchrony was found among the three southern species within both the larval and juvenile abundance time series. These results provide support for using larval ingress measures as indices of abundance for these and other species with similar geographic ranges and winter shelf-spawning life-history strategies.

Development of kelp rockfish, Sebastes atrovirens (Jordan and Gilbert 1880), and brown rockfish, S. auriculatus (Girard 1854), from birth to pelagic juvenile stage, with notes on early larval development of black-and-yellow rockfish, S. chrysomelas (Jordan and Gilbert 1880), reared in the laboratory (Pisces: Sebastidae).

Larval kelp (Sebastes atrovirens), brown (S. auriculatus), and blackand-yellow (S. chrysomelas) rockfish were reared from known adults, to preflexion stage, nine days after birth for S. chrysomelas, to late postflexion stage for S. atrovirens, and to pelagic juvenile stage for S. auriculatus. Larval S. atrovirens and S. chrysomelas were about 4.6 mm body length (BL) and S. auriculatus about 5.2 mm BL at birth. Both S. atrovirens and S. auriculatus underwent notochord flexion at about 6–9 mm BL. Sebastes atrovirens transform to the pelagic juvenile stage at about 14–16 mm BL and S. auriculatus transformed at ca. 25 mm BL. Early larvae of all three species were characterized by melanistic pigment dorsally on the head, on the gut, on most of the ventral margin of the tail, and in a long series on the dorsal margin of the tail. Larval S. atrovirens and S. auriculatus developed a posterior bar on the tail during the flexion or postflexion stage. In S. atrovirens xanthic pigment resembled the melanistic pattern throughout larval development. Larval S. auriculatus lacked xanthophores except on the head until late preflexion stage, when a pattern much like the melanophore pattern gradually developed. Larval S. chrysomelas had extensive xanthic pigmentation dorsally, but none ventrally, in preflexion stage. All members of the Sebastes subgenus Pteropodus (S. atrovirens, S. auriculatus, S. carnatus, S. caurinus, S. chrysomelas, S. dalli, S. maliger, S. nebulosus, S. rastrelliger) are morphologically similar and all share the basic melanistic pigment pattern described here. Although the three species reared in this study can be distinguished on the basis of xanthic pigmentation, it seems unlikely that it will be possible to reliably identify field-collected larvae to species using traditional morphological and melanistic pigmentation characters. (PDF file contains 36 pages.)

Guide to the Identification of Larval and Early Juvenile Poachers (Scorpaeniformes: Agonidae) from the Northeastern Pacific Ocean and Bering Sea

Developmental stages of 22 species representing 16 genera of agonid fishes occurring in the northeastern Pacific Ocean from San Francisco Bay to the Arctic Ocean are presented. Three of these species also occur in the North Atlantic Ocean. Larval stages of nine species are described for the first time. Additional information or illustrations intended to augment original descriptions are provided for eight species. Information on five other species is provided from the literature for comparative purposes. The primary objective of this guide is to present taxonomic characters to help identify the early life history stages of agonid fishes in field collections. Meristic, morphometric, osteological, and pigmentation characters are used to identify agonid larvae. Meristic features include numbers of median-fin elements, pectoral-fin rays, dermal plates, and vertebrae. Eye diameter, body depth at the pectoral-fin origin, snout to first dorsal-fin length, and pectoral-fin length are the most useful morphological characters. Presence, absence, numbers, and/or patterns of dermal plates in lateral rows or on the ventral surface of the gut are also useful. Other important characters are the presence, absence, numbers, and ornamentation of larval head spines. Lastly, distinct pigmentation patterns are often diagnostic. The potential utility of larval characters in phylogenetic analysis of the family Agonidae is discussed. (PDF file contains 92 pages.)

Larval Fish Recruitment and Research in the Americas: Proceedings of the Thirteenth Annual Larval Fish Conference Merida, Mexico, 21-26 May 1989

The 13th Annual Larval Fish Conference and Annual Meeting of the American Fisheries Society Early Life History Section cohosted by Mote Marine Laboratory, United States, and the Instituto Nacional de la Pesca, Mexico, were held 21-26 May 1989, in Merida, Yucatan, Mexico. The purpose of holding the meeting in Mexico was to encourage the participation of our Latin American and Caribbean colleagues and to provide a forum for the exchange of ideas and information among researchers working in the Americas. More than 150 participants represented 24 U.S. states, the District of Columbia, and 13 foreign countries including Mexico, Canada, Puerto Rico, Costa Rico, Panama, Cuba, Columbia, Chile, Peru, Brazil, Argentina, South Africa, and West Germany. The Conference began with registration and a social in the courtyard patio of the Merida Holiday Inn. Fresh red grouper, the most important commercial finfish species of the State of Yucatan, was prepared and served by the hotel staff, courtesy of CPI, Itzamex, and the Terramar Trading Company. (PDF file contains 146 pages.)

