U.S. GLOBEC: Stage-specific distribution and abundance of the copepods, Pseudocalanus moultoni and P. newmani, on Georges Bank: life history comparisons using molecular identification of sibling species

Ann Bucklin
Ocean Process Analysis Laboratory
University of New Hampshire
Durham, NH 03824

The calanoid copepod, Pseudocalanus sp., is thought to be endemic to Georges Bank: the species reproduces on the Bank and its life history is apparently adapted to ensure retention in the cyclonic gyre patterns of the Bank. A revision of the genus by Frost (1989: Can. J. Zool. 67:525) concluded that two sibling species of Pseudocalanus occur sympatrically on Georges Bank. The species, P. moultoni and P. newmani, differ in geographic distribution and some aspects of their reproductive ecology - but they cannot be reliably distinguished using morphological characteristics. Both species are thought to be present in waters over the Bank throughout much of the year, and may reproduce nearly continuously on the Bank. The primary source of recruitment may be local reproduction of Bank populations, in contrast to another copepod, Calanus finmarchicus, that re-populates the Bank each spring from surrounding waters. The proposed work will use sequence-specific gene amplification by the polymerase chain reaction (PCR) to identify and discriminate all life stages of the two species.

Goals of the proposed research:

1. To determine the temporal and spatial patterns of reproduction of the copepods, Pseudocalanus newmani and P. moultoni, over Georges Bank, by determining the distribution and abundance of larval, juvenile, and adult stages. Species discrimination will be done using a molecular techniques: polymerase chain reaction (PCR) amplification using species- specific oligonucleotide primers.

2. To distinguish between the life history patterns of the sympatric species of Pseudocalanus over Georges Bank in terms of the seasonal abundance of adults, the timing of reproduction, and the retention of larvae and juveniles in the circulation patterns over the Bank. A number of features of the reproductive ecology of the species will be determined, including size-at-stage to estimate developmental rates for each species.

Research approach:

Temporal and spatial distributions of larval, juvenile, and adult stages of both species will be determined from zooplankton samples of the Georges Bank Study Broadscale Survey cruises. Patterns of distribution will be characterized monthly from January to June of 1997 and 1998 to determine how stage-specific distributions interact with circulation patterns on the Bank. It is hypothesized that the life histories of Pseudocalanus spp. optimize their use of the circulation patterns on the Bank to maximize the likelihood of retention and successful recruitment. Comparison between the stage-specific distributions of the two species in time and space over Georges Bank will help reveal the mechanisms of population maintenance for each species, and whether these differ between species that are morphologically indistinguishable at larval and juvenile stages.

Size-at-stage measurements: All larvae, juveniles, and adults will be measured before molecular assay. Length of the cephalothorax will be determined in staged individuals using a dissecting microscope, and the image analysis software, NIH Image Version 1.52, which the P.I. has used for other studies involving large numbers of individuals (Bucklin et al., 1996b). All size measurements will be made in UNH's Image Analysis Laboratory (C.W. Walker, Director). The proposed research assistant on this project is currently engaged in a related project, and is becoming expert in the identification and staging of copepods. The student research assistant will receive further training to discriminate stages by the Georges Bank Study technical group, at the NOAA/NMFS Narragansett Laboratory and the University of Rhode Island Narragansett campus (E. Durbin, University of Rhode Island, personal communication). Any errors in taxonomic identification are revealed immediately upon molecular analysis.

The goal of this portion of the study is to determine and compare the size frequency distributions of the developmental stages for both species and to document their rates of growth and development. Changes in the size and stage frequency of each species will be documented for samples collected during Broadscale Survey cruises from January to June, 1997 and 1998. The monthly patterns will be evaluated for each species, to determine whether P. moultoni and P. newmani differ in characteristic size-at-stage and in their rates of development.

Molecular analyses: In order to characterize intra- and interspecific patterns of base sequence variation in the mitochondrial 16S rRNA gene, the DNA sequence of a 220 bp region will be determined for up to 100 female individuals of each of P. moultoni and P. newmani. This analysis will ensure that there is no significant intraspecific variation within the priming sites selected. The goal is to identify regions of 14 to 20 bases that differ significantly between species but are conserved within species. One such region has tentatively been identified; it's reliability as a species-specific oligonucleotide will be confirmed.

At the simplest level, PCR reactions will be done with one of the two alternate allele-specific primers paired with a "common" primer. In amplification reactions with appropriate stringencies (i.e., high annealing temperatures; Charlieu, 1994), the primers will not amplify mismatching templates. Discrimination of the species will require two PCR reactions; results of the reactions will be assessed by electrophoresis on an agarose gel. At a more technically-challenging level, both species-specific primers will be placed in one reaction tube with the "common" primer. In this competitive reaction, stringency will be controlled so that the vast majority of the products will result from the perfect-fit primer. Fluorescent labelling of both primers will allow discrimination of the species, and identification of an individual copepod may be determined by a single PCR reaction.

Rationale for technical approach: The technical approach is rapid and inexpensive; it will be possible to assay hundreds to thousands of individuals, and thus to document the distribution and abundance patterns of the two species on Georges Bank and in adjacent regions at sufficiently high spatial resolution. This can only be done if a single, inexpensive step is sufficient to determine an individual's genotype. Since the individual copepods are so small, an obvious approach is one based in gene amplification.

Revisions of work statement in response to budget reduction:

The budget reduction will reduce the planned scope of the sample analysis effort. In Year I, the proposed high-volume Polymerase Chain Reaction (PCR) machine will not be purchased. Instead, we will use two slower PCR machines currently in the P.I.'s laboratory. Since use of slower machines will require more time for the technical assistant, funds recovered (over and above the cost of the PCR machine) will be used to pay a portion of the salary of an undergraduate laboratory assistant. In years II and III, the amount of time the P.I. will spend on this project the will be reduced. The primary impact of this will be that the P.I. will not participate in any of the planned U.S.GLOBEC Broadscale Survey cruises, as she had done during Phase I of the Georges Bank Study.