Analysis of skeletal muscle development in Drosophila.

The Drosophila system has been invaluable in providing important insights into mesoderm specification, muscle specification, myoblast fusion, muscle differentiation, and myofibril assembly. Here, we present a series of Drosophila protocols that enable the researcher to visualize muscle precursors and differentiated muscles, at all stages of development. In doing so, we also highlight the variety of techniques that are used to create these findings. These protocols are directly used for the Drosophila system, and are provided with explanatory detail to enable the researcher to apply them to other systems.

Bithorax complex genes control alary muscle patterning along the cardiac tube of Drosophila.

Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. We found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. Double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles.

Sex change by gene conversion in a Caenorhabditis elegans fog-2 mutant.

Caenorhabditis elegans primarily reproduces as a hermaphrodite. Independent gene conversion events in mutant obligately outcrossing populations of C. elegans [fog-2(lf)] spontaneously repaired the loss-of-function mutation in the fog-2 locus, thereby reestablishing hermaphroditism as the primary means of reproduction for the populations.

Urban habitat evaluation for West Nile virus surveillance in mosquitoes in Albuquerque, New Mexico.

As part of an ongoing mosquito surveillance program, 27 sites in the greater metropolitan Albuquerque area (Bernalillo County, New Mexico) were trapped from May through September 2004. Each site was sampled for 1 night weekly, using a standard CO2-baited Centers for Disease Control and Prevention light trap and a gravid trap. Captured mosquitoes were catalogued by location, species, and date, and selected pools were tested for West Nile virus (WNV) by reverse transcription-polymerase chain reaction. Based on previous surveillance, WNV was already established in the state of New Mexico. Surveillance during 2003, the 1st year of WNV detection in New Mexico mosquitoes, was focused on the bosque forest of the Rio Grande river valley. Surveillance during summer of 2004 was extended to additional areas around the city of Albuquerque, the state's largest population center. In addition to the standard surveillance objectives, a secondary goal was to determine whether foci of WNV activity were detectable in other habitats besides the riparian ecosystem of the Rio Grande, and in other species not previously identified as vectors. There was no demonstrable advantage to extending the traditional trapping area outside of the Rio Grande valley. Sites in the valley area had WNV-positive mosquitoes earlier in the season, and for a longer period than the added sites. In addition, riparian sites had the highest diversity of species, the largest numbers of Culex spp. captured, and the largest proportion of the WNV-positive mosquito pools from the study. Species found in other areas of the metropolitan area were also represented in the valley. Although WNV activity was detected in other areas of the city, its activity began later and ended earlier than in the river valley. We surmise that the greatest benefit to mosquito surveillance could be achieved by focusing on the river valley area.

Comparison of mosquito trapping method efficacy for West Nile virus surveillance in New Mexico.

As part of the West Nile virus surveillance program for the state of New Mexico, 13 sites along the Rio Grande River were sampled for mosquitoes during spring and summer 2003. We evaluated 3 different trapping procedures for their effectiveness at capturing selected species of mosquitoes. The 3 methods used were a dry ice-baited Centers for Disease Control and Prevention (CDC) light trap set 1.5 m above the ground (standard method), a CDC light trap suspended within the forest canopy, and a gravid trap set on the ground. Thirteen sites were sampled for 10 1-night periods biweekly from May through September. The relative numbers of captured Culex tarsalis, Cx. salinarius, Cx. quinquefasciatus, and Aedes vexans as well as the numbers of total recorded captures of all species were compared for each trapping method. Significant differences were observed for each species by location and by trapping method. Culex tarsalis was most commonly caught in canopy or standard CDC traps, especially in cottonwood bosque. Culex salinarius was found most frequently in association with marshy water, and was most often caught in gravid or standard light traps. Culex quinquefasciatus was captured almost exclusively in gravid traps within urban areas. Aedes vexans was primarily sampled in standard CDC light traps and found most frequently in wooded areas near floodplains. With the exception of Cx. Quinquefasciatus, no species was collected significantly more frequently in gravid or canopy traps than in the standard CDC light trap. Our findings do not support altering the methods currently used in New Mexico, namely, the use of 1.5-m CDC light traps and gravid traps. An increased use of gravid traps seems to be warranted in monitoring urban vector populations (specifically Cx. quinquefasciatus and Cx. salinarius) that may be involved in human transmission.

Emergence of West Nile virus in mosquito (Diptera: Culicidae) communities of the New Mexico Rio Grande Valley.

The first appearances of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in New Mexico were reported in late summer to early fall 2002. Several dead birds tested positive for WNV, and 78 equine cases were confirmed. All mosquito pools tested (n = 268) were negative. A statewide surveillance program was launched in May 2003 to study the emergence and spread of this new arbovirus in mosquitoes from the Rio Grande valley. Mosquitoes were trapped at 32 sites along a 750-km stretch of the Rio Grande valley. Sites were trapped for one night either weekly or biweekly, by using CO2-baited CDC light traps and gravid traps. Pools of captured mosquitoes were tested for WNV by reverse transcription-polymerase chain reaction. By mid-July 2003, WNV levels in the mosquito population had reached levels that were detectable by the surveillance program. Positive pools of mosquitoes were found in the Rio Grande valley from mid-July through late September. In total, 75 positive pools were found, from sites throughout the study area. The predominant species infected with WNV in this region were Culex tarsalis (Coquillett) in rural areas, and Culex salinarius (Coquillett) and Culex pipiens quinquefasciatus (Say) in urban areas. There were 202 human cases and 438 equine cases of WNV in New Mexico in 2003, which corresponded well in time with the positive mosquitoes. Our results seemed to be consistent with introduction of WNV in late summer 2002, followed by a period of transmission and amplification cycles between local avian hosts and mosquito vectors.