A new giant species of placented worm and the mechanism by which onychophorans weave their nets (Onychophora: Peripatidae)
December 2010

Bernal Morera-Brenes
Laboratorio de Genética Evolutiva
Escuela de Ciencias Biológicas
Universidad Nacional
Heredia, Costa Rica

Centro de Investigaciones en Estructuras Microscópicas (CIEMIC)
Universidad de Costa Rica
Costa Rica. 3. Vicerrectoría de Investigación2060San José

Universidad Estatal a Distancia
San JoséCosta Rica

Julián Monge-Nájera
A new giant species of placented worm and the mechanism by which onychophorans weave their nets (Onychophora: Peripatidae)

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN
584December 2010Received 17-II-2010. Corrected 20-VI-2010. Accepted 22-VII-2010.1127new speciesnet weaving mechanismlargest onychophoranfossil and extant velvet wormsPeripatidaetaxonomyCosta RicaPeripatus
Onychophorans, or velvet worms, are poorly known and rare animals. Here we report the discovery of a new species that is also the largest onychophoran found so far, a 22cm long female from the Caribbean coastal forest of Costa Rica. Specimens were examined with Scanning Electron Microscopy; Peripatus solorzanoi sp. nov., is diagnosed as follows: primary papillae convex and conical with rounded bases, with more than 18 scale ranks. Apical section large, spherical, with a basal diameter of at least 20 ranks. Apical piece with 6-7 scale ranks. Outer blade 1 principal tooth, 1 accessory tooth, 1 vestigial accessory tooth (formula: 1/1/1); inner blade 1 principal tooth, 1 accessory tooth, 1 rudimentary accessory tooth, 9 to 10 denticles (formula: 1/1/1/9-10). Accessory tooth blunt in both blades. Four pads in the fourth and fifth oncopods; 4th. pad arched. The previously unknown mechanism by which onychophorans weave their adhesive is simple: muscular action produces a swinging movement of the adhesive-spelling organs; as a result, the streams cross in mid air, weaving the net. Like all onychophorans, P. solorzanoi is a rare species: active protection of the habitat of the largest onychophoran ever described, is considered urgent. Rev. Biol. Trop. 58 (4): 1127-1142. Epub 2010 December 01.

Onychophorans, or velvet worms, are small invertebrates that most biologists study in theory but due to their rarity, never see in the real life (Bouvier 1905, 1907, New 1995. There are two living families: the egg-laying Peripatopsidae occuring in Chile, South Africa and Oceania, and the Peripatidae that bears live young and occurs in the Neotropics, and in isolated tropical areas of Africa and Asia (Bouvier 1905, 1907, Ruhberg 1985.

Onychophorans are predators that hunt for small invertebrate prey that they capture with an adhesive net mainly composed of water and protein (Bouvier 1905, Read & Hughes 1987, Mora et al. 1996a. The phylum has been considered a landmark of the evolutionary process, sharing important features with both the annelids and the arthropods (Bouvier 1905, Ballard et al. 1992). In addition, the onychophorans are an ancient group that is known in fossil records from the mid Cambrian (Dzik & Krumbiegel 1989, Hou & Bergstrom 1995 and are regarded as the first animals that could raise their bodies from the substrate and walk (Monge-Nájera & Hou 2000). They originally were a marine taxon with varied body shapes, often protected by spiculae and armor, but the nearly 180 named extant species (Reid 1996, Trewick 1998 are all terrestrial, showing no spiculae or armor and presenting the same simple body plan (Monge-Nájera & Hou 2000).

Previously known onychophorans range in body length from 10mm through 15cm (Read 1988a, b;Ruhberg 1985) and it has been suggested that growth is limited by their tracheal respiratory system and by their lack of a hard skeleton (Monge-Nájera & Lourenço 1995). No formal studies were made, so far, dealing with size constraints caused by the hydraulic skeleton of Onychophora.

The scientific information about most species is limited to the species description and minimal collection data; mating behavior in the wild has not been properly documented (Ruhberg 1985, Tait & Briscoe 1995 and there are many taxonomical problems (Read 1988a, 1988band Reid 1996. Here we describe the uncommonly sized Peripatus solorzanoi sp. nov. from the Caribbean coastal forest of Costa Rica, and provide additional information regarding its taxonomy, behaviour and conservation.

MATERIALS AND METHODS

Scanning Electron Microscopy: Specimens were prepared for SEM according to standard procedures (Morera-Brenes & Monge-Nájera 1990).

DNA isolation and sequencing: DNA was extracted from tissue samples using the Wizard genomic DNA purification kit (Promega) according to the manufacturer's instructions. DNA concentration in aqueous solutions was measured spectrophotometrically and adjusted to 50ng/µl with TE. Polymerase chain reaction (PCR) amplification and sequencing conditions were previously described by Podsiadlowski et al. (2008). A 658 bp fragment (region) of the cytochrome c oxidase subunit I (COX1/ COI) gene was amplified using the primer pair LCO1490/HCO2198 (Folmer et al. 1994). Sequences were obtained from two specimens and confirmed by double-check.

BLAST searches at GenBank provide useful mitochondrial cytochrome oxidase I (COI) gene sequences. Relevant species from every Onychophoran region were included at DNA analysis, including the few Peripatidae available: accession Ns.: Epiperipatus biolleyi, NC_009082-DQ666064 (Podsiadlowski et al. 2008), Oroperipatus corradoi, U62429 (Gleeson et al. 1998), and Peripatopsidae: Metaperipatus inae, NC_010961-EF624055 (Braband et al. 2010).

Sequences were analyzed using Geneious software (Biomatters). Default parameters settings for DNA global alignment with free end gaps (Blosum 62 cost matrix, gap opening 12, extension penalties 3) were used.

The protein-coding sequence of P. solorzanoi n.sp. was inferred by translation in silico using the invertebrate mitochondrial Genetic Code. The COI protein was compared to relevant taxa from the America's: Peripatidae: Epiperipatus biolleyi (ACCESSION ABF93293, Podsiadlowski et al. 2008), Oroperipatus corradoi (ACCESSION AAC95414, Gleeson et al. 1998), and Peripatopsidae: Metaperipatus inae (Mayer 2007;ACCESSION ABQ95564, Braband et al. 2010).

Phylogenetic analysis: This analysis was performed with Geneious Tree Builder. A genetic distance matrix was constructed using the Jukes-Cantor model, and the tree build method was based on the Neighbor Joining Algorithm (Biomatters 2009). A tree was drawn using the Tree View program (Page 1998).

Following Ruhberg (1985) we call the legs "oncopods". Whenever we use a term that is not used by all authors, we include the equivalent. Etymology: Peripatus solorzanoi sp. nov. is dedicated to Costa Rican herpetologist Alejandro Solórzano, who discovered the species, in consideration of his extensive work on the Central American herpetofauna and for his frequent contribution of onychophoran specimens to the University of Costa Rica.

SPECIES DESCRIPTION

Diagnosis:

The following combination of characters: Dorsal primary papillae: Convex and conical with rounded bases; more than 18 scale ranks. Basal piece separated from apical piece by a sligh constriction. Apical section dilated, spherical and symmetric, with a basal diameter of 20 ranks; 6 or 7 scale ranks on apical pieces. Sensory bristle central, thorn-shaped, straight or slightly curved with ornamented basis. Outer blade 1 tooth, 1 accessory tooth, 1 vestigial accessory tooth (formula: 1/1/1); inner blade 1 tooth, 1 accessory tooth, 1 rudimentary accessory tooth, 9 to 10 denticles (formula: 1/1/1/9-10). Accessory tooth blunt in both blades. Four pads in the fourth and fifth oncopods (oncopods="legs"). Nephridial tubercle free from third and fourth pads, in lateral posterior position. The 4th. pad is arched.