The feeding ecology of some zooplankters that are important prey items of larval fish

Diets of 76 species of fish larvae from most oceans of the world were inventoried on the basis of information in 40 published studies. Although certaln geographlc, size- and taxon-specific patterns were apparent, certain zooplankton taxa appeared in the diets of larvae of a variety of fish species in numerous localities. Included were six genera of calanoid copepods (Acartia, Calanus, Centropages, Paracalanus, Pseudocaianus, Temora), three genera of cyclopoid copepods (Corycaeus, Oilhona, Oncata), harpacticoid copepods, copepod nauplii, tintinoids, cladocerans of the genera Evadne and Podon, barnacle nauplii, gastropod larvae, pteropods of the genus Limacina, and appendicularians. Literature on feeding habits of these zooplankters reveals that most of the copepods are omnivorous, feeding upon both phytoplankton and other zooplankton. Some taxa, such as Calanus, Paracalanus, Pseudocalanus, and copepod nauplii appear to be primarily herbivorous, while others, such as Acartia, Centropages, Temora, and cyclopoids exhibit broad omnivory or carnivory. The noncopepod zooplankters are primarily filter-feeders upon pbytoplankton and/or bacterioplankton. Despite the importance of zooplankters in larval fish food webs, spectic knowledge of the feeding ecology of many taxa is poor. Further, much present knowledge comes only from laboratory investigations that may not accurately portray feeding habits of zooplankters in nature. Lack of knowledge of the feeding ecology of many abundant zooplankters, which are also important in larval fish food webs, precludes realistic understanding of pelagic ecosystem dynamics. (PDF file contains 34 pages.)

The distribution, abundance, and ecology of larval tunas from the entrance to the Gulf of California

ENGLISH: This study is based on collections of larvae of Thunnus albacares, Euthynnus llneatus, and Auxis sp. obtained from surface and oblique net tows made during seven cruises, each along a comparable track in the entrance of the Gulf of California and each during a different month. Concomitant measurements of surface temperature, salinity, and zooplankton were made at each of the plankton stations. The catches of larval Auxis sp. were examined by analysis of variance techniques to determine which environmental features were associated with the spawning of this tuna as indicated by the distribution of larvae and to gain some insight into the behavior of the larvae themselves. The testing indicated that the spawning of Auxis sp. varied significantly among the different months of the cruises. The testing also indicated that if the larvae were capable of avoiding the sampling apparatus, this ability was not related to features associated with time of day such as light conditions. The analysis did not detect any change in the vertical distribution of the larvae among the months of the experiment. It was concluded that the larvae did not exhibit a diel vertical movement. The measurements of temperature, salinity, and zooplankton volumes were treated as covariates in the analysis. The surface temperature proved to be a highly important factor in explaining the distribution of larvae, but salinity and zooplankton volumes were not. Catches of Thunnus albaeares and Euthynnus lineatus were rare during the course of the study; these are discussed in qualitative terms with respect to the time of the year and the surface temperature. The distribution of larval tunas in the area of study was compared with the distribution of surface water masses. It appeared that these masses had no influence per se on the distribution of larvae. SPANISH: Este estudio está basado en las recolecciones de larvas de Thunnus albacares, Eutbynnus lineatus, y Auxis sp. obtenidas según los arrastres superficiales y oblicuos de la red, realizados durante siete cruceros, cada uno a la entrada del Golfo de California a lo largo de un derrotero comparable, y cada uno durante distintos meses. Las mediciones correspondientes de la temperatura superficial, salinidad y de zooplancton se realizaron en cada una de las estaciones de plancton. Las capturas de larvas Auxís sp. fueron examinadas mediante el análisis de la varianza para determinar cuales características ambientales se encontraban asociadas con el desove de este atún según lo indicaba la distribución de las larvas, y para obtener alguna idea del comportamiento de las larvas en sí mismas. Las pruebas indicaron que el desove de Auxis sp. varió significativamente entre los diferentes meses de los cruceros; indicaron también que si las larvas eran capaces de evitar el aparato de muestreo, esta habilidad no se relacionaba a las características asociadas con la hora del día de acuerdo a las condiciones de luz. El análisis no demostró ningún cambio en la distribución vertical de las larvas durante los meses del experimento. Se determinó que las larvas no exhiben un movimiento vertical diario. Las mediciones de temperatura, salinidad, y de los volúmenes de zooplancton fueron tratadas como covariables en el análisis. La temperatura superficial demostró ser un factor altamente importante en la explicación de la distribución de las larvas, pero la salinidad y los volúmenes de zooplancton no lo fueron. Las capturas de Thunnus albacares y Eutbynnus lineatus fueron pocas durante el curso de este estudio; éstas se discuten en términos cualitativos respecto a la época del año y a la temperatura superficial. La distribución de los atunes larvales en el área de estudio fue comparada con la distribución de las masas superficiales de agua. Parece que estas masas no tienen influencia per se en la distribución de las larvas. (PDF contains 40 pages.)