Oncopods: Males with 34 leg pairs (n=2) and females with 39-41 leg pairs (39, n=1; 40, n=2; 41, n=11). Total observed animals n=16.

Foot papillae: Three foot papillae, two anterior and one posterior (Fig. 1).

Soles on foot (also known as creeping pads): Four complete creeping pads, without presence of the vestigal fifth one in all oncopods.

Nephridial tubercle: Present in the 4 th and 5 th oncopod pairs, anteriorly displaced and opening between the 3 rd and 4 th creeping pads, free from the 3 rd and indenting the proximal margin of 4 th pad, which is crescent-shaped around it. Fourth creeping pad not divided by the nephridial tubercle (Figs. 8,9).

Integument: Structure of papillae. Dorsal primary papillae convex and conical with rounded bases; without grooves parallel to the main body axis between them. Primary papillae of dorsal surface all of one type, conical, usually 5 to 12 accessory papillae between two of the larger ones. Basal pieces height of >18 scale ranks (Fig. 2). Accessory papillae oriented both to ridge and borders of each fold (Fig. 3).

Apical pieces symmetric, globular-shaped and dilated, with a basal diameter of about 20 ranks, and 6 to 7 scale ranks tall (Fig. 2, 3).

Sensory bristles. Thorn-shaped bristle, little developed, straight or slightly curved. Sensory bristles placed centrally on the apical pieces and bearing an ornamented basis (Fig. 2).

Plicae. Dorsal integument with 12 plicae per segment, arranged in rings separated by straight grooves perpendicular to the main body axis. Seven plicae pass to the ventral side between oncopods.

Mid-line (also known as dorsomedian furrow): The dorsomedian furrow is conspicuous, forming a channel which splits the folds anteroposteriorly (Fig. 3). Hyaline organs absent along the dorsomedian furrow.    two accessory teeth, the first one well developed and blunt and the second less developed or vestigial (Fig. 4B), 9 to 10 denticles (Fig. 4C).

Body size:

In life, holotype body length was 22cm including antennae (reduced to 13cm after preservation in 70% alcohol). Other specimens: adults 7.1-11.7cm, young 2.2-4.0cm (in 70% alcohol).

Color in life:

No dorsal ornamentation but greater dorsal primary papillae may look like dark dots. Oncopods pale or light yellow, contrasting with the darker body. Holotype was light brown (Fig. 5), her newborns were red. Apparently there are two color morphotypes present: light brown or red wine color. We also observed deep brown onychophorans in the area but they might represent a different species.

Molecular analysis:

The COI DNA sequences of the two P. solorzanoi's specimens differ in 15 bases (2.13%) ( Note that E. biolleyi presents a deletion of 1 amino acid (at shown position no. 50) with respect to the other studied species. Unfortunately the comparison between P. solorzanoi and O. corradoi is possible just in a length of 151 amino acids, because the studied sequenced fragment is shorter in the last species. They show a pairwise identity of 92.7% (140 identical sites). Phylogenetic analysis: The available information on the COI protein sequence of the species: P. solorzanoi, E. biolleyi, O. corradoi and M. inae was used to estimate the genetic distances between such taxa. Genetic distance matrix (substitutions per site) is shown at Table 3.

The phylogenetic analysis at the full comparison 151 amino acids COI fragment is shown at Fig. 6. The Caribbean Peripatidae species Peripatus and Epiperipatus are close to each other in a node. The Peripatopsidae M. inae was used as outgroup.

Behavior and net weaving: Individuals of this new species actively forage for prey at night in rivulet banks. They expel adhesive when touched; the amount of adhesive is larger than in other Costa Rican species. They move away from a common flashlight beam. Some individuals were born in captivity, probably prematurely as a result of the mother's postcollecting stress. A high speed film (Fig. 7) shows the animal touching prey with the antennae before expelling two streams of liquid adhesive. Muscular action produces a swinging movement of the adhesive-spelling organs; as a result, the streams cross in mid air, weaving the net.

DISCUSSION

Placement in the Caribbean Group, genus Peripatus:

The new species, P. solorzanoi belongs to the Caribbean group, sensu Bouvier 1905, by presenting three foot papillae arranged laterally, two anterior and one posterior (Figs. 1, 8) and four complete creeping pads with nephridial tubercle opening between the 3 rd and 4 th pad (Figs. 8, 9). These features are shared currently by five neotropical genera: Peripatus Guilding 1826, Epiperipatus (Clark 1913), Macroperipatus (Clark 1913), Plicatoperipatus (Clark 1913) and Speleoperipatus (Peck 1975).

The presence of eyes and only twelve plicae per body segment exclude the possibility of such species being to Speleoperipatus or Plicatoperipatus, respectively. The rounded bases of dorsal primary papillae exclude also Macroperipatus, that bears papillae with quadrangular basis.   (1975), but the type is similar to Epiperipatus (all of one type, Peck 1975). The new species is unique in having 5 to 12 accessory papillae between two of the larger ones. This is somehow similar to Peripatus (the primary papillary tubercles separated by rather broad intervals where the accessory papillae occur, Peck 1975). Read (1988a) believed that the distinction between Peripatus and Epiperipatus was invalid. According to Read's classification (1988a), the number of scale ranks at apical piece in this new species classifies it in the genus Peripatus (sensu strictu).  Fig. 7. Prey capture and feeding behavior of the new species: the onychophoran walks at night looking for prey; (A) touches the potential prey with the antennae, and (B) expels two streams of liquid adhesive from specialized organs that (C) oscillate so that the streams cross in mid air and produce the net. D. Finally, when the prey is secured with the adhesive, it is processed with external digestion. P. solorzanoi is a normal value for intraspecific variation, often sister species differ by >8%, while intraspecific variation is up to 5%. In contrast to the DNA variation, the inferred amino acid sequence is conserved in P. solorzanoi, suggesting that the majority of amino acid positions are constrained within this region of the COI gene.  Differences with similar species: As an aid to identification, we present here some information on how to distinguish the new species from others that occur in the same region. Some of these characteristics may be highly variable within the family, but are reliable within the geographic region considered.

DNA diversity and phylogenetics:

The position of the nephridial tubercle on the fourth and fifth foot distinguishes P. solorzanoi from P. ruber and P. bouvieri because they have "the tubercle largely fused with the third arc" as stated by Bouvier (1905Bouvier ( , 1907. Additionally, P. solorzanoi does not have the fifth pad that P. bouvieri has.

The other species recorded from Costa Rica also differ from P. solorzanoi. The new species differs from E. isthmicola, E. hilkae and E. nicaraguensis because its fourth arc is complete. It also differs from M. valerioi "where the fourth creeping pad is thin and twists around the urinary tubercle, which is completely free and outerly bound" (Morera-Brenes & León 1986). If the size is not considered, the new species resembles Epiperipatus biolleyi (Fig. 9), but it can be separated because the third pad is not indented in P. solorzanoi.

The mandible's outer blade formula in P. solorzanoi (1.1.1) is different from all confirmed and unconfirmed species of Peripatus (as detailed by Read 1988a, b); and it is also different from the other Costa Rican onychophorans (Morera-Brenes & León 1986). Only Plicatoperipatus from Jamaica has a similar outer blade formula. The outer blade shape resembles that of Macroperipatus torquatus from Trinidad, except that the accessory teeth have a more pointing shape in M. torquatus (Read 1988a, b).

The inner blade formula of P. solorzanoi (1/1/1(9-10) is similar to that of Macroperipatus valerioi: (1/1/1/11) (Morera-Brenes & León 1986). The shape of the inner blade also resembles that of M. valerioi, except that in M. valerioi has more denticles and the accessory tooth is more acute (Morera-Brenes & León 1986). It distantly resembles the accessory tooth in the inner blade of "E. brasiliensis" from Venezuela (in quotation marks because we believe this is a misidentification). However, both M. valerioi and "E. brasiliensis" have a well developed second accessory tooth that is lacking in the new species. The bilobular shape of the inner blade diastema resembles those of P. sedgwicki from Venezuela and P. swainsonae from Jamaica.

Both males and females of the new species have more oncopods than other Neotropical Peripatus (sensu stricto) and other Costa Rican onychophorans (Epiperipatus and Macroperipatus).

Geographic distribution: These are the species of Peripatus that have been corroborated with SEM: P. juliformis Guilding from St. Vincent, P. d. dominicae Pollard from Dominica, P. antiguensis Bouv. from Antigua and P. antiguensis from Montserrat, P. d. lachauxensis Brues from Haiti, P. swainsonae Cockerel from Jamaica, P. sedgwicki Bouv. from Venezuela, and the new P. solorzanoi from Costa Rica (Fig. 10). When only these species are considered, Peripatus has a circum-Caribbean distribution, reaching northward from South America into the Central American bridge and Antillean arc. All the Antillean species occur on continental islands, so we hypothesize that the genus colonized the current islands in times of a lower sea level, as suggested by Monge-Nájera (1996). The exception to this hypothesis was Barbados, an oceanic island reported to have a Peripatus species, but Read (1988a, b) concluded that it was misidentified and relocated the species in another genus (as Epiperipatus barbadensis).

The unconfirmed continental species of Peripatus (P. ruber from Costa Rica, P. bouvieri from Colombia, P. brolemani from Venezuela, P. torrealbai from Venezuela (Scorza 1953), P. bavaysi from Guadalupe, P. daniscus from St. Tomas, P. daniscus juanensis from Porto Rico, P. manni from Haiti and P. haitienisis Brues from Haiti) fit our hypothesis. If they were removed from Peripatus in the future, it would not affect our hypothesis.

In contrast with Peripatus, the genus Epiperipatus seems to be more widely distributed in South America. Nevertheless, both genera have the same circum-Caribbean distribution, suggesting they originated from a common ancestor before their parallel expansion (Monge-Nájera 1995). We must note, however, that maybe Peripatus is not a monophyletic genus.

Body size:

The body size has been regarded as of little taxonomic use due to the contraction capacity of Onychophora. Thus, it is difficult to obtain a precise measurement of body length, mainly after fixation, when several degrees of contraction may occur (Read 1988a). Despite Read's statement (1988a), the body size of P. solorzanoi is quite uncommon among extant and also extinct onychophorans and should be regarded as a diagnostic feature in addition with other morphological characters. The size has been already used to help the diagnosis of Metaperipatus inae Mayer 2007 and is very useful to characterize adults of P. solorzanoi. Read (1988a) has previously commented: "The largest onychophorans include the large species of Oroperipatus (section 1 of Peripates Andicoles in Bouvier 1905), and Macroperipatus torquatus. Certain Epiperipatus, including E. lewisi, reach a large size (Arnett 1961)". Of these, Macroperipatus torquatus was, so far, the largest living species with a total length of 15cm (Read 1985). The new species, P. solorzanoi, is even longer with 22cm and its size can be only compared with extinct taxa, namely as the fossil Xenusion Pompeckj, 1927 from the Baltic, with 20cm (Dzik & Krumbiegel 1989) and the not fully identified fossil Jianshanopodia decora that may reach an equal size if confirmed as being an onychophoran (Liu et al. 2006 of the animal's length, but the correlation has important exceptions. The Onychophora's ancestors were a few millimeters long, lived under water and had few but long oncopods (Monge-Nájera 1994, so we suspect that at the extreme size of 22cm, P. solorzanoi is close to the practical limit for a functional onychophoran because they lack a hard skeleton and cannot excavate their own burrows (Monge-Nájera et al. 1993).

Conservation:

The Onychophora's conservation has been previously discussed (Mesibov 1990, New 1995 and as all other species of Onychophora, P. solorzanoi is rare. Despite additional effort to find the species during fieldwork in the Caribbean of Costa Rica, it has never been found outside the limited area where the type was collected. Furthermore, much of the original tropical rainforest in that area has been deforested for farming and housing development. So far, the only known population is limited to a few kilometers of riparian vegetation. So the species is at least under the Vulnerable IUCN category (IUCN 2000). Under these circumstances, we believe that active protection of the habitat of the largest onychophoran ever described, is urgent.

ACKNOWLEDGMENTS

We appreciate the assistance and support of Alejandro Solórzano, who brought the new species to our attention and provided the photograph in Fig. 5-B, José A. Vargas Z. for the photograph in Fig. 5-A, and the British Broadcasting Corporation, through Tim Haynes, for allowing us to reproduce frames from the high speed video of P. solorzanoi (Fig. 7). We are indebted to Lars Podsiadlowski for his great support with DNA sequencing and critical reading of the paper. Francisco Hernández helped greatly with the SEM. We also thank Hilke Ruhberg, Randall Rubí Chacón, Robert Mesibov, V.M.S.J. Read and three anonymous reviewers for suggestions to improve an earlier draft, as well as Sergio Aguilar for preparing the final version of figures. RESUMEN Los onicóforos o "peripatos" son animales escasos y poco conocidos. Aquí informamos el descubrimiento del onicóforo más grandes conocido, una hembra de 22cm de longitud del bosque costero caribeño de Costa Rica. Analizamos los especímenes con microscopia electrónica de barrido. La nueva especie, Peripatus solorzanoi, sp. nov. se caracteriza así: papilas primarias convexas y cónicas, con bases redondeadas y más de 18 filas de escamas de alto. Sección apical grande y esférica, con diámetro basal de al menos 20 filas. Parte apical con 6-7 filas de escamas de alto. Fórmula de lámina mandibular externa 1/1/1, lámina interna: 1/1/1/9-10. Diente accesorio romo en ambas láminas. Cuatro almohadillas en cuarto y quinto oncopodios; cuarta almohadilla arqueada. Describimos el mecanismo, previamente desconocido, mediante el cual tejen su red los onicóforos, usando fotografías para mostrar que es de naturaleza muscular. Como todos los demás onicóforos, P. solorzanoi es una especie escasa: consideramos urgente la protección del hábitat del onicóforo más grande del mundo.

Fig. 1 .
1Oncopod tip of P. solorzanoi in ventral view: foot papillae, (a) two anterior and (b) one posterior. (c) claws. Scale bar = 20 µm.

Fig. 2 .
2Jaw:Outer jaw blade with one principal and one well developed accessory blunt tooth; presence of a vestigial second accessory tooth (Fig. 4A). Inner jaw blade with one Left. Apical section of the primary papillae, with large spherical shape. The apical piece has 7 rank scales. Right: primary papillae with a basal piece >18 scale ranks (rows of scales), from the base to the apical piece. Scale bar = 0.35 mm.