Post-larval feeding of Clarias gariepinus (Burchell, 1802) on cultured zooplankton and artemia diets

The findings are presented of a study undertaken to provide baseline information that may be employed to develop a simplified hatchery technique for postlarval feeding of Clarias gariepinus on zooplankton cultured indoors in small aquaria. Three series of experiments were undertaken to identify the type and quantity of organic manure that would produce maximum densities of zooplankton in the shortest time in indoor cultures. Results suggest the possibility of the culture of natural zooplankton populations for use in larval feeding of C. gariepinus fry. Its effective hatchery application would bring about reduction in the unit cost per fingerling and ensure continuous hatchery operations even in the absence of Artemia cysts

Validation of daily growth increments and estimation of growth rates of larval and early juvenile black skipjack, Euthynnus lineatus, using otoliths

ENGLISH: Increments in otoliths (sagittae) were examined, using light and scanning electron microscopy, to determine ages and estimate growth rates of larval and early-juvenile black skipjack, Euthynnus lineatus. Larvae and juveniles were collected between 1987 and 1989 from coastal waters of Panama in the eastern Pacific Ocean. Results from a laboratory experiment indicated that immersion for 6 and 12 hours in a 200 mg/L solution of tetracycline hydrochloride adequately marks otoliths and that increments are formed daily in the sagittae of postflexion larvae and early juveniles. Further, survival rates of tetracycline-treated fish were not significantly different from those of control fish. Growth rates were derived from length-age relationships of 218 field-collected specimens ranging in size from 5.7 to 20.3 mm SL. A growth rate of 0.70 mm/d was estimated from the weighted regression of standard length on age for all specimens. This rate lies within the range reported for larvae and early juveniles of other species of subtropical and tropical scombrids. Growth rates of postflexion larvae and early juveniles were not significantly different between the rainy season in July-August 1988 and the dry, upwelling season in January-February 1989. Growth was, however, significantly more variable for older individuals in July-August than in January-February, and may correspond, in part, to seasonal patchiness of prey. The growth rates of the otoliths relative to fish length were also not significantly different between seasons; however, the otoliths were larger relative to the lengths of fish collected in the rainy season, which may reflect slower growth during earlier larval stages. SPANISH: Se examinaron incrementos en otolitos (ságitas), usando microscopia de luz y de barrido electrónico, a fin de determinar la edad y estimar las tasas de crecimiento de barriletes negros, Euthynnus lineatus, larvales y juveniles tempranos. Entre 1987 y 1989 se capturaron larvas y juveniles en las aguas costeras de Panamá en el Océano Pacífico oriental. Los resultados de un experimento de laboratorio indicaron que una inmersión de 6 a 12 horas de duración en una solución de 200 mg/L de hidrocloro de tetraciclina marca los otolitos adecuadamente y que los incrementos se forman a diario en las ságitas de larvas en postflexión y juveniles tempranos. Además, las tasas de supervivencia de los peces tratados con tetraciclina no fueron significativamente diferentes a aquellas de los peces de control. Se calcularon las tasas de crecimiento a partir de las relaciones de talla-edad de 218 especímenes de TE entre 5.7 y 20.3 mm capturados en el mar. Se estimó.una tasa de crecimiento de 0.70 mm/día a partir de la regresión ponderada de talla estándar sobre edad para todos los especímenes. Esta tasa cae dentro del rango reportado para larvas y juveniles tempranos de otras especies de escómbridos subtropicales y tropicales. Las tasas de crecimiento de larvas en postflexión y juveniles tempranos no fueron significativamente diferentes entre la temporada de lluvias en julio-agosto de 1988 y la temporada de sequía y afloramiento en enero-febrero de 1989. Sin emoargo, el crecimiento fue significativamente más variable para los individuos de mayor edad en julio-agosto que en enero-febrero, y quizás corresponda parcialmente a la irregularidad temporal de la abundancia de presas. Las tasas de crecimiento de los otolitos en relación a la talla de los peces tampoco fueron significativamente diferentes entre temporadas; sin embargo, los otolitos eran más grandes en relación a la talla en peces capturados en la temporada de lluvias, lo cual podría reflejar crecimiento más lento durante las etapas larvales más tempranas. (PDF contains 42 pages.)