Fig. 3 .
3Upper: apical piece of the primary papillae (pp), with large spherical shape, and a basal diameter of ~20 ranks. The apical piece has 5-6 scale ranks. ( ) The base of the apical seta (sensory bristle) is ornamented. Lower: ( ) the middle dorsal line forms a channel which splits the folds antero-posteriorly (primary papillae). Scale bar= 38 mm. Right: another view of middle dorsal line ( ). Accesory papillae (ac). Scale bar= 200 µm.

Fig. 4 .
4Jaws. (A) Outer blade of mandible with (pt) one principal tooth, (at) one well developed blunt accessory tooth, and (rt) rudiments of a second accessory tooth. Scale bar= 30mm. (B) Inner blade of mandible with two accessory teeth, the first one blunt and well developed, and a rudimentary second tooth. Scale bar= 30 mm. (C) Inner blade of the mandibule, with 9-10 denticles (dt). Scale bar= 150 µm.

Fig. 5 .
5Brown specimen of Peripatus solorzanoi Morera-Brenes and Monge-Nájera 2010.

Fig. 9 .
9Oncopod tip of the 4 th and 5 th leg pairs (ventral view) of the known Costa Rican onychophorans, and Epiperipatus brasiliensis and E. edwardsi.

Species account: Peripatus solorzanoi, n. sp. Holotype: Female. Guayacán de Siquirres (Costa Rica, Limón Province, 10º02'58" N, 83º32'31" W, 400-500m.a.s.l.), 19 February 1996, Alejandro Solórzano. Museo de Zoología, Universidad de Costa Rica, San José (UCRMZ-59-01). Distribution: Guayacán and Liverpool de Siquirres and Barbilla National Park, Limón Province, Costa Rica.Paratypes: Four young that were born
to holotype shortly after capture and other
collected by Miguel Solano, Norberto Solano
and Alejandro Solórzano, 13 August 2000.
Museo de Zoología, Universidad de Costa
Rica, San José (UCRMZ-60-01).

Table 1). Iden-tity=644/658 (97%), gaps=0. Nevertheless, all the differing bases at DNA level do not affect amino acid coding, so the translated aminoacid sequences of 219 length show identical COI proteins in both P. solorzanoi's individuals (Table 2). Such length comparison show a pairwise identity of 97.7% (214 identical sites) between P. solorzanoi and E. biolleyi, both from the same family, but just 89.5% (196 identical sites) between P. solorzanoi and M. inae, species from different families. Coherently E. biolleyi and M. inae show a pairwise identity of 88.6% (194 identical sites).

TABLE . 1
.Nucleotide alignment of two P. solorzanoi individuals' sequences of the cytochrome coxidase subunit I (COI) mitochondrial gene

PE11
1 AACAATATATTTTATTTTTGGTGTATGGTCTGCTATAGTTGGGACTTCGTTAAGTTTATT 60
PE12
AACAATATATTTTATTTTTGGTGTATGGTCTGCTATAGTTGGGACTTCGTTAAGTTTATT

PE11
61 AATTCGAACAGAATTAATAGTTATGGGGAATTTATTAGGTGATGATCAATTGTTTAATGT 120
PE12
AATTCGAACAGAATTAATAGTTATGGGGAATTTATTAGGTGATGATCAATTGTTTAATGT

PE11
121 TATTGTTACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCAATTATAATTGG 180
PE12
TATTGTTACGGCTCATGCTTTTGTTATAATTTTTTTTATAGTTATACCAATTATAATTGG

PE11
181 TGGTTTTGGGAATTGGTTAGTTCCTTTAATGTTAGGTGCACCTGATATGGCATTTCCTCG 240
PE12
TGGTTTTGGGAACTGGTTAGTTCCCTTAATGTTAGGTGCGCCTGATATGGCATTTCCTCG

PE11
241 TTTAAATAATTTGAGTTTTTGATTATTACCTCCTTCTTTTTTTTTGTTGTTGAGATCTTC 300
PE12
TTTAAATAATTTAAGGTTTTGATTATTACCTCCTTCTTTTTTTTTATTGTTAAGATCTTC

PE11
301 AATGGTTGAAAGAGGAGCCGGAACAGGGTGAACTGTATATCCTCCTTTGTCAAGGAATTT 360
PE12
AATGGTTGAAAGAGGAGCTGGAACAGGGTGAACTGTATATCCTCCTTTGTCAAGGAATTT

PE11
361 GACTCATAGTGGGGGATCTGTTGATTTAACTATTTTTTCTTTACATTTGGCTGGTATTTC 420
PE12
AACTCATAGTGGGGGATCTGTTGATTTAACTATTTTTTCTTTACATTTGGCTGGTATTTC

PE11
421 ATCAATTTTGGGTGCATTAAATTTTATTACTACTGTGATTAATATGCGAACTTTTGGGAT 480
PE12
ATCAATTTTGGGTGCGTTAAATTTTATTACTACTGTAATTAATATGCGAACTTTTGGGAT

PE11
481 GGTTTTTGAACGTGTTCCTTTATTTGTTTGATCTGTAAAGATCACGGCTATTTTATTGTT 540
PE12
GGTTTTTGAACGTGTTCCTTTATTTGTTTGATCTGTAAAGATTACGGCTATTTTATTGTT

PE11
541 ATTATCGTTACCTGTTTTGGCAGGTGCTATTACTATGTTATTAACTGATCGAAATTTAAA 600
PE12
ATTATCGTTGCCTGTTTTGGCAGGTGCTATTACTATGTTATTAACTGATCGAAATTTAAA

PE11
601 TACTTCATTTTTTGATCCTGCTGGGGGGGGTGATCCTATTTTATATCAACATTTATTT
658
PE12
TACTTCATTTTTTGACCCTGCTGGGGGGGGTGATCCTATTTTATATCAACATTTATTT

Individuals: PE11(Holotype), PE12 (Paratype).

TABLE 2
2Alignment of amino acid sequences of a portion of the mitochondrial cytochrome c oxidase subunit I (COI) solorzanoi ...........................................A.....I..M....... inae .......S.A........F......TQT...M.....Y..V..A.....I..M....... solorzanoi ............................................................ O. corradoi ????????...............M.........M..IA...................... M. inae .......................M.........L..IG.....................I E. biolley THSGGSVDLTIFSLHLAGISSILGALNFITTVINMRTIGMVFERVSLFVWSVKITAILLL P. solorzanoi .....................................F.......P.............. O. corradoi S...A.............V............I.....Y...M...P.............. M. inae S...............................L....Y..TM..IS..........V... solorzanoi ....................................... O. corradoi ................................L...... M. inae .A..................................... Amino acid sequences of a portion of the mitochondrial cytochrome c oxidase subunit I (COI) inferred from the gene nucleotide sequences in relevant taxa. Dots indicate identity with E. biolleyi amino acids used here as reference protein, (-) delection, (?) unstudied segment. No. 1 corresponds to position #14 at E. biolleyi. Peripatidae: E. biolleyi (ABF93293), P. solorzanoi (Code PE11), O. corradoi (AAC95414), Peripatopsidae: M. inae (ABQ95564)..
1
10
20
30
40
50
60
|
|
|
|
|
|
|
E. biolley
TMYFIFGVWSAMVGTSLSLLIRTELMVMGNLLGDDQLFNVIVTGHAFVM-FFLVMPIMIG
P. O. corradoi
????????????????????????????????????????????????????????????
M. E. biolley
GFGNWLVPLMLGAPDMAFPRLNNLSFWLLPPSFFLLLSSSMVESGAGTGWTVYPPLSSNL
P. E. biolley
LSLPVLAGAITMLLTDRNLNTSFFDPAGGGDPILYQHLF
P.

TABLE 3
3Genetic distance matrix for the relevant species compared in Fig 1 The distribution of dorsal papillae in the new species does not fit the old definition of Peripatus and Epiperipatus made by PeckE. biolley
M. inae
O. corradoi
P. solorzanoi

E. biolley
-

M. inae
0.090226
-

O. corradoi
0.082974
0.090226
-

P. solorzanoi
0.013338
0.097533
0.075777
-

The observed DNA sequence diversity (2.13%) inFig. 6. Neighbor-Joining tree of the Onychophoran Peripatidae: Epiperipatus biolleyi, Peripatus solorzanoi, Oroperipatus corradoi and Peripatopsidae: Metaperipatus inae based on Jukes-Cantor's genetic distances.O. corradoi
U62429

P. solorzanoi

E. biolleyi
DQ666064
0.01

M. inae
EF624055

The phylogenetic analysis at the COI protein showed that the Central American Caribbean Peripatidae species P. solorzanoi and E. biolleyi are close to each other, far from the Andean South American O. corradoi. The South American Peripatopsidae M. inae was used as appropriate outgroup.P. solorzanoi
P. swainsonae
P. sedgwicki

P. juliformis
P. bouvieri
P. ruber

P. brolemanni
P. dominicae
P. evansi

Fig. 8. Oncopod tip of the 4 th and 5 th leg pairs (ventral view) of the genus Peripatus species (confirmed by Scanning Electron
Microscopy).
P. solorzanoi
P. ruber
M. valerioi

E. hilkae
E. isthmicola
E. nicaraguensis

E. brasiliensis (from Venezuela)

E. edwardsi (holotype)
E. brasiliensis
E. biolleyi

). Based on a morphometric analysis, Monge-Nájera & Morera (1994) suggested that the number of oncopods is simply a function Peripatus spp. SEM tested Peripatus spp. Non-testedFig. 10. Geographic distribution of Peripatus. Open circles (yellow): species of Peripatus that have been confirmed with Scanning Electron Microscopy. Closed circles (orange): other described species of Peripatus.P. solorzanoi

P. swainsonae

P. d. lachauxensis

P. antiguensis?

P. antiguensis

P. dominicae

P. juliformis

P. ruber

P. brolemani

P. sedgwicki

P. brolemani

P. sedgwicki?

P. manni

P. haitiensis
P. juanensis P. daniscus

P. bavaysi

P. bouvieri

Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 58 (4): 1127-1142, December 2010

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Computer-assisted resilience training to prepare healthcare workers for pandemic influenza: a randomized trial of the optimal dose of training

Robert G Maunder
William J Lancee
Reet Mae
Leslie Vincent
Nathalie Peladeau
Mary Agnes Beduz
Jonathan J Hunter
Molyn Leszcz
Computer-assisted resilience training to prepare healthcare workers for pandemic influenza: a randomized trial of the optimal dose of training
R E S E A R C H A R T I C L E Open Access
Background: Working in a hospital during an extraordinary infectious disease outbreak can cause significant stress and contribute to healthcare workers choosing to reduce patient contact. Psychological training of healthcare workers prior to an influenza pandemic may reduce stress-related absenteeism, however, established training methods that change behavior and attitudes are too resource-intensive for widespread use. This study tests the feasibility and effectiveness of a less expensive alternative -an interactive, computer-assisted training course designed to build resilience to the stresses of working during a pandemic. Methods: A "dose-finding" study compared pre-post changes in three different durations of training. We measured variables that are likely to mediate stress-responses in a pandemic before and after training: confidence in support and training, pandemic-related self-efficacy, coping style and interpersonal problems.Results: 158 hospital workers took the course and were randomly assigned to the short (7 sessions, median cumulative duration 111 minutes), medium (12 sessions, 158 minutes) or long (17 sessions, 223 minutes) version. Using an intention-to-treat analysis, the course was associated with significant improvements in confidence in support and training, pandemic self-efficacy and interpersonal problems. Participants who under-utilized coping via problem-solving or seeking support or over-utilized escape-avoidance experienced improved coping. Comparison of doses showed improved interpersonal problems in the medium and long course but not in the short course. There was a trend towards higher drop-out rates with longer duration of training. Conclusions: Computer-assisted resilience training in healthcare workers appears to be of significant benefit and merits further study under pandemic conditions. Comparing three "doses" of the course suggested that the medium course was optimal.

Background

This report concerns pilot testing of a computer-assisted training course which was intended to build resilience to stress in healthcare workers facing an influenza pandemic. In particular, this study aimed to determine the optimal "dose" of such training by comparing three versions of the course (differing in duration) on pre-post changes in variables that were considered proximal determinants of resilience.

Well before the onset of the H1N1 influenza pandemic in 2009, pandemic preparedness plans recognized that psychological support of healthcare workers would be necessary during an influenza pandemic [1][2][3][4]. Observations made during and after the 2003 outbreak of severe acute respiratory syndrome (SARS) suggested that an emerging infectious disease causes stress in healthcare settings because of fear of contagion [5][6][7], concern for family health [6,8,9], job stress [6,7], interpersonal isolation [6,7], quarantine [10] and perceived stigma [7,10,11].

Moving beyond plans to support affected healthcare workers after exposure to a pandemic, evidence suggests that psychosocial support and training should be provided to healthcare workers prior to a pandemic to build resilience [12], thereby reducing the impact of stress after exposure. This may be important because predictive models often assume high absenteeism due to illness in healthcare workers and their families, and the stress of dealing with an extraordinary outbreak of infectious disease may further increase absenteeism [13]. Stress-related absenteeism, which may be avoidable with intervention, provides a "second-hit" on a healthcare system which is compromised by other unavoidable losses of human resources. The importance of discretionary absenteeism was highlighted by a report that up to 53% of healthcare workers would refuse to attend work if multiple patients infected with pandemic influenza were admitted to their hospital [14].

We designed an educational intervention to improve resilience to pandemic-related stress. Resilience has been defined as overcoming stress or adversity or, more precisely, as having a good outcome after an adverse experience [15]. The resilience literature is large and varied [16], ranging from studies of individual differences between children that promote healthy outcomes after early life adversity [15] to the factors that protect individuals exposed to extreme traumatic stressors [17,18] or protect adults from mental illness [19]. Nurses' resilience to workplace stresses may be bolstered by processes that enhance the quality of professional relationships and that foster the development of emotional insight, life balance, spirituality, and reflective thinking [20]. Our intention was to design and implement a training intervention which healthcare workers could take prior to an influenza pandemic that would reduce the adverse stress-related effects of a subsequent pandemic exposure.

We first considered the educational goals. Outcomes of pandemic-related resilience, such as reduced eventrelated absenteeism and psychological stress, cannot be measured until exposure to a pandemic occurs. For this pilot we focused instead on proximal outcomes which could facilitate resilience. Studies of the individual and health-system variables which mediated the outcomes of stress related to SARS suggested that the best goals of training would be to (i) increase confidence in being well-supported by the hospital and well-prepared for the pandemic and (ii) enhance adaptive strategies of coping (increasing problem solving and seeking support and decreasing escape-avoidance) [13]. Since the purpose of effective training is to change the behavior of healthcare workers in a pandemic situation, we also proposed, based on the theory of social learning [21], that enhancing self-efficacy (expectations about personal ability to respond adaptively to pandemic-related stresses) should be a goal of training because it is expected to be a proximal predictor of behavior [22]. Finally, since many stressful aspects of an infectious disease outbreak are directly or indirectly of an interpersonal nature (e.g. concern for family health, interpersonal job stresses, interpersonal isolation and perceived stigma) and because interpersonal problems are associated with job stress in healthcare workers [23], reducing interpersonal problems was a goal. These pandemic related stressors could be expected to amplify the stress and strain which is commonly experienced by health care workers in the contemporary health care environment [24].

We next considered the optimal format of training. Since hundreds of thousands of healthcare workers around the world are affected by an influenza pandemic, resilience training should be available at reasonable cost on a very large scale. This is a challenge. Those modes of continuing professional education that demonstrate strong effectiveness regarding behavioral change are resource-intensive interventions designed for the training of individuals and small groups [25]. On the other hand, computer-assisted and Internet-based learning technologies have been promoted as effective strategies to facilitate learning, which may overcome barriers related to timing and cost [26]. Computer-assisted learning also has the advantages of standardization of the course material for all learners and enhanced opportunities for personalization of timing, pace and opportunities for review. It can be provided to large and widely distributed groups of people and, beyond the costs of developing the course, the incremental cost per learner may be modest.

Despite a growing body of literature on computerassisted and Internet-based continuing professional education for health professionals, most studies are methodologically weak [27,28]. Computer-assisted learning has been shown to be a feasible option for educating professionals when compared with printed and/or lecture based continuing education [29]. Compared to no intervention, Internet-based training has a large effect on learner satisfaction, acquisition of knowledge, skills and behavioral change [28]. Compared to other learning modalities the effects on these outcomes have been small, non-significant and inconsistent [28,[30][31][32]. Thus, there is no strong evidence base for the superiority of any particular modality. The application of computerassisted learning to the goals and context of pandemic preparation is untested. Thus one purpose of our research study was to determine if the goals of pandemic resilience training could be accomplished with a computer-assisted educational course that could be widely distributed and self-administered.

Finally, we considered the content of the course and the types of learning experience that might achieve the identified goals. This consideration was guided by experience counseling healthcare workers during and after SARS [9], and by knowledge from other learning resources. We assumed that information about influenza and about stress and coping should be provided but would not, in itself, achieve the goals of training. Based on research in continuing professional education, we expected that the format of the course should address both objective and perceived learning needs, and include both cognitive interactivity and practicing of skills [25,33]. The effect of these modes of training on educational outcomes of Internet-based training has been inconsistent, although practice of prescribed exercises more consistently contributes to skills acquisition [28]. Based on the experience of counseling workers during and after exposure to SARS, we expected that exercises should be provided that not only involve cognitive interactivity but also exposure to affectively charged interpersonal events. The goal of these exercises is to enhance reflective, as opposed to immediately reactive responses to acute interpersonal stressors. The principle of increasing reflection had been valued by our colleagues during facilitated group discussion of SARS-related stressors [34]. We also drew upon previous work on psychological first aid [35,36], stress management [37], and coping [38].

The resulting course was named the Pandemic Influenza Stress Vaccine. We hypothesized that the course would lead to improvements in satisfaction with support and training, coping, pandemic-related self-efficacy and interpersonal problems. It was not known at the time of developing the course what the ideal "dose" of this intervention would be, in terms of the time spent on the course by learners and the comprehensiveness of its curriculum. Previous studies of Internet-based learning have shown that longer duration is positively associated with behavioural outcomes [28]. We expected that a course that was too brief might not achieve the teaching goals while a course that was too long might be unnecessarily burdensome and lead to dropping out. The purpose of this study was to test three versions of the course (short, medium and long) with respect to improvements at the end of the course in the hypothesized outcomes and drop-out rates.

Methods

The setting for this study was Mount Sinai Hospital, a teaching hospital in Toronto, Canada which was directly affected by the 2003 SARS outbreak. All employees and professional staff at were eligible to participate in the study, which was conducted from September 2008 to January 2009. Information about the study was presented at staff meetings and rounds in departments throughout the hospital. Employees and staff who consented to participate were randomized to one of three course lengths: short, medium or long. In order to reduce technological challenges, for the sake of this pilot study the course was accessed on a computer flash drive rather than via the Internet. Participants were provided with a computer flash drive containing the course and instructions for self-administering the training. Participants were instructed to complete the course in several sittings at their own pace, working alone in a quiet setting that allowed them to focus on learning. Participants used the course on computers connected to the Internet, which allowed information provided by the participants (when answering questions or providing information in interactive exercises) to be transmitted back to the researchers and entered in the study database. This process allowed researchers to monitor the progress of participants through the course and the timing of completion. Neither the research assistant administering the course, nor the participant knew which course length was assigned. However, assignment could not be truly blinded because participants were informed that different lengths of the course were being compared and that the typical cumulative duration of these courses was 1.75 hr, 3 hr and 4.5 hr respectively. Those who completed the course received CPE credit. Except for physicians, course completers also received paid educational time. Staff (except physicians) who were not usually entitled to paid educational time were reimbursed at the same rate as staff nurses. The study was approved by the Mount Sinai Hospital Research Ethics Board.

The Pandemic Influenza Stress Vaccine

The course consisted of modules incorporating different modalities of learning. Knowledge-based modules addressed the topics listed below using audio and video mini-lectures accompanied by onscreen notes and printed fact sheets. Quizzes and games provided brief diversions and reinforced knowledge. Relaxation skills were taught with audio modules guiding participants in progressive muscle relaxation, relaxation breathing, imagery, and combined techniques. Self-assessment modules used psychological questionnaires to characterize interpersonal problem and coping style. Feedback was provided that was both individualized and relevant to the context.

List of topics addressed in the Pandemic Stress Vaccine

What to expect during a pandemic. What is resilience? Normal stress responses. How to help others with stress (psychological first aid). Working outside your comfort zone. Moral and ethical dilemmas. An approach to coping. Active listening.

Expressing yourself constructively. Balancing family and work. How to talk to children about disasters and emergencies. Personal and home preparation. Managing drugs and alcohol. Danger signals & resources for getting help.

Interactive reflective exercises consisted of four parts. First, participants watched a 1-3 minute video dramatization of a pandemic-related scenario designed to be realistic and stressful. For example, in one video dramatization (Additional file 1) a nurse is confronted in the hallway by a patient's family member who demands to know why her family member is not receiving a scarce medication and is clearly not satisfied with the nurse's reply. Participants are asked to imagine themselves in a character's role while they watch. Second, participants described responses to the scenario by completing the stems of 3 sentences: "On a good day I might...", "On a bad day I might..." and "Somebody else might..." Third, participants reflected on their immediate response to the scenario and considered a highly individualized menu of explanations for their response, alternative responses and constructive next steps. There are more than 3000 possible logical paths through this exercise, but a sense of the experience can be conveyed with an example. We illustrate the first few steps of one possible path through reflecting on the scenario described above.

Q: What is your initial reaction? A: It makes me angry.

Q: In what way does this make you angry? A: I deserve more respect.

Q: You have answered, "I deserve more respect". How can you make this difficult situation work? A: I will be clear and open in communicating with others.

Q: You recognize the importance of clear communication in this situation. What can you do to facilitate effective communication? Etc. (the reflective exercise continues).

The menu items available for responses, and thus the possible paths through the exercise, are specific to the context of each scenario. Fourth, the participant receives feedback in the form of a brief narrative description of their "reflective path." Each reflective exercise can be completed in less than 5 minutes.

Instruments

Confidence in training and support was measured with a questionnaire derived to measure responses of healthcare workers to SARS [39] that was found to predict long-term effects of SARS [13], modified to apply to influenza. Nine items (e.g. "If I have problems using equipment in an influenza pandemic, I am confident that I will have someone to ask for help") are rated on a 5-point scale from 1 (strongly disagree) to 5 (strongly agree). Summed item scores were normally distributed. Cronbach's alpha was 0.85.

Perceived efficacy to adapt to pandemic conditions was measured with the Pandemic Self-Efficacy Scale, authored for this study. This 24-item scale measures attitudes toward working in a pandemic (e.g. "How confident are you now that in the event of an influenza pandemic you will be able to do your job effectively, even if you are stressed or tired?") rated on a 5-point scale from 1 (Not confident at all. I don't think I can do this) to 5 (Very confident. I am sure of it). Scores were normally distributed. Cronbach's alpha was 0.93.

Interpersonal problems were measured with the 32item Inventory of Interpersonal Problems (IIP-32), an abbreviated version of the IIP-64. Participants rate the degree to which they experience interpersonal problems on a 5-point scale from 0 (not at all) to 4 (extremely). Each of the eight subscales of the IIP (Controlling, Self-Centered, Cold/Distant, Socially Inhibited, Nonassertive, Overly Accommodating, Self-Sacrificing, and Intrusive/ Needy) are calculated as the mean of 4 items. The IIP has adequate test-retest reliability, convergence to an established measure of interpersonal styles, and responsiveness to changes in psychotherapy [40,41]. Total interpersonal problems (sum of 32 item scores) were distributed as a truncated normal curve (skewed toward 0, range 6 -75). Cronbach's alpha was 0.90.

Coping via problem-solving, seeking support from others and escape-avoidance were measured with subscales of the Ways of Coping Inventory [42,43] a widely used instrument which yields eight subscales of coping strategies, which are supported in clinical and non-clinical samples [44]. In this study coping scales were selected that have predictive power with respect to longterm stress-related outcomes of working during the SARS outbreak [13]. Coping scales were calculated as the mean of item scores on a 4 point scale from 0 ("Not used") to 4 ("Used a great deal"). The reliability of the subscales has been adequate in many studies (typically 0.60 to 0.75) [45]. Problem-solving and seeking support were both normally distributed. Cronbach's alpha was 0.76 for problem-solving and 0.77 for seeking support. Escape-avoidance was skewed toward zero. Cronbach's alpha 0.73.

Analysis

Drop-out rates were calculated as proportions and between-group differences in proportions were tested with Pearson's chi-square. An intention to treat analysis of outcome variables was used comparing pre-course (T1) and post-course (T2) values of outcome variables using paired T-tests for all subjects who started the course. Subjects who dropped out between T1 and T2 were assigned T1 values of outcome variables at T2 (i.e. were assigned a pre-post change of zero). Pre-post differences were expressed as mean and 95% confidence intervals of means.

Results

Most participants (86%) were women and more than half (54%) were nurses (Table 1). Of 265 healthcare workers who consented to participate, 158 (59.6%) started the course (recruitment and retention flowchart, Figure 1). The proportion of people dropping out, either before or after starting the course, differed by job type and gender (Table 1). Among nurses, 58% who consented to participate started the course (84 of 144) compared to 87% (45 of 52) of other professionals and 42% (29 of 69) of non-professional staff. Women were more likely to start the course (62%) than men (44%).

Of the 158 hospital workers who started the course, 127 (80%) completed it. There was no significant difference between mid-course drop-outs and course completers with respect to gender or job type. Failure to complete the course after starting occurred in 6 of 51 participants (11.8%) assigned to the short course, 10 of 54 participants (18.5%) assigned to the medium course and 15 of 53 participants (28.3%) assigned to the long course. The trend to greater drop-outs with longer course was nonsignificant (Chi-square = 4.6, df = 2, p = 0.10). The cumulative time required to complete the course is illustrated in Table 2.

Participants showed significant improvements from the start to the end of the course in pandemic self-efficacy, confidence in training and support and interpersonal problems ( Table 3). Dimensions of interpersonal problems which improved included problems with being socially inhibited, nonassertive, overly accommodating, self-sacrificing and intrusive/needy. Dimensions of interpersonal problems which did not improve were being controlling, self-centered and cold/distant (Table 3). It is noteworthy that the severity of interpersonal problems at T1 was mild, with mean scores near a score of 1 on a 0 to 4 scale. For the dimensions of interpersonal problems which did not show a significant overall change, the median score at T1 was 0.25 to 0.5. We therefore tested if there was a change in these dimensions among participants who reported problems (score > 0) at T1. Among these participants there was a significant pre-post decrease in problems with being cold-distancing (n = 104, pre: 0.9 ± SD 0.8; post 0.8 ± 0.8; 95% CI of difference -0.2 -0.0, p = 0.02) and selfcentered (n = 99, pre: 1.0 ± SD 0.9; post 0.8 ± 0.9; 95% CI of difference -0.4 --0.1, p = 0.001) but no significant change in controlling behavior.

Coping with stress using problem-solving, seeking support from others or through escape-avoidance did not change over the course (Table 3). Since coping with problem-solving and seeking support was common at the start of the course, we tested if there was a pre-post improvement among people who were using less of these strategies (score <1.5). Among those who were under-utilizing problem-solving at T1, there was a significant pre-post increase (n = 72, pre: 0.9 ± SD 0.9; post 1.2 ± 0.6; 95% CI of difference 0.2 -0.4, p < 0.001). Similarly, among those who were under-utilizing support-seeking at T1, there was a pre-post increase (n = 72, pre: 0.9 ± 0.9; post 1.0 ± 0.5; 95% CI of difference 0.1 -0.3, p = 0.003). Among participants who reported any use of escape-avoidance at T1 (score > 0), there was a pre-post decrease in this mode of coping (n = 135, pre: 0.7 ± SD 0.5; post 0.6 ± 0.5; 95% CI of difference -0.1 -0.0, p = 0.01).

Comparing the outcomes among the three doses of the Pandemic Influenza Stress Vaccine (Table 4) reveals that each course length resulted in improved self- Difference in drop-out status by gender, Chi-square = 6.8, df = 2, p = 0.03 efficacy and confidence in training and support. Total interpersonal problems decreased for participants of the medium and long courses but not for those who took the short course. Although there were trends toward a decrease in improvement in self-efficacy with increased course length, and an increase in improvement in interpersonal problems with increased course length, between-group differences in change scores were not statistically significant.

Discussion and Conclusions

This study suggests that interactive computer-assisted training for healthcare workers is feasible and may facilitate improvement in psychological variables that predict resilience to the stress of an outbreak of an emerging infectious disease. Improvements were obtained in three of the four targeted domains of psychological function: pandemic self-efficacy, confidence in support and training, and interpersonal problems. Improvements in interpersonal problems were similar to those achieved with brief dynamic psychotherapy, in which improvements in socially avoidant, nonassertive and overly accommodating problems are more readily achieved than improvements in the cold, self-centered and controlling dimensions [46]. In this study, there were specific improvements for all participants in most dimensions of interpersonal problems. For problems of being cold or distant there was an improvement for those who reported any such problem at the start of the course. We attribute this success to two factors. First,   developing skills to respond to interpersonal difficulties was an explicit focus of the course. Second, participants differ from psychotherapy patients in that they are not selected for having psychiatric disorder and, in fact, report mild interpersonal problems.

Coping by the use of problem-solving, seeking support and escape-avoidance did not change over the course of the Pandemic Influenza Stress Vaccine. This may be because the use of coping strategies can be contextdependent. Since the specific stressful events that require coping at T1 and T2 may differ, contextual changes may affect coping more than changes that result from training. On the other hand, the lack of change in these variables may be because prior to training most participants tended to use problem-solving and support seeking frequently and escape-avoidance  infrequently, and thus there was only room for improvement in a subset of the sample. Among those who report under-utilizing support-seeking or problem solving or over-utilizing escape-avoidance at T1, the training was associated with an improvement. There was a substantial attrition of about 40% of healthcare workers who consented to participate in the course but did not take the training. Since these people did not commence taking the course, the attrition is not attributable to their reactions the course itself, and may rather represent an insufficiently considered intention to take the training, obstacles to finding time (such as high competing demands for healthcare workers' time) or a lack of motivation (due to low perceived salience of pandemic stress prior to an actual pandemic or other factors). Since there was no follow-up with drop-outs in this study to determine reasons for attrition, future research is required to identify barriers to learning and the extent of perceived need for resilience training, especially in nonprofessional hospital staff for whom drop-out rates were highest. Empirically demonstrated efficacy would likely bolster the appeal of this training. Once healthcare workers commence the course, dropping out is less common (20%).

Comparison of different doses of the course suggest that the medium length course is sufficient for significant improvements in pandemic self-efficacy, confidence in support and training, and interpersonal problems. Although there was a trend toward larger improvements in interpersonal problems in the long course, there was also a trend toward a higher drop-out rate. The lack of a statistically significant difference in these measures between groups may indicate that the groups are truly similar or may reflect a lack of power. For example, it would require two groups of 182 participants each to confirm the null hypothesis for a between-group difference in the change in interpersonal problems of the magnitude that we found between the short and long course. To the extent that differences between courses can be detected, and following the principle that the shortest course that meets the training goals is the most desirable, the evidence supports the medium length course as the optimal duration.

Although statistically significant benefits were consistently found, the practical significance of these improvements is untested. Bearing in mind that we measured the mediators of stress-outcomes rather than direct measures of pandemic stress, this study leaves unanswered the question of whether modest improvements in self-efficacy, confidence in support and training, and interpersonal problems are sufficient to reduce pandemic-related stress and absenteeism due to stress. A test of this mode of training which directly measures stress and absenteeism under real-world pandemic conditions could answer this question. This course mixes several modalities of teaching. While using mixed modalities is consistent with best practices in continuing professional education [47], it does not allow us to identify which component, if any, is the "effective ingredient" of the Pandemic Influenza Stress Vaccine. In particular, it would be useful to test the specific impact of the reflective exercises, both because this mode of teaching is innovative and because these segments are by far the most resource-intensive to design and implement, and are highly specific to the context of the training.

The conclusions of this study are limited by weaknesses in its design. First there was no comparison between computer-assisted learning and face-to-face educational techniques and so this study provides no information about the relative benefits of these approaches. Second, there was no long-term follow up to determine the stability of the benefits that were found. Third, it was only possible to measure proximal predictors of resilience rather than the participants' actual responses to an influenza pandemic. Further research would benefit from attending to these unanswered questions.

These results suggest that further testing of the Pandemic Influenza Stress Vaccine is warranted. The H1N1 influenza pandemic provides both motivation and opportunity for a randomized controlled trial (RCT) of this training under real-world pandemic conditions. An RCT is now underway in which a modified version of the medium length course is compared to (i) a knowledge-based course with similar content and duration but without the reflective interactive exercises and (ii) a waiting condition. This trial will allow testing of the hypothesis that reflective exercises are necessary for the course's benefits and will allow for longer term (6 month) follow-up. The RCT also expands the outcomes measured to include absenteeism, perceived personal risk and subjective stress. Through this RCT, participating in the Pandemic Influenza Stress Vaccine is available free of charge to any English-speaking hospital-based healthcare worker in the world http://www.msh-healthyminds.com/stressvaccine.

Maintaining the well being of health care workers is essential in sustaining the systems in which care is provided. It is also humane, decent and mirrors core values of health care. We expect that resilience training even independent of a influenza pandemic warrants attention to the development and evaluation of interventions.

Additional file 1: Example of interactive reflective exercise video. Reflective exercises take < 4 min to complete in addition to the time spent viewing the video. The path through the exercise is highly personalized with more than 3000 possible pathways within the program's logic. The goal is not prescriptive, but rather to increase reflection, diminish non-reflective reactive responses to stress and to increase awareness of multiple possible responses to particular situations. The first few steps of one possible pathway is illustrated below. Q: What is your initial reaction? A: It makes me angry. Q: In what way does this make you angry? A: I deserve more respect. Q: You have answered, "I deserve more respect". How can you make this difficult situation work? A: I will be clear and open in communicating with others. Q: You recognize the importance of clear communication in this situation. What can you do to facilitate effective communication? Etc. (the reflective exercise continues).

Figure 1
1Recruitment and participation flowchart.

Table 1
1Distribution of course completers and drop-outs by job type and gender.Post-consent
drop out
(did not start)

Mid-course
drop out
(did not finish)

Completed course
Total

n
%
n
%
n
%
n
%

Job type

Health professional -nurse
60
56%
18
58%
66
52%
144
54%

Health professional -other
7
7%
6
19%
39
31%
52
20%

Other staff
40
37%
7
23%
22
17%
69
26%

Gender

Female
87
81%
25
81%
117
92%
229
86%

Male
20
19%
6
19%
10
8%
36
14%

Total
107
31
127
265

Difference in drop-out status by job type, Chi-square = 26.7, df = 4, p < 0.001

Table 2
2Total (cumulative) duration of the Pandemic Stress Vaccine course.Number of sessions assigned
Cumulative time spent on course (min)

Minimum
Maximum
Median

Short course
7
55
173
111

Medium course
12
75
362
158

Long course
17
124
414
223

Table 3
3Changes in outcome variables from start to finish of course.Pre-Post Difference 1

Table 4
4Course outcomes among three lengths of the Pandemic Influenza Stress Vaccine.Pre-Post Difference

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Authors' contributions RGM conceived of the study, performed the analysis and drafted the manuscript. WJL conceived of the study, authored the interactive training and participated in the analysis. RM produced the reflective videos and coordinated the study. LV, NP, JJH and ML participated in the design of the study and helped to draft the manuscript. MAB consulted on principles of continuing professional education and helped to draft the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Pre-publication history The pre-publication history for this paper can be accessed here. 10.1186/1472-6963-10-72Pre-publication history The pre-publication history for this paper can be accessed here: http://www. biomedcentral.com/1472-6963/10/72/prepub doi:10.1186/1472-6963-10-72

